CN115227708A - Application of LncRNA IFITM4P targeted small interfering RNA in treatment of oral leukoplakia and/or oral cancer - Google Patents

Abstract

The invention discloses an application of a small interfering RNA targeting LncRNA IFITM4P in treatment of oral leukoplakia and/or oral cancer, wherein the LncRNA IFITM4P nucleotide sequence is shown as Seq ID NO:1 is shown in the specification; the sequence of the small interfering RNA is at least one of the sequence shown in SEQ ID NO.2 and the sequence shown in SEQ ID NO. 3. The invention provides an application of a targeted LncRNA IFITM4P small interfering RNA in oral leukoplakia and/or oral cancer treatment, and also provides an application of a PD-1 monoclonal antibody in preparing a medicine for treating high-expression LncRNA IFITM4P oral leukoplakia and/or oral cancer, provides a new way for treating human oral leukoplakia and/or oral cancer, and provides a new method for developing medicines for treating oral leukoplakia and antitumor medicines.

Description

Small interfering RNA targeting LncRNA IFITM4P in the treatment of oral leukoplakia and/or oral cancer application in therapy

technical field

The present invention relates to the technical field of biomedicine, in particular, to the application of small interfering RNA targeting LncRNA IFITM4P in the treatment of oral leukoplakia and/or oral cancer.

Background technique

Oral cancer (OSCC) is a common malignant tumor with poor prognosis, with more than 300,000 new cases worldwide every year. Oral precancerous lesions refer to certain clinical and histological changes in the oral and maxillofacial region with a tendency to cancer, including leukoplakia, erythema, lichen planus, discoid lupus erythematosus, submucosal fibrosis, papilloma, Chronic ulcers, mucosal black spots and pigmented nevus, etc. Among them, oral leukoplakia (OL) is the most typical type of oral potential malignant disorder (OPMD).

At present, surgical resection and radiation therapy are still the two most effective methods for the treatment of oral leukoplakia and/or oral cancer. Although the survival rate of patients has been greatly improved after surgery, the overall prognosis is still not optimistic, and the main cause of death of patients is The reason is tumor recurrence and metastasis. Therefore, the development of new drugs for the treatment of oral leukoplakia and/or oral cancer has important economic and social benefits.

SUMMARY OF THE INVENTION

In view of the defects in the prior art, the purpose of the present invention is to provide the application of a small interfering RNA targeting LncRNA IFITM4P in the treatment of oral leukoplakia and/or oral cancer.

The first aspect of the present invention provides the application of small interfering RNA targeting LncRNA IFITM4P in the treatment of oral leukoplakia and/or oral cancer, and the nucleotide sequence of the LncRNA IFITM4P is shown in Seq ID NO:1.

In one embodiment of the present invention, the sequence of the small interfering RNA is at least one of the sequence shown in SEQ ID NO.2 and the sequence shown in SEQ ID NO.3.

The second aspect of the present invention provides an RNA drug for the treatment of oral leukoplakia and/or oral cancer, comprising a small interfering RNA targeting LncRNA IFITM4P, the nucleotide sequence of the LncRNA IFITM4P is shown in Seq ID NO: 1.

In one embodiment of the present invention, the sequence of the small interfering RNA is at least one of the sequence shown in SEQ ID NO.2 and the sequence shown in SEQ ID NO.3

A third aspect of the present invention provides a method for screening a drug and/or an anticancer drug for the treatment of oral leukoplakia, comprising the following steps:

S1. Determine the expression level of LncRNA IFITM4P in oral tissue cells, the LncRNA IFITM4P nucleotide sequence is shown in Seq ID NO: 1;

S2, contacting the drug candidate with the cells in step S1;

S3. Determine the expression level of LncRNA IFITM4P in the cells after step S2;

S4. Comparing the expression levels of LncRNA IFITM4P determined in step S1 and step S3, wherein a decrease in the expression level of LncRNA IFITM4P indicates that the candidate drug has the potential to treat leukoplakia and/or anti-cancer.

The fourth aspect of the present invention provides the application of PD-1 monoclonal antibody in the preparation of a drug for treating oral leukoplakia and/or oral cancer with highly expressed LncRNA IFITM4P, the LncRNA IFITM4P nucleotide sequence is shown in Seq ID NO: 1. Compared with the prior art, the embodiments of the present invention have the following beneficial effects: the application of the reagents for reducing or inhibiting the expression of LncRNA IFITM4P provided in the embodiments of the present invention in the preparation of medicines for the treatment of oral leukoplakia and/or oral cancer, proposing PD- 1 The application of monoclonal antibody in the preparation of oral leukoplakia and/or oral cancer drugs for the treatment of highly expressed LncRNA IFITM4P provides a new way for the treatment of human oral leukoplakia and oral cancer, and provides a new way for the development of oral leukoplakia therapeutic drugs and anti-tumor drugs. new method.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Figure 1 shows the expression characteristics of IFITM4P in patient tissues; wherein, Figure 1A is the clinical photos and pathological diagnosis photos of normal human oral mucosa, oral leukoplakia and oral squamous cell carcinoma, Figure 1B is the heat map of the chip detection results, Figure 1C is List of the top ten differentially expressed LncRNAs. Figure 1D shows the results of LncRNA IFITM4P qRT-PCR. Figure 1E shows the pathological diagnosis photos of normal human oral mucosa, oral leukoplakia and oral squamous cell carcinoma and the results of LncRNA IFITM4P in situ hybridization. Figure 1F shows the results Quantitative results of LncRNA IFITM4P in the TCGA database Figure 1G is a schematic diagram of the mouse leukoplakia/squamous cell carcinoma animal model, and Figure 1H is the general photo, pathological diagnosis photo and LncRNA IFITM4P in situ hybridization results of the normal mucous membrane of the mouse tongue and the leukoplakia/squamous cell carcinoma animal model , Figure 1I is the qRT-PCR results of the normal mucous membrane of the mouse tongue and leukoplakia/squamous cell carcinoma LncRNA IFITM4P Figure 2A is the qRT-PCR results of Leuk-1 cell line after RNA interference;

Fig. 2 is that IFITM4P promotes the proliferation and colony formation of OL and OSS; among them, Fig. 2B is the result of CCK8 cell proliferation, Fig. 2C is the result of CCK8 cell proliferation, Fig. 2D is the photo of the culture dish of colony formation, Fig. 2E is the result of colony formation, Fig. 2F Figure 2G is the result of CCK8 cell proliferation, Figure 2H is the result of CCK8 cell proliferation, Figure 2I is the photo of the culture dish of colony formation, Figure 2J is the result of colony formation, Figure 2K is the nude mouse Tumor photos, Figure 2L tumor volume diagram of nude mice;

Figure 3 shows that PD-L1 is a new downstream target of IFITM4P in OL and OSCC; Figure 3A is the RNA sequencing heat map, Figure 3B is the gene set enrichment analysis map, Figure 3C is the gene set enrichment analysis map, Figure 3 3D is the Venn diagram, Figure 3E is the qRT-PCR result, Figure 3F is the western blotting result, Figure 3G is the qRT-PCR result, Figure 3H is the western blotting result, Figure 3I is the qRT-PCR result, and Figure 3J is the TCGA database PD -L1 quantification results, Figure 3K is the pathological diagnosis photos of normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma, LncRNA IFITM4P in situ hybridization results, PD-L1 immunohistochemical results and immunofluorescence photos, Figure 3L is Correlation analysis of qRT-PCR results, Figure 3M is the correlation analysis of qRT-PCR results;

Figure 4 is LPS/TLR4 activation of IFITM4P/PD-L1 signaling pathway to promote signal immunity; Figure 4A is the workflow of the in vitro tumor formation test, Figure 4B is the in vitro tumor formation result graph, and Figure 4C is the in vitro tumor formation volume line graph, Figure 4D is a line graph of mouse body weight, Figure 4E is the qRT-PCR result, Figure 4F is the qRT-PCR result, Figure 4G is the qRT-PCR result, Figure 4H is the luciferase report result, and Figure 4I is the modified mouse leukoplakia /Squamous cell carcinoma model workflow, Figure 4J is the qRT-PCR results and western blotting results, Figure 4K is the correlation analysis of qRT-PCR results, Figure 4L is the mouse leukoplakia PD-1 monoclonal antibody treatment workflow Figure, Figure 4M is the naked eye view of the living tongue of the mouse, and 4N is the score result of the mouse before and after the curative effect;

Figure 5 shows that the interaction between IFITM4P and SASH1 promotes the expression of PD-L1 through the TAK-1-NF-KB signaling pathway; Figure 5A shows the results of RNA pull-down and western blotting, and Figure 5B shows the results of RNA binding protein immunoprecipitation Fig. 5C is the working flow chart of the truncated body, Fig. 5D is the result of RNA-binding protein immunoprecipitation, Fig. 5E is the result of qRT-PCR and western blotting, Fig. 5F is the result of co-immunoprecipitation and qRT-PCR, and Fig. 5G is the result of immunoprecipitation and qRT-PCR. qRT-PCR and western blotting results, Figure 5H is the western blotting results, Figure 5I is the qRT-PCR and western blotting results, Figure 5J is the qRT-PCR results and western blotting results, Figure 5K is the luciferase reporter method result graph;

Figure 6 shows that the ectopic expression of IFITM4P effectively enhances the binding of KDM5A to the Pten promoter and increases the abundance of PD-L1: Figure 6A shows the results of fluorescence confocal microscopy; Figure 6B shows the results of RNA pull-down and western blotting Fig. 6C is the result of RNA binding protein immunoprecipitation; Fig. 6D is the result of chromatin immunoprecipitation; Fig. 6E is the result of western blotting; Fig. 6F is the result of qRT-PCR; Fig. 6G is the mechanism diagram;

Figure 7 is a signal pathway enrichment map;

Figure 8A is the result of western blotting;

FIG. 8B is a CCK8 result diagram;

Figure 8 Cwestern blotting results;

FIG. 8D is a CCK8 result diagram;

Figure 8E is a crystal violet staining image of cell viability;

Figure 8F is a crystal violet staining image of cell viability;

Figure 9A is a graph of qRT-PCR results;

Figure 9B is a qRT-PCR result graph;

Figure 10 is the result of western blotting;

Figure 11 shows the results of laser confocal microscopy and qRT-PCR results;

Figure 12 is a graph showing the results of immunoprecipitation of RNA-binding protein;

Figure 13 is a graph of qRT-PCR results;

Figure 14 shows the results of bioinformatics analysis of protein interactions

Figure 15A is a graph of qRT-PCR results;

Figure 15B is a graph of qRT-PCR results;

Figure 15C shows the results of western blotting and qRT-PCR;

Figure 15D is a graph of qRT-PCR results;

Figure 16A is a graph of qRT-PCR results;

Figure 16B is a graph of qRT-PCR results;

Figure 16C is a graph of qRT-PCR results;

Figure 17A is a flow chart of RNA pull-down experiment;

Figure 17B is a graph of immunoprecipitation results;

Figure 18 is a graph of qRT-PCR results;

Figure 19 is a graph of qRT-PCR results;

Figure 20 is a graph showing the results of correlation analysis at the level of TCGA public genes.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several changes and improvements can be made without departing from the inventive concept. These all belong to the protection scope of the present invention.

Oral cancer (OSCC) is a common malignant tumor with poor prognosis, with more than 300,000 new cases worldwide every year. Oral precancerous lesions refer to certain clinical and histological changes in the oral and maxillofacial region with a tendency to cancer, including leukoplakia, erythema, lichen planus, discoid lupus erythematosus, submucosal fibrosis, papilloma, Chronic ulcers, mucosal black spots and pigmented nevus, etc. Among them, oral leukoplakia (OL) is the most typical type of oral potential malignant disorder (OPMD). The development of oral leukoplakia (OL) to oral cancer (OSCC) is a typical multi-stage carcinogenesis process. However, effective markers for predicting oral carcinogenesis and their mechanisms remain unknown.

The present study found that LncRNA IFITM4P was highly expressed in OSCC compared to normal tissues, and ectopic expression or knockdown of IFITM4P resulted in increased or decreased cell proliferation in vitro and in xenograft tumors, respectively. Mechanistically, in the cytoplasm, IFITM4P acts as a scaffold in the cytoplasm to promote the binding and phosphorylation of SASH1 to TAK1 (Thr187), which in turn increases the phosphorylation of NF-κB (Ser536), and concomitantly induces the expression of PD-L1, resulting in Activation of an immunosuppressive program allows OL cells to evade anticancer immunity in the cytoplasm. In the nucleus, IFITM4P upregulates PD-L1 in OL cells by reducing Pten transcription by enhancing KDM5A binding to the Pten promoter.

The present invention will be further described in detail below with reference to specific embodiments.

The following materials and methods used in each embodiment are as follows:

1. OL/OSCC patients and specimens

Tissue samples for lncRNA chip experiments were obtained from human oral NM (n=3), to OL (n=5), to OSCC (n=4) in a stepwise setup (Table 1).

Table 1 Clinicopathological information of patients in LncRNA microarray experiment

Among them, NM is normal oral mucosa, OL is oral leukoplakia, and OSCC is oral cancer.

Tissue samples used for QRT-PCR validation are shown in Table 2, including NM (n=23), OL (n=67), and OSCC (n=46).

Table 2 Essential characteristics of patients involved in qRT-PCR validation of IFITM4P and PD-L1 expression

Note that all research procedures involving human participants comply with institutional and/or national research council ethical standards, as well as the 1964 Declaration of Helsinki and subsequent revisions or similar ethical standards. Histological examination of all subjects was performed by the applicant according to WHO criteria. "All patients diagnosed with primary OL or OSCC were not treated prior to biopsy or surgery.

2. Cell Culture and Drugs

Leuk-1 cells were cultured in keratinocyte serum-free medium (cat. no. 10744, Gibco, Waltham, MA, USA) and HN4 cells were cultured in DMEM supplemented with 10% fetal bovine serum. Information on the drugs used is shown in Table 3.

table 3

3. lncRNA microarray analysis, RNA-seq and qRT-PCR validation

Total RNA was extracted from cultured cells and tissue samples using TRIzol reagent according to the manufacturer's protocol (TaKaRa, Dalian, Japan). For microarray analysis, Affymetrix (Santa Clara, CA, USA) GeneChip Human Transcriptome Array 2.0 was used according to the manufacturer's protocol.

Sample RNA-seq sequencing was performed using the Illumina HiSeq X Ten sequencing system and detected by Novigen (China Pvt Bioinformatics Company).

For expression verification, cDNA was synthesized using the PrimeScript RT kit with gDNA Eraser (TaKaRa). The mRNA expression level was detected by SYBR green (TaKaRa) RT-PCR method according to the manufacturer's instructions. The primers used for qRT-PCR are shown in Table 4.

Table 4

4. Immunohistochemistry and RNAscope

PD-L1-IHC staining was described previously. OL and OSCC sections were assayed for IFITM4P expression using RNAscope red manual assay (Advanced Cell Diagnostics, Newark, CA, USA) according to the manufacturer's recommendations. The probes were IFITM4P, hs-PPIB-1ZZ (positive control, 701041) and dapB (negative control, 701021) (Advanced Cell Diagnostics).

5. Cell viability and colony formation assays

Cell viability was determined by enzyme-linked immunosorbent assay in 96-well plates (1x10 ⁴ per well). The cell proliferation reagent CCK-8 was as previously described. Absorbance was measured at a background of 480 nm with a microplate reader.

High expression of IFITM4P in Leuk-1 and HN4 cells, as well as IFITM4P-knockout cells and corresponding control cells (1x10 ³ ) in 12-well plates for 14 days, then count more than 50 cells under a dissecting microscope

6. Co-immunoprecipitation, RNA pull-down assay and RNA immunoprecipitation assay

A detailed method description for co-immunoprecipitation (co-IP). IFITM4P-binding protein was studied by RNA pull-down assay analysis using Pierc Magnetic RNA-Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Biotinylated IFITM4P and antisense sequences were synthesized using the Transcription Assisted T7 High Yield Transcription Kit (Thermo Fisher Scientific). Cytoplasmic fractions obtained using the NE-PER protein extraction kit (Thermo Fisher Scientific) were incubated with biotinylated IFITM4P overnight and then precipitated with streptavidin magnetic beads. Extracted proteins were eluted from RNA-protein complexes and analyzed by immunoblotting or silver staining. Silver staining was performed using a silver staining kit (Beyotime, China) according to the manufacturer's instructions. Ubiquitination sites were investigated by MS (Novogene Bio-informatics Technology, Beijing, China).

RIP detection was performed using the EZ-Magna RIP kit (Microwell). Leuk-1 cells ( ^4x108 ) were lysed with complete RIP lysis buffer. Lysates were immunoprecipitated in RIP buffer with anti-HuR antibody coupled to magnetic beads (Abeam, Cambridge, UK), SASH1 antibody or IgG. Precipitated RNA was subjected to qRT-PCR analysis. Mouse IgG and HuR RNA were used as negative and positive control groups, respectively.

7. Chromatin immunoprecipitation assay

ChIP detection was performed using a ChIP detection kit (Cell Signaling Technology [CST], Danvers, MA, USA). Briefly, Leuk-1 cells ( ^5x108 ) were fixed with a final concentration of 1% formaldehyde, cross-linked and sonicated. KDM5A antibody (10 mg/mL, Abcam), IgG control antibody (2 mg/mL, Abcam) were added to the sonicated lysate overnight at 4°C, and then mixed with protein A/G bead mixture (1:1 ratio, CST) at 4°C. Incubate for another >7 hours at °C. Chromatin was DNA eluted, reverse cross-linked and recovered using the achipassay Kit (CST). Input DNA and immunoprecipitated DNA were analyzed by qPCR using the promoter DNA specific primers listed in Table 4.

A modified mouse model of leukoplakia/squamous cell carcinoma

8-week-old male C57Bl/6J mice were used for PD-1 Marble therapy and were purchased from Shanghai Lingchang Biotechnology Co., Ltd. (China). The carcinogen 4NQO (Sigma, St. Louis, MO, USA) solution was placed in 100 μg/mL double distilled water (ddH2O) in advance and stirred overnight at room temperature, and the drinking water was replaced after 1 day. LPS (Solarbio, Beijing, China) was put in advance at a solution concentration of 10 μg/mL ddH2O. A total of 20 mice were assigned to 4 cages (I-IV) of 5 mice each. The drinking regimen and PD-1 mAb treatment for each cage are shown in Figures 4I and 4L.

To examine the effect of IFITM4P against pd-1 treatment, another in vivo mouse model was used in the present invention. For the immunocompetent mouse model, B16F10 cells ( ⁵ x 105 cells in 100 mL of medium) were injected subcutaneously with IFITM4P expressing cells or empty vector into C57BL/6 mice. Tumor growth was measured using digital calipers, and tumor size was recorded. All animal experiments were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals (SCXK [Shanghai 2007-0005]), approved by the Shanghai Institutional Animal Care and Use Committee.

Example 1

RNA extraction and RT-PCR

(1) RNA extraction steps

a) When extracting tissue RNA, use 1 ml of Trizol reagent per 50-100 mg of tissue to dissolve the tissue, add 2-3 iron beads to the 1.5 ml EP tube added to the tissue, and use a cryogenic freezer automatic grinder to break the tissue; extract cells For RNA, centrifuge the cells at 800 g for 5 min, add 1 ml of Trizol to every 5 to 106 cells, and mix with a 1 ml pipette to lyse the cells.

b) The above-mentioned Trizol lysis solution of the tissue or cells was placed at room temperature for 5 minutes to fully lyse the tissue and cells.

c) In the above EP tube, add chloroform (trichloromethane) in an amount of 0.2 ml per 1 ml of Trizol, cover the EP tube, shake it on a vortex shaker for 15 seconds, and place it at room temperature (about 22°C). After 3 minutes, centrifuge at 12000g for 15 minutes at 4°C to separate the organic phase and the RNA-containing aqueous phase.

d) Transfer the upper RNA-containing aqueous phase to a new EP tube, about 400 μl, add an equal volume of isopropanol at the same time, shake on a vortex shaker for 15s, centrifuge at 12000g for 10 minutes at 4°C, and precipitate RNA.

e) Discard the supernatant, add 1 ml of -20°C pre-cooled 75% ice ethanol to the RNA precipitate for washing, shake on a vortex shaker for 10 seconds, centrifuge at 12000g for 10 minutes at 4°C.

f) Discard the supernatant and let the precipitated RNA dry naturally at room temperature for 5-10 min.

g) Dissolve the RNA precipitate with high-pressure RNase-free distilled water, measure the RNA concentration by Nandrop, and then reverse the mRNA into cDNA.

(2) RNA reversed into cDNA

A two-step method was used to reverse the mRNA into a cDNA system. The first system was as follows:

Table 5. The first step system of mRNA reversal into cDNA system

reagent dose RNA 2μg dNTPs 1μl oligo-dT 1μl DEPC water 13μl

Put the above mixed system into the PCR instrument, react at 65°C for 5 minutes, and then immediately set it on ice for 2 minutes. After the first step, prepare the second step system, and add on the basis of the first step system:

Table 6. The second step system of mRNA reverse to cDNA system

reagent dose DTT(0.1M) 2μl reverse transcriptase buffer 4μl RT reverse transcriptase 1μl

Put the second-step system into the PCR instrument, and the PCR system is as follows:

60min extension at 42℃

Inactivate reverse transcriptase at 85°C for 5min

Store at 4°C

After the PCR reaction is completed, collect cDNA for subsequent RT-PCR target gene expression detection or amplify the target gene. The cycle number of the amplified target gene is determined by the specific target gene. The general PCR reaction system is as follows:

(3) Real-time quantitative PCR detection of target gene expression

The real-time quantitative PCR reaction system is as follows:

Table 7. Real-time quantitative PCR system

reagent dose cDNA 0.5μl Upstream primer (10μM) 0.5μl Downstream primer (10μM) 0.5μl SYBR Green 2x mix 5μl distilled water 3.5μl

The reaction conditions were set as follows:

1. 95℃ for 10min

2. 95℃ for 30s

3. 60℃ for 30s

4. 72℃ for 30s (2-4 steps for 30 cycles)

5. Store at 4°C

Make 3 duplicate wells for each test sample, use ABI 7500 real-time fluorescence quantitative PCR instrument to detect the expression of the target gene, calculate the number of cycles required to amplify the specified fluorescence intensity of the target gene, and then calculate the expression intensity of the target gene. The specific data The calculation method is analyzed by the 2-△CT method: ΔCt=Ct(Target gene)–Ct(GAPDH)

(4) Real-time quantitative PCR detection of PD-L1 expression in patient samples. PCR primers are as follows:

Embodiment 2

To identify differentially expressed lncRNAs in oral normal mucosa (NM), OL and OSCC, in this example NM (n=3), OL (n=4) and OSCC (n=5) samples from Chinese patients were subjected to microarray analysis. Array analysis, as shown in Figure 1A and Table 1.

The analysis revealed 3109 interactions between lncRNAs and mRNA transcripts in the NM, OL and OSCC groups (p<0.05, fold difference >2); focusing on 10 differentially expressed lncRNAs (5 genes were up-regulated, 5 genes were down-regulated), the fold difference was >2, ***p<0.0001 (Fig. 1C). The expression of IFITM4P was highest in OSCC/OL and OL/NM groups. To validate these observations, qRT-PCR was used to detect the expression of IFITM4P in samples of oral normal mucosa (NM) (n=23), OL (n=64) and OSCC (n=43), respectively, in contrast to OL and NM In contrast, IFITM4P was most highly expressed in OCSS and least expressed in NM (Fig. 1D), and these results were validated by RNA fluorescence in situ hybridization (FISH) staining of samples from OL, OSCC, and adjacent NM from the same patient, respectively.

In step sampling from the same patient, IFITM4P was not detected in NM, however, IFITM4P was progressively enhanced as OL progressed to early-onset aggressive OSCC (Fig. 1E). In addition, data from The Cancer Genome Atlas (TCGA) indicated that IFITM4P expression was higher in head and neck squamous cell carcinoma (HNSC) tissues (n=519) than in normal tissues (n=44) (**p<0.001) (Fig. 1F). The 4-nitroquinoline-1-oxidation (4NQQ)-induced OL/OSCC model allowed us to study oral epithelial carcinogenesis in vivo. 4NQO-induced immunocompetent mice (C57BL/6) developed OL at 14-16 weeks and OSCC at 22-24 weeks.

To evaluate the effect of IFITM4P on 4NQO-induced oral carcinogenesis in mice, a modified C57/B6J mouse leukoplakia/squamous cell carcinoma (SCC) model was used in this example (Fig. 1G). Macroscopically, the tongues of the mice in the PBS group did not develop any lesions (Fig. 1H), while typical leukoplakia and tumors appeared in the 4NQO group (Fig. 1H). To confirm the results obtained by naked eye observation, H&E staining, qRT-PCR staining and IFITM4P-FISH staining were performed on the tongue lesions of the two groups, respectively. Histopathological diagnosis also proved that the 4NQO group had leukoplakia on the back and local early invasive tongue SCC. FISH staining showed that IFITM4P staining was stronger in the 4NQO group, but not in the PBS group (Figure 1H). In addition, qRT-PCR results showed that, Compared with the PSB group, IFITM4P was increased in the 4NQO group (Fig. 1I).

In conclusion, the expression of lncRNA IFITM4P increases with the development of normal mucosa to OL and OSCC, and IFITM4P is a biomarker in the process of oral carcinogenesis.

Embodiment 3

To determine the role of IFITM4P in oral carcinogenesis, Leuk-1 (OL) and HN4 (OSCC) cells were selected for this experiment, and were manipulated by stably transducing Leuk-1 cells with a lentiviral vector carrying a cDNA encoding the full length of IFITM4P Expression of IFITM4P (FIG. 2A), and cell growth and colony formation in these cells were analyzed. The results showed that exogenous IFITM4P increased cell growth and colony formation of Leuk-1 cells (Figures 2B, 2D and 2E).

To verify the opposite results, the present invention depleted IFITM4P by using IFITM4P-specific short hairpin RNA (shRNA) in Leuk-1 cells (Fig. 2A), and observed a significant reduction in cell growth and colony formation (Fig. 2B, 2D and 2E). Similar results were seen in HN4 cells with high expression (Figures 2F, 2G, 2I and 2J) and deletion (Figures 2F, 2H, 2I and 2J) of IFITM4P. In order to study the effect of IFITM4P in OSCC in vivo, in this example, HN4-Vector and HN4-IFITM4P cells were transplanted into BALB/C nude mice. High expression of IFITM4P in cells increased OSCC growth in vivo compared to the vehicle group (Figures 2K and 2L). Collectively, these data suggest that IFITM4P promotes the proliferation of OL and OSCC cells and that IFITM4P acts as a novel oncogene during oral carcinogenesis.

Embodiment 4

To identify the downstream targets of IFITM4P in regulating Leuk-1 cell proliferation, RNA sequencing (RNA-seq) was performed in this example to identify differentially expressed genes between highly expressed IFITM4P and the vector (Fig. 3A). Gene ontology analysis of RNA-seq data revealed that IFITM4P affects many biological processes, including immune response, innate immune response and inflammatory response (Figure 7). At the same time, the gene signatures regulated by IFITM4P were revealed by Gene Set Enrichment Analysis (GSEA), showing the enrichment of adhesion molecules (Figure 3B and 3C). In this example, the OL group was further compared with the OSCC group by GSEA (Figure 3D), and it was found that PD-L1 was significantly enriched in both groups. In order to verify the effect of IFITM4P on PD-L1 in the process of oral carcinogenesis, this study In the Examples, Leuk-1 and HN4 cells were stably transduced with shRNA targeting IFITM4P or cDNA encoding full-length IFITM4P. IFITM4P was confirmed to induce PD-L1 in Leuk-1 (Figure 3E and 3F) and HN4 cells (Figure 3G and 3H) by qRNA and western blot (WB).

To further evaluate the role of PD-L1 in oral carcinogenesis, this example performed qRT-PCR on human NM (n=23), OL (n=67) and OSCC (n=37) samples (Figure 3I) Detection, qRT-PCR assay showed that the expression of PD-L1 was significantly increased in OSCC samples compared with OL and NM (*p<0.05) (Fig. 3I). TCGA data showed that PD-L1 expression was higher in HNSC tissues (n=519) than in normal tissues (n=44) (*p<0.05) (Fig. 3J).

In addition, PD-L1 immunohistochemistry (IHC), PD-L1 immunofluorescence (IF), and IFITM4P-FISH staining were performed on OL, OSCC, and NM samples in this example, respectively, and PD-L1 and IFITM4P were not detected. As OL progressed to early invasive OSCC, PD-L1 and IFITM4P staining was enhanced (Fig. 3K), analysis showed that in OL (Fig. 3L, *p<0.05, r ² =0.443) and OSCC (Fig. 3M, *p<0.05 , r2=0.623) samples, IFITM4P and PD-L1 levels were positively correlated.

Taken together, PD-L1 is a novel target of IFITM4P in OL and OSCC.

Embodiment 5

In this example, it was verified that IFITM4P/PD-L1 induced by the LPS/TLR4 pathway promotes immune escape in mouse tongue cancer.

To determine the role of PD-L1 downregulation in IFITM4P-mediated cell proliferation, vector (control) or IFITM4P were stably transduced into shPD-L1 and negative control (NC) Leuk-1/HN4 cells in this example (Figures 8A and 8B).

In this example, CCK-8 cell counting kit (cell counting kit-8, CCK-8) was used to detect the effect of PD-L1 on cell growth. Significant reduction of PD-L1 inhibited the growth of IFITM4P Leuk-1/HN4 cells to levels similar to control Leuk-1 and HN4 cells (Figures 8C and 8D).

In order to further clarify that IFITM4P is functionally involved in regulating the expression of PD-L1, a T cell killing experiment was also performed in this example. Cell death was significantly higher than in the NC group. Furthermore, increased PD-L1 levels in shIFTM4P reduced cell death compared to the NC group (Figures 8E and 8F).

To test the effect of IFITM4P on anti-pd-1 treatment, anti-pd-1 mAb was used in this example to treat C57BL/6 mice inoculated with melanoma (B16F10) cells overexpressing control vector or IFITM4P (Figure 4A). Tumor-bearing mice were treated with PD-1 mAb or immunoglobulin G (IgG) isotype (IgG2a). Compared with the vehicle group, the tumor volume of the IFITM4P group was significantly increased (*p<0.05) (Figure 4B, Figure 4C); compared with the control group IgG group, PD-1 mAb-treated mice showed a significant reduction in tumor volume (*p<0.05). 4B and 4C). Figure 4B and Figure 4C show that the anti-tumor effect of PD-1 mAb treatment in the IFITM4P group was significantly better than that in the control group (*p<0.05) (Figure 4B and Figure 4C), but it was observed between the IFITM4P group and the vehicle group There were no significant differences in mouse body weight (Fig. 4D).

Existing studies have found that oral inflammation promotes the progression of OSCC through TLR4. In this example, in order to study the role of TLR4 in the process of oral carcinogenesis through immune escape by IFITM4P, the TLR4 ligand LPS was used as a stimulus. First, the effect of LPS on IFITM4P expression was examined, and it was found that LPS efficiently induced IFITM4P transcription in Leuk-1 cells in a dose-dependent manner (Figures 4E and 9A). Polymyxin B (PmB) normally neutralizes contaminated LPS by preventing the binding of LPS to TLRs. In order to investigate whether the activating factor is LPS, PmB was used to bind and inactivate LPS in the culture medium in this example. It should be noted that the induction of IFITM4P by LPS could be effectively eliminated by adding PmB in Leuk-1 cells, while the addition of PmB alone did not affect the expression of IFITM4P (Fig. 9B); the effect of TLR4 was further verified in this example, It can specifically recognize LPS. In IFITM4P induction, TLR4 deficiency was found to inhibit LPS-induced IFITM4P expression in Leuk-1 cells using TLR4-specific shRNA (Figures 4F and 10).

Next, the effects of TAK-242 (resatorvid), a small molecule that can inhibit the TLR4 signaling pathway and suppress the inflammatory response, were evaluated in this example, and LPS induced changes in IFITM4P mRNA levels in Leuk-1 cells. LPS stimulation resulted in increased IFITM4P mRNA levels compared to controls, and this response was abolished upon addition of TAK-242 (Figure 4G). These findings were confirmed by a luciferase reporter assay showing that IFITM4P is associated with IFITM4P upon LPS stimulation. The luciferase activity of TAK-242 was significantly increased, while the IFITM4P-related luciferase activity was significantly decreased after TAK-242 treatment (Fig. 4H).

In this example, in order to study the effect of LPS-mediated IFITM4P on 4NQO-induced oral carcinogenesis in mice, an improved C57/B6J mouse tongue carcinogenesis model was used (Fig. 4I). The results showed that the mice in cages ⅲ and ⅳ had heavier leukoplakia, dorsal leukoplakia and local early invasive tongue squamous cell carcinoma. The PD-L1 and IFITM4P staining of the tongue SCCs of the III and IV cage mice was stronger than that of the II cage mice's leukoplakia, while the I cage mice's normal tongue mucosa was not stained (Fig. 4J). WB and qRT-PCR analyses consistently showed that the expression of IFITM4P and PD-L1 was similar to IHC and FISH staining (Fig. 4K). In addition, to verify the effect of IFITM4P on anti-PD-1 treatment, PD-1 mAb was used in this example to treat early leukoplakia in mice induced by 4NQO+LPS or 4NQO alone (Fig. 4L). The results showed that after 12 days of PD-1 monoclonal antibody treatment, the leukoplakia of the tongue mucosa of mice was significantly relieved. Compared with the 4NQO-induced group, PD-1 mAb was more effective in the 4NQO+LPS-induced group (Figures 4M and 4N).

Taken together, LPS accelerates tongue carcinogenesis in mice, upregulates IFITM4P expression, and induces tumor immunosuppression by upregulating PD-L1. In addition, elevated expression of IFITM4P increased the sensitivity of PD-1 mAb therapy. Therefore, high IFITM4P may be an indicator of the sensitivity of PD-1 mAb therapy during oral carcinogenesis.

Embodiment 6

In this example, biotin-labeled RNA pull-down assay analysis was performed by mass spectrometry (MS) to identify proteins that interact with IFITM4P.

In order to reduce the non-specificity of RNA pull-down assay/MS results, FISH was used in this example to map the expression of IFITM4P in Leuk-1 cells. FISH staining showed that IFITM4P was mainly expressed in the cytoplasm (Figure 11). Protein 1 containing functional sites SAM and SH3 (SASH1) is a scaffold protein in the TLR4 signaling pathway that assembles signaling complexes downstream of TLR4 to activate early endothelial cell responses to receptor activation. In this example, SASH1 was found to be a potential interacting protein of IFITM4P. In addition, biotin-hur was used as a positive control in this example, and a pull-down experiment of biotin-labeled IFITM4P was performed, and its interaction with SASH1 was verified by WB (Fig. 5A). RNA immunoprecipitation (RIP) assays revealed that SASH1 was significantly enriched for IFITM4P compared to controls (Figure 5B and Figure 12).

The SASH1 protein can be divided into SH3 (amino acids 554-615), SAM1 (amino acids 633-697) and SAM2 (amino acids 1177-1241) functional sites (Fig. 5C). To determine the functional site mediating the interaction with IFITM4P, a 554- to 615-Myc-labeled SASH1 truncation was first generated in this example and RIP detected using Myc antibody. The full-length protein, as well as the SAM1 and SAM2 functional sites of SASH1, bound to IFITM4P, but not the SH3 functional site (Fig. 5D), suggesting that amino acid residues 554–615 are critical for SASH1 binding. Downregulation of SASH1 resulted in the inhibition of PD-L1 expression (Figures 5E and 13). In addition, PD-L1 expression was also significantly decreased after SASH1 knockdown in IFITM4P-overexpressing cells (Fig. 5E). Next, the interaction of SASH1-related proteins and TAK1 (MAP3K7) as a potential interactor of SASH1 was analyzed in this example (Figure 14).

To determine whether there is a physical interaction between SASH1 and TAK1 and whether it is IFITM4P-dependent, we transiently expressed Sh-IFITM4P and performed co-immunoprecipitation (co-IP) assays using TAK1. In Leuk-1 cells, SASH1 could not bind to TAK1 after IFITM4P knockdown (Fig. 5F). To further understand the mechanism of PD-L1 regulation mediated by IFITM4P, we detected the mRNA levels of TAK1 and SASH1 after IFITM4P overexpression and downregulation, respectively. The results showed that IFITM4P had no significant effect on TAK1 and SASH1 (Figures 15A and 15B). Next, we examined the phosphorylation status of TAK1 (Thr187) and TAK1 (Thr412) with increased IFITM4P expression by WB (Figure 15C). Phosphorylation at Thr187 of TAK1 was positively correlated with IFITM4P expression, while phosphorylation at Thr412 of TAK1 was not significantly changed (Figure 15C). In addition, deletion of IFITM4P resulted in decreased phosphorylation of TAK1 (Thr187), whereas elevation of IFITM4P resulted in increased phosphorylation of TAK1 (Thr187) (Fig. 5G). TAK1 is involved in the activation of nuclear factor kB (NF-kB). GSEA showed that features of malignancy, including the NF-kB signaling pathway, were significantly enriched in Leuk-1 cells with a risk score higher than the median risk score (Fig. S1A). Consistent with these findings, stable expression of IFITM4P was found in this example to increase the phosphorylation of NF-kB (Ser536), whereas downregulation of IFITM4P decreased the phosphorylation of NF-kB (Ser536) (Fig. 5G). Furthermore, deletion of SASH1 resulted in reduced expression of NF-kB, p-NF-kB (Ser536) and p-tak1 (Thr187) in vector and IFITM4P expressing Leuk-1 cells (Figures 5H and 15D); NF-kB It was also reduced in shIFITM4P-Leuk-1 cells and vice versa (Figures 5G, 5H and 16A). BAY 11-7082 is an inhibitor of IkBa phosphorylation, which can stabilize IkBa and specifically block the NF-kB signaling pathway. BAY 11-7082 treatment resulted in decreased PD-L1 expression in vector and IFITM4P expressing cells (Figures 5I and 16B). Furthermore, knockdown of TAK1 resulted in reduced (Figures 5J and 16C) PD-L1 levels in IFITM4P-leuk-1 cells.

NF-kB-driven luciferase activity is enhanced in HN4 and LPS-stimulated Leuk-1 cells. However, transfection of these cells with shTAK1, shSASH1 or shIFITM4P inhibited LPS-induced NF-kB signaling, indicating that the activity of NF-kB is completely dependent on the expression of TAK1/SASH1/IFITM4P (Fig. 5K). Taken together, these data suggest that IFITM4P promotes immune evasion in OLs by regulating the SASH1-TAK1-NF-kB-PD-L1 axis.

Taken together, cytoplasmic IFITM4P interacts with SH3, the functional site of SASH1, to promote PD-L1 transcription through the TAK-1/NF-kB pathway in OL.

Embodiment 7

In the aforementioned Example 5, FISH assay was performed to detect the subcellular localization of IFITM4P under LPS stimulation. Upon LPS stimulation, IFITM4P was significantly translocated from the cytoplasm to the nucleus in Leuk-1 (Fig. 6A). In the current study, increasing the amount of histone 3 lysine 4 demethylase KDM5A significantly improved the response to PD-1 antibody therapy in a mouse tumor model. The interaction of IFITM4P with KDM5A was confirmed according to the biotin-labeled RNA pull-down experiments and MS analysis of the present invention (FIGS. 17A and 17B). In addition, biotin-labeled IFITM4P pull-down experiment was performed in the present invention, and biotin-HuR was used as a positive control to verify its interaction with KDM5A by WB. The results showed that the interaction of IFITM4P and KDM5A was dependent on LPS stimulation (Fig. 6B). Furthermore, RIP assay showed that KDM5A could significantly enrich IFITM4P compared with LPS control (Fig. 6C). KDM5A increases the abundance of PD-L1 in tumor cells by inhibiting the PTEN expression pathway and inducing the PI3K-AKT-S6K signaling pathway, and directly interacts with the PTEN promoter (the transcription start site is nearly 3kb) to inhibit Pten transcription. We performed chromatin immunoprecipitation (ChIP) assays to determine whether IFITM4P regulates KDM5A binding to the Pten promoter. The results showed that KDM5A preferentially binds to P1 (2,929-2,819) and P2 (2,751-2,533). Deletion of IFITM4P alone significantly reduced KDM5A binding to Pten, while stable knockout of IFITM4MP-WT (wild-type) transfected with plasmid IFITM4MP-WT reduced the inhibitory effect of KDM5A binding to Pten (Fig. 6D). In the present invention, it was also confirmed by qPCR and WB that overexpression of KDM5A reduced the abundance of PTEN at the transcriptional and protein levels, and knockdown of IFITM4P could reduce this inhibition (Figures 6E and 18). In addition, qRT-PCR showed that the expression of PD-L1 was significantly decreased after KDM5A and NF-kB knockdown, whereas PD-L1 expression was increased in Leuk-1 and HN4 cells after KDM5A or NF-kB knockdown (Fig. 6E) (Figure 19). In order to explore the clinical correlation between IFITM4P and PTEN, TAK1, SASH1 and NF-kB, the expression of HNSC in 519 cases of TCGA was analyzed in the present invention. Among them, IFITM4P was negatively correlated with PTEN expression (p=0.001, r=0.14, n=519; Figure 20), but there was no correlation between other genes. Taken together, the above data suggest that LPS partially induces the entry of IFITM4P into the nucleus, enhances the binding of KDM5A to the Pten promoter, decreases Pten transcription, and thus upregulates PD-L1 in OL.

In conclusion, LPS partially induces IFITM4P to enter the nucleus, enhances the binding of KDM5A to the Pten promoter, and reduces Pten transcription.

Embodiment 8

In this example, the sequence of shRNA was designed by the method of bioinformatics,

Sh1-GCCCAAACCTTCTTCATTCCT

Sh2-TGTCCACCATGATCCATATCT.

The nucleotide sequences of shRNA are also shown in Seq ID NO: 2 and Seq ID NO: 3.

The vectors for knocking down IFITM4P in this example all use PLKO vectors, and the PLKO, PMD2G, and PSPAX vectors used for packaging viruses. The specific steps are as follows:

(1) The 293T cells overgrown in a 6cm dish were passaged into a new 6cm dish by trypsinization, and the cells were passaged at a ratio of 1:3.

(2) Plasmid transfection was started when the cell density reached 80%-90%. The viral packaging plasmids include PMD2G and PSPAX2. During transfection, these two viral packaging plasmids and the PCDH (overexpression)/PLKO (knockdown) plasmid containing the target gene were transferred into 293T cells. The specific transfection process was the same as above, in a 6cm dish , the doses of the three plasmids transfected were PCDH/PLKO:PMD2G:PSPAX2=2μg:1ug:1ug, and 4-6h after transfection, the basal medium of cell culture was replaced with a complete medium of 10% FBS and 1% PS.

(3) Complete medium (4ml) was replaced after 24h.

(4) After 48 hours, the supernatant medium was collected gently, and new complete medium (4 ml) was added to the cells.

(5) After 72h of transfection, continue to collect the supernatant medium, and filter the virus supernatant collected at 48h with a 0.22 μM filter to obtain the virus supernatant, so as to carry out the next step of cell infection or subpackage and store at -80 °C refrigerator.

(6) When the density of Leuk-1 cells or HN4 cells reaches 60%, add the collected 48h or 72h virus supernatant for infection. When virus infection, mix and dilute the virus supernatant and the corresponding cell complete medium (the dilution ratio is: Virus/complete medium = 1:1), discard the medium of the cells to be infected, replace with diluted virus, and add 10 μg/ml Polybrene (1000X dilution) to increase the virus infection efficiency.

(7) After 24 hours of infection, supplement the appropriate medium to continue the culture.

(8) After 48 hours of infection, discard the virus supernatant, add complete medium containing 0.5-2 μg/ml puromycin (different cells need to explore the appropriate screening concentration), screen the infected cells, and screen out the stable The expression/knockdown of cells was verified by RT-PCR or WB for gene overexpression/knockdown.

In this example, IFITM4P was depleted in Leuk-1 cells by using IFITM4P-specific short hairpin RNA (shRNA) (Figure 2A), and a significant reduction in cell growth and colony formation was observed (Figures 2B, 2D, and 2E) .

Overexpression/knockdown of IFITM4P can promote or inhibit the proliferation and clone formation of OL cells.

In this example, two cell lines, the OL cell line Leuk-1 and the OSCC cell line HN4, were selected for experiments. First, we used IFITM4P-overexpressing lentivirus to construct stable transfectants in Leuk-1 cells (see Figure 18). Figure 18 Through lentivirus transfection, IFITM4P was stably overexpressed in Leuk-1 cells, or by specific shRNA Knock out IFITM4P and establish Leuk-1 cells stably overexpressing or knocking down IFITM4P by puromycin selection, and further study the cell proliferation and colony formation ability.

According to the above examples, the present invention discloses a LncRNA IFITM4P, which is activated by LPS/TLR4 in the process of oral carcinogenesis, and up-regulates PD-L1 through dual mechanisms.

According to the above examples, in the nucleus, IFITM4P enhances the binding of KDM5A to the Pten promoter, reduces the transcription of Pten, and thus upregulates PD-L1 in OL cells. KDM5A may have multiple mechanisms to promote PD-L1 abundance. KDM5A increases the abundance of PD-L1 in tumor cells by inhibiting PTEN expression and inducing PI3K-AKT-S6K signaling. In the present invention, it is found that LPS can induce the IFITM4P part to enter the nucleus, enhance the binding of KDM5A to the Pten promoter and reduce the transcription of Pten, thereby up-regulating PD-L1 in OL cells.

SASH1 is a large protein with a predicted molecular mass of 137 kDa and belongs to the SAM and SH3 adaptor families of proteins. It consists of an SH3 functional site (SLY1) expressed in lymphocytes and a hematopoietic adaptor (HACS1, also known as SLY2) containing SH3 and SAM functional site 1. Activation and cleavage of SASH1 by caspase-3 has been shown to mediate a NF-kB-dependent apoptotic response. These findings reveal that SASH1 may be an adaptor protein for multiple signaling pathways. In the present invention, it is found that the IFITM4P/SASH1 complex acts as a scaffold molecule by binding the TAK1 complex, resulting in the activation of NF-kB p65 in OL and OSCC cells. NF-kB has been shown to be a key positive regulator of PD-L1 expression in multiple cancers. This process is largely dysregulated in cancer. Upregulation of PD-L1 in cancer cells is controlled through downstream signaling of NF-kB, including oncogene and stress-induced pathways and inflammatory cytokines. TLR4 has been shown to protect tumors from immune attack in HNSC/OSCC. In the present invention, the TLR4 ligand LPS35 promotes the carcinogenesis of leukoplakia in mice by increasing the levels of IFITM4P and PD-L1. The above examples in the present invention also show that in the process of oral carcinogenesis, the LPS/TLR4 pathway significantly induces IFITM4P.

In conclusion, IFITM4P is gradually induced from OL to OSCC cells through the LPS/TLR4 pathway, and high expression of IFITM4P leads to increased proliferation and enhanced immune escape of OSCC cells by inducing PD-L1 expression. Mechanistically, IFITM4P induces PD-L1 through a dual pathway.

In the cytoplasm, IFITM4P acts as a scaffold to promote the aggregation and binding of SASH1, phosphorylate TAK1 (Thr187), further increase the phosphorylation of NF-kB (Ser536), and induce PD-L1 transcription; in the nucleus, IFITM4P enhances the interaction between KDM5A and the Pten promoter Binding decreased Pten transcription, thereby upregulating PD-L1 in OL cells. In addition, the tumor mice with high expression of IFITM4P showed obvious therapeutic sensitivity to PD-1 monoclonal antibody treatment. In conclusion, IFITM4P can be used as a new therapeutic target for blocking oral carcinogenesis, and PD-1 monoclonal antibody can be used as an effective reagent for the treatment of OSCC with high expression of IFITM4P.

Further detailed analysis and description of the accompanying drawings in the description are as follows: Figure 1A shows typical macroscopic and microscopic findings (H&E staining) of NM, OL, and OSCC (100x); Array analysis, OL (n=4) and OSCC (n=5) samples from Chinese patients were based on differentially expressed RNA transcripts in microarray data (p<0.05, fold change >2), each column represents one sample, each Rows represent a transcript, and the expression level of each gene in a single sample is described according to color scale; shown in Figure 1C, regulated lncRNAs were identified in OL/NM and OSCC/OL, and 10 folds are listed Differentially expressed lncRNAs with a change >2 and p<0.001; in Figure 1D, qRT-PCR analysis showed that IFITM4P had the highest expression in OSCC samples and the lowest expression in NM samples compared to OL and NM samples, **p < 0.001; in Figure 1E, in stepwise samples from the same patient, IFITM4P staining became stronger during OSCC development at OL (200x), and IFITM4P staining was negative in adjacent NMs; in Figure 1F, TCGA data showed, The expression of IFITM4P in HNSC tissues (n=519) was higher than that in normal tissues (n=44), **p<0.001; Figure 1G is a schematic time table of the leukoplakia/squamous cell carcinoma model; Figure 1H appeared in the 4NQO group Typical leukoplakia and squamous cell carcinoma. Histopathological diagnosis also confirmed leukoplakia on the back of the tongue and local early invasive tongue squamous cell carcinoma in the 4NQO group, and FISH showed strong IFITM4P staining in the 4NQO group, but no staining was found in the PBS group (n=6); Figure In II, qRT-PCR results showed that IFITM4P was elevated in the 4NQO group compared with the PBS group, (n=6), **p<0.001.

In Figure 2A, IFITM4P is stably overexpressed and knocked out in white Leuk-1 cells by viral transduction using a specific shRNA; stable cells are established after puromycin selection; in Figure 2B, CCK-8 analysis shows IFITM4P The overexpression of Leuk-1 significantly promoted cell proliferation; in Figure 2C, CCK-8 analysis showed that knockout of IFITM4P significantly inhibited cell proliferation of the cell line Leuk-1; Figure 2D represents the culture dish, and the overexpression of IFITM4P on the surface was quantitatively analyzed in Figure 2E Significantly increased colony formation, whereas knockdown inhibited colony formation in Leuk-1; shown in Figure 2F, in HN4 cells, by viral transduction, stable overexpression of IFITM4P and knockdown of IFITM4P using specific shRNA; purine Stable cells were established after screening with mycin; in Figure 2G, CCK-8 analysis showed that overexpression of IFITM4P significantly promoted the proliferation of HN4 cells; in Figure 2H, CCK-8 analysis showed that knockdown of IFITM4P significantly inhibited cell proliferation in HN4 Figure 2I represents the culture dish and quantitative analysis in Figure 2J showed that overexpression of IFITM4P significantly increased colony formation in HN4, whereas knockdown inhibited colony formation; Figure 2K, IFITM4P promoted HN4 cell growth in vivo (n=6); In Figure 2L, overexpression of IFITM4P in HN4 significantly increased tumor volume. Wherein, the data of panel A, panel B, panel C, panel F, panel G and panel H are shown as the mean ± SD*p<0.05 of three independent experiments. Data in panels E, J and L are shown as the mean±SD*p<0.05 of six independent experiments.

Figure 3A, heatmap of frontier cell adhesion-related genes (analyzed by GSEA) showing the strongest upregulation in Leuk-1 cells by the IFITM4P group; Figure 3B, by GSEA analysis of aligned gene expression data (left, upregulation [ Red]; right, down-regulated [blue]), enrichment map of the resulting vector compared to IFITM4P-expressing Leuk-1 cells. The enrichment fraction is shown as the green line (enrichment fraction = 0.52; **p<0.001); Figure 3C, gene expression data (left, up-regulated [red]; right, down-regulated [blue]) generated by GSEA analysis of OL and Comparison of OSCC enrichment plots, enrichment fraction shown as green line (enrichment fraction = 0.49; **p<0.001); Figure 3D, Venn plot showing overlap between vector and IFITM4P expressing Leuk-1 cells and OL and OSCC Figure 3E and Figure 3F, induction of PD-L1 in leukemia cells by IFITM4P was confirmed by qRT-PCR (E) and WB (F); Figure 3G and Figure 3H, in HN4 cells by qRT-PCR (G) and WB (H) Confirmation that IFITM4P induces PD-L1; Figure 3I, qRT-PCR analysis showed that compared with OL and NM samples, PD-L1 expression was highest in OSCC samples, while PD-L1 expression was lowest in NM samples; Figure 3J , TCGA data analysis showed that the expression of PD-L1 was higher in HNSC tissues (n=519) than in normal tissues (n=44), *p<0.05; Figure 3K, in samples from patients, not observed in NM PD-L1 and IFITM4P staining. However, PD-L1 and IFITM4P staining became stronger as OL progressed to early invasive OSCC (a–l, 200). (L and M) OL (*p<0.05) (L) and OSCC (*p<0.05); Figure 3M, positive correlation between IFITM4P and PL-D1 levels in samples. Among them, the data of Figure 3E, Figure 3G and Figure 3I are shown as the mean ± SD of three independent experiments, *p<0.05.

Figure 4A, 5x 10 ⁵ B16F10 cells expressing IFITM4P or vector were implanted into C57BL/6J mice and treated with PD-1 mAb or an IgG isotype control (IgG2a), with tumor induction and treatment schedules shown; Figure 4B, tumor volume is shown; Figure 4C, introduction of IFITM4P into B16F10 cells significantly increased tumor volume in C57BL/6J mice, and PD-1 mAb significantly reduced tumor volume in C57BL/6J mice bearing B16F10 cells expressing IFITM4P Tumor volume; Figure 4D, the body weight of mice was measured every 3 days, and there was no significant difference in body weight between groups; Figure 4E, qRT-PCR showed a dose-dependent increase in LPS-induced IFITM4P transcription in leukemia cells; Figure 4F, qRT-PCR showed that TLR4 shRNA inhibited LPS-induced (100 mg/mL) IFITM4P expression in Leuk-1 cells; Figure 4G, qRT-PCR showed that TAK-242 (1 mM) inhibited LPS-induced (100 mg/mL) IFITM4P expression in Leuk-1 cells Figure 4H, Inhibition of IFITM4P promoter-driven luciferase activity in 293T cells by TAK-242 (1 mM) in response to LPS (100 mg/mL); Figure 4I, Modified mouse leukoplakia/squamous cells Schematic timeline of the cancer model; Figure 4J, (a) normal tongue (cage I) and (bd) typical leukoplakia (cage II-IV), (c) and (d) larger leukoplakia lesions and rougher texture ( 3rd and 4th cages), histopathological diagnosis: NM (e) (cage I), OL with moderate dysplasia (f) (cage II), OL with severe dysplasia and localized early invasive squamous cell carcinoma (g) and (h) (cage III and IV), (i–l) IHC staining of PD-L1, negative staining (i) (cage i), staining for localized early invasive squamous cell carcinoma (k) and (l) (cage III and IV) are stronger than leukoplakia (j) (cage II), and the early invasive squamous cell carcinoma area is more stained than the adjacent OL area (k). (m–p) FISH staining for IFITM4P, negative staining (m) (cage I), staining (o) and (p) for localized early invasive squamous cell carcinoma (cage III and IV) stronger than leukoplakia (n ) (cage II), the early invasive squamous cell carcinoma area stained more strongly than the adjacent OL area (e–p, 200); Figure 4K, qRT-PCR and WB confirmed that in 4NQO-induced leukoplakia/SCC mice In the model, the expression of IFITM4P increased with the progression of the disease. Compared with the ddH2O control group, LPS significantly increased the expression of IFITM4P and promoted the carcinogenesis of leukoplakia; Figure 4L, PD-1 monoclonal antibody treatment of early leukoplakia mouse model. Timeline schematic; Figure 4M, PD-1 mAb is effective in vitiligo treatment, especially 4NQO and LPS-induced vitiligo. Macroscopic observations before and after PD-1 mAb treatment; Figure 4N, the ratio of tongue lesion scores before and after PD-1 mAb treatment was assessed; wherein, the data in Figure 4E, Figure 4F, and Figure 4G are shown as the mean ± the mean of three independent experiments SD*p<0.05. Data from (Fig. 4B, Fig. 4C, Fig. 4D), (Fig. 4H), (Fig. 4J, Fig. 4K) and (Fig. 4M, Fig. 4N) are shown as mean ± SD from six independent experiments, *P< 0.05.

Figure 5A, Biotin-labeled IFITM4P pull-down and WB showed that IFITM4P specifically co-precipitates with SASH1 in leukemia cells, with biotin HuR and antisense serving as positive and negative controls, respectively. Figure 5B, RIP analysis validated the association of SASH1 with IFITM4P in Leuk-1 cells, with antibodies against GAPDH or control IgG as controls; Figure 5C and Figure 5D, different truncated forms of SASH1 (Figure 5C) and their binding to IFITM4P , using RIP analysis in Leuk-1 cells (Figure 5D); Figure 5E, WB showed that PD-L1 expression was significantly reduced in IFITM4P-overexpressing cells after knockdown of SASH1 using ShSASH1; Figure 5F, by co-IP and qRT-PCR analysis detected the endogenous interaction between IFITM4P, TAK1 and SASH1 in Leuk-1 cells; Figure 5G, WB analysis showed that the expression of pTAK1(Thr187) and pNF-kB p65(Ser536) increased with IFITM4P in Leuk-1 1 cells were increased by ectopic expression, but decreased by knockout of IFITM4P; Figure 5H, WB analysis showed that after SASH1 deletion in control and IFITM4P-expressing Leuk-1 cells, NF-kB, pNF-kB (Ser536 ) and pTAK1(Thr187) levels decreased; Fig. 5I, WB analysis showed that the expression of PD-L1 decreased after BAY 11-7082 (10 mM) treatment in control and IFITM4P-expressing cells; Fig. 5J, WB analysis showed that the expression of Leuk in IFITM4P In -1 cells, shTAK1 knockdown of TAK1 inhibited PD-L1 transcription; Figure 5K, NF-kB-driven luciferase activity was enhanced in LPS-stimulated HN4 and Leuk-1 cells. Transfection of these cells with shTAK1, shSASH1 or shIFITM4P inhibited LPS-induced NF-kB signaling, pGL3.0 served as a control; data in Figure 5B and Figure 5D are shown as the mean±SD*p<0.05 of three independent experiments , data in Figure 5K are shown as the mean ± SD of six independent experiments, *p < 0.05.

Figure 6A, Confocal microscopy showed that FISH analysis (400x) showed that IFITM4P was significantly translocated from the cytoplasm to the nucleus of Leuk-1 under LPS stimulation (100 mg/mL), and the nuclear morphology was assessed by DAPI staining; Figure 6B, Biotinylated IFITM4P pull-down and WB showed that IFITM4P specifically co-precipitated with KDM5A in Leuk-1 cells after LPS stimulation (100 mg/mL) for 12 h, with magnetic beads serving as a negative control. Figure 6C, RIP analysis verified the correlation between KDM5A and IFITM4P in Leuk-1 cells after 12 hours of LPS stimulation (100 mg/mL), and IgG antibody was used as a control; Figure 6D, Chip analysis of the Pten promoter in Leuk-1 cells, Figure In the upper panel in 6D, the KDM5A binding site, and in the lower panel in FIG. 6D, microarray analysis shows that KDM5A specifically binds P1 and P2; deletion of IFITM4P alone significantly reduces the binding of KDM5A to Pten, while transfection of the IFITM4MP-WT plasmid Staining into stable knockout-IFITM4P-Leuk-1 reduced inhibition of KDM5A binding to Pten; chip detection with anti-KDM5A antibody or IgG as controls; detection of P1 and P2 flanks by RT-PCR using a specific primer set of enriched DNA fragments; Figure 6E, WB analysis of relative expression of Pten mRNA in vector and KDM5A-overexpressing Leuk-1 cells under LPS stimulation (100 mg/mL). Figure 6F, IFITM4P-Leuk-1 and Vector-Leuk-1 transiently transfected with ShRNA into NF-kB p65 or KDM5A or disordered control (ShRNA NC), KDM5A or NF-kB p65 vectors treated with LPS (100 mg/mL) cells, qRT-PCR showed that PD-L1 expression was significantly decreased after KDM5A and NF-kB p65 were knocked out, whereas WT-KDM5A or WT-NF-kB p65 increased PD-L1 expression. Figure 6G, Model describing the role of IFITM4P as an oncogene by increasing PD-L1 abundance in IL; where data in panels C, D, and F are shown as mean ± SD*p< 0.05; NS means no significant difference, WL means wild type.

Figure 8A shows the stable expression of PD-L1 in Leuk-1 cells treated with IFITM4P and shPD-L1, and Figure 8B shows the effects of FITM4P and shPD-L1 on the growth of Leuk-1 cells overexpressing empty vector or IFITM4P-Leuk-1, Cell proliferation was determined using the CCK-8 assay, Figure 8C shows the stable expression of PD-L1 in HN4 cells treated with IFITM4P and shPD-L1, and Figure 8D shows the effect of IFITM4P and shPD-L1 on overexpressing empty vector or IFITM4P-Leuk-1. The effect of HN4 cell growth was measured by CCK assay. Figure 8E and Figure 8F show the results of T cell-mediated cancer cell killing assay. Leuk-1 cells (E) or HN4 (HN4 (E) co-cultured with activated T cells for 48 hours F) The cells were stained with crystal violet, and the ratio of Leuk-1 or HN4 to T cells was 1:3;

In Figure 9A, qRT-PCR showed a dose-dependent increase in LPS-induced apoptosis, and IFITM4P was transcribed in Leuk-1 cells after 12 hours; in Figure 9B, LPS alone (400 ng/mL) and LPS (400 ng/mL) were compared ) effect of ng/mL) + PMB (10 μg/mL) treated on IFITM4P after 12 hours of culture, data are shown as mean ± SD of three independent samples. NS = not significantly different, P<0.05.

Western blot in Figure 10 shows that shTLR4 is stably expressed in Leuk-1 cells.

In the upper panel of Fig. 11, images taken by confocal microscopy show the expression of IFITM4P in Leuk-1 cells by localized FISH, and in the lower panel of Fig. 11, qRT-PCR shows that IFITM4P is located in the nucleus of Leuk-1 cells and cytoplasmic expression, data derived from the mean of three independent values, *P<0.05.

The RIP analysis in Figure 12 verifies the correlation of the HuR probe with HuR in Leuk-1 cells, HuR is used as a positive control, (n=3), the data is derived from the mean of three independent values, *P<0.05.

In Figure 13, qRT-PCR showed that PD-L1 expression in IFITM4P-overexpressing cells was significantly reduced after SASH1 knockdown using ShSASH1, data derived from the mean of three independent values, *P<0.05.

Figure 14 shows the SASH1 interaction network bioinformatics analysis related proteins.

According to the results shown in Figure 15A and Figure 15B, IFITM4P had no significant effect on the expression of TAK1 and SASH1.

In Figure 15C, IFITM4P significantly enhanced the phosphorylation of TAK1 (Thr187) at doses of 0-4 ng in Leuk-1 cells, but the phosphorylation of TAK1 (Thr412) was not affected. In Figure 15D, knockdown of SASH1 in Leuk-1-IFITM4P inhibits NF-κB mRNAp65. Data from A, B, D are shown as mean ± standard deviation of three independent samples.

In Figure 16A, ectopic expression of IFITM4P increased the expression level of NF-κB p65 in Leuk-1 cells, which was decreased by its knockdown. In Figure 16B, qRT-PCR showed that PD-L1 transcription was inhibited in Leuk-1 cells treated with BAY 11-7082 (10 μM), expressing the vector or IFITM4P. In Figure 16C, qRT-PCR analysis showed that shTAK1 knockdown TAK1 inhibited PD-L1 transcription in IFITM4P-expressing Leuk-1 cells, data from A, B, D are shown as the mean ± standard of three independent samples Difference.

Figure 17A is a schematic diagram of the RNA pull-down experiment used to identify IFITM4P-related proteins; in Figure 17B, Leuk-1 cells stably overexpressed IFITM4P were treated with LPS for 12 hours, and IFITM4P and its related complexes were IP-enriched with streptavidin magnetic beads set.

In Figure 18, the relative expression of Pten mRNA in vector and KDM5A overexpressing Leuk-1 cells was analyzed by RT-PCR under LPS (100 μg/ml) treatment, data from A, B, D are shown for three independent samples Mean ± standard deviation.

In Figure 19, IFITM4P-HN4 cells and Vector-HN4 cells transfected with ShRNA to NF-κB p65 or KDM5A or scrambled control (ShRNA NC), KDM5A or NF-κB p65 vectors were treated with LPS (100 μg/ml). qRT-PCR showed that PD-L1 expression was significantly decreased after KDM5A and NF-κB p65 were knocked out, whereas KDM5A or NF-κB p65 increased PD-L1 expression. Data are shown as mean ± standard deviation of three independent samples *P < 0.05. NS = no significant difference. WT = wild type.

In Figure 20, data from The Cancer Genome Atlas (TCGA) showed that IFITM4P and PTEN (A) levels were inversely correlated (P<0.05) in HNSC samples (n=518), but IFITM4P levels were significantly correlated with SASH1 (B), NR2C2 ( There was no correlation between TAK1) (C) and NFKB1 (D) levels.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which do not affect the essential content of the present invention. The embodiments of the present application and features in the embodiments may be combined with each other arbitrarily, provided that there is no conflict.

sequence listing

<110> Shanghai Jiaotong University School of Medicine

<120> Small interfering RNA targeting LncRNA IFITM4P in the treatment of oral leukoplakia and/or oral cancer

<160>3

<210>1

<211>342

<212> DNA

<213> Artificial sequences

<400>1

atgaaccaca ctgcccaaac cttcttcatt cctgccaaca gtggctgccc tcccccgccc 60

cccaacccca gctatgagat gctcaaggag gagcatgagg tggctgtgct gggggcgccc 120

cacaaccctg ctcccccaat gtccaccatg atccatatct gcagcgagac ctccgtgtct 180

gactatgttg tctggtccct gtccaacatc ctcttcatga acccccactg cctgggattc 240

atagcattca cctactccct gaagtctagg gacaggaaga tggttggaga cctgactggg 300

gcccaggcct atgcctccac tgccaagtac ctgaacatct ga 342

<210>2

<211>21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

gcccaaacct tcttcattcc t 21

<210>3

<211>21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

tgtccaccat gatccatatc t 21

<110> Shanghai Jiaotong University School of Medicine

<120> Small interfering RNA targeting LncRNA IFITM4P in the treatment of oral leukoplakia and/or oral cancer

<160> 3

<210> 1

<211> 342

<212> DNA

<213> Artificial sequences

<400> 1

atgaaccaca ctgcccaaac cttcttcatt cctgccaaca gtggctgccc tcccccgccc 60

cccaacccca gctatgagat gctcaaggag gagcatgagg tggctgtgct gggggcgccc 120

cacaaccctg ctcccccaat gtccaccatg atccatatct gcagcgagac ctccgtgtct 180

gactatgttg tctggtccct gtccaacatc ctcttcatga acccccactg cctgggattc 240

atagcattca cctactccct gaagtctagg gacaggaaga tggttggaga cctgactggg 300

gcccaggcct atgcctccac tgccaagtac ctgaacatct ga 342

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcccaaacct tcttcattcc t 21

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgtccaccat gatccatatc t 21

Claims (6)

1. Use of a small interfering RNA targeting LncRNA IFITM4P for the treatment of oral leukoplakia and/or oral cancer, wherein the LncRNA IFITM4P nucleotide sequence is as defined in Seq ID NO:1 is shown.

2. The use of the LncRNA IFITM 4P-targeting small interfering RNA according to claim 1 in the treatment of oral leukoplakia and/or oral cancer, wherein the sequence of the small interfering RNA is at least one of the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No. 3.

3. An RNA agent for the treatment of oral leukoplakia and/or oral cancer comprising a small interfering RNA targeting LncRNA IFITM4P, said LncRNA IFITM4P nucleotide sequence being as defined in Seq ID NO:1 is shown.

4. The drug for treating oral leukoplakia and/or oral cancer according to claim 3, wherein the sequence of the small interfering RNA is at least one of the sequence shown as SEQ ID No.2 and the sequence shown as SEQ ID No. 3.

5. A method for screening a drug for treating oral leukoplakia and/or an anticancer drug, comprising the steps of:

s1, determining the expression level of LncRNA IFITM4P in the oral tissue cells, wherein the nucleotide sequence of LncRNA IFITM4P is as defined in Seq ID NO:1 is shown in the specification;

s2, contacting the candidate medicine with the cell in the step S1;

s3, determining the expression level of LncRNA IFITM4P in the cells after the step S2;

s4, comparing the expression levels of LncRNA IFITM4P determined in step S1 and step S3, wherein a decreased expression level of LncRNA IFITM4P indicates that the drug candidate has the potential to treat vitiligo and/or to prevent cancer.

Use of a pd-1 mab in the manufacture of a medicament for treating a high expression LncRNA IFITM4P leukoplakia and/or oral cancer, wherein the LncRNA IFITM4P nucleotide sequence is as defined in Seq ID NO:1 is shown.

Images