CN114832105B - Use of an inhibitor of C7orf50 gene or protein - Google Patents

Abstract

The invention discloses the use of an inhibitor of C7orf50 gene or protein. The invention provides that RNAi knockdown of C7orf50 gene can effectively inhibit the proliferation, invasion and migration of liver cancer cells and the lung metastasis of liver cancer. Through animal experiments (mouse original Tumor model) to verify that the liver tumor volume of RNAi knockdown C7orf50 gene mice was significantly smaller than that of the control group, and RNAi knockdown C7orf50 gene can significantly inhibit the lung metastasis of mouse liver cancer. The invention provides a molecular target for effective treatment of liver cancer, and provides a new direction for the development of new drugs and clinical diagnosis and treatment of liver cancer.

Description

Use of an inhibitor of C7orf50 gene or protein

technical field

The invention relates to the use of an inhibitor of C7orf50 gene or protein, and belongs to the field of molecular biology technology.

Background technique

Although liver cancer is currently the sixth most common tumor and the third leading cause of cancer death in the world, only 5–10% of liver cancer patients can be surgically removed. Because symptoms are not apparent in the early stages, most patients are diagnosed at an advanced stage of the disease, for which treatment options are extremely limited. The pathogenic mechanism of liver cancer is complex, and its occurrence and development are a multifactorial process driven by the accumulation of various genetic and non-genetic factors. Abnormal inactivation of tumor suppressor genes (such as p53), abnormal activation of proto-oncogenes (such as K-ras), abnormal changes of signaling pathways (such as PI3K, MAPK, JAK/STAT, NF-κB, Wnt/β-catenin) , abnormal regulation of epigenetic events, etc. can all participate in the development and process of liver cancer. The progression of liver cancer from abnormal growth to life-threatening metastatic tumors is also accompanied by the accumulation of various factor alterations. Therefore, there is an urgent need in this field to study the molecular mechanism of liver cancer development and regulation, so as to better understand the pathogenesis of liver cancer and develop drugs that can effectively treat liver cancer or inhibit liver cancer metastasis.

Contents of the invention

The technical problem solved by the present invention is: how to develop molecular targets for effective treatment of liver cancer and drugs that can effectively treat liver cancer or inhibit liver cancer metastasis.

In order to solve the above problems, the present invention provides a use of an inhibitor of C7orf50 gene or protein, which is characterized in that it is used for preparing a drug for treating liver cancer and/or for preparing a drug for inhibiting liver cancer metastasis.

Preferably, the medicament includes a medically acceptable carrier and an effective amount of an active ingredient, and the active ingredient is an inhibitor of C7orf50 gene or protein.

Preferably, the C7orf50 gene or protein inhibitor includes C7orf50 gene-specific RNAi, C7orf50 gene-specific microRNA, C7orf50 gene shRNA, C7orf50 gene siRNA, C7orf50 protein antibody or C7orf50 protein activity inhibitor.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention discloses the function of the C7orf50 gene or protein with unknown function, and based on the regulation of the C7orf50 gene or protein in the proliferation and metastasis of liver cancer, knocking down the C7orf50 gene by RNAi can effectively inhibit the proliferation, invasion and migration of liver cancer cells, and inhibit Lung metastasis of liver cancer provides a molecular target that can effectively treat liver cancer, and provides a new direction for the development of new drugs and clinical diagnosis and treatment of liver cancer.

Description of drawings

Fig. 1A is a graphical representation of the expression of the C7orf50 gene extracted from the TCGA database in liver cancer tissues;

Fig. 1B is a graphical representation of the relationship between the expression of C7orf50 gene extracted from the TCGA database and the prognosis of liver cancer;

Fig. 1C is a graphic representation of the expression level of C7orf50 gene in 20 pairs of clinical liver cancer samples, where T is cancer tissue and P is paracancerous tissue;

Figure 1D is a graph showing the results of PCR detection of the expression level of the C7orf50 gene in liver cancer cell lines;

Fig. 1E is a graph showing the results of detecting the expression level of C7orf50 gene in liver cancer cell lines by western blotting;

Fig. 2A is a graphical representation of the expression of C7orf50 mRNA in the virus stable transfection strain of the constructed liver cancer cell line;

Fig. 2B is a graphical representation of the expression of C7orf50 protein in the virus stable transfection strain of the constructed liver cancer cell line;

Fig. 2C is a graphical representation of the effect of C7orf50 knockdown or overexpression on the proliferation ability of liver cancer cell lines detected by cell proliferation assay (CCK-8);

Figure 2D is a graph showing the effect of C7orf50 knockdown or overexpression on the proliferation ability of liver cancer cell lines detected by cell clone formation assay;

Figure 2E is a graph showing the effect of C7orf50 knockdown or overexpression on the migration and invasion ability of liver cancer cell lines detected by cell migration and invasion assay;

Figure 2F is a graph showing the effect of C7orf50 knockdown or overexpression on the migration ability of liver cancer cell lines detected by cell scratch test;

Figure 3A is a graph of the in vivo imaging results of a liver cancer mouse model (in situ tumor) after C7orf50 knockdown or overexpression;

Figure 3B is the results of lung metastases in the liver cancer mouse model (in situ tumor) after C7orf50 knockdown or overexpression (observation of HE staining results with 100X and 200X microscopes);

Figure 3C is a diagram of the fluorescence quantification results of liver tumor in situ after C7orf50 knockdown or overexpression;

Figure 3D is a diagram of the fluorescence quantification results of lung metastases after C7orf50 knockdown or overexpression;

Figure 3E is a graph showing the weight and quantification results of liver tumor in situ after knockdown or overexpression of C7orf50;

Fig. 3F is a graph showing the quantitative results of lung metastatic nodules after C7orf50 knockdown or overexpression.

Detailed ways

In order to make the present invention more comprehensible, preferred embodiments are described in detail below with accompanying drawings.

In the following examples of the present invention, experimental material and general experimental method are as follows:

1. Patients and follow-up

Snap-frozen tumor tissues from patients undergoing radical resection at the Liver Cancer Institute, Zhongshan Hospital, Fudan University (Shanghai, China) were collected and used for western blot (WB) analysis. All patients received a histopathological diagnosis of HCC according to the World Health Organization criteria. Informed consent was obtained from each patient for inclusion in the study, and the use of human subject tissue was approved by the Research Ethics Committee of Zhongshan Hospital, Fudan University. All patients underwent uniform follow-up monitoring after radical resection.

2. Experimental cell lines

The Liver Cancer Institute of Zhongshan Hospital Affiliated to Fudan University developed human liver cancer cell lines MHCC97L, MHCC97H and HCCLM3, which showed progressively increased lung metastatic potential, respectively. The normal liver cell line L02 and the non-metastatic human liver cancer cell lines HepG2 and PLC/PRF/5 were purchased from the American Type Culture Collection. All cell lines were cultured in DMEM (Invitrogen) with 10% fetal bovine serum (Gibco) at 37°C with 5% _CO2 .

3. Western Blotting

Protein lysates were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. After blocking with 5% skimmed milk, they were probed with primary antibodies and species-specific secondary antibodies.

4. RT-qPCR detection method

Total RNA samples were isolated from tissue specimens and cell lines in this study using TRIzol reagent (Invitrogen, USA). cDNA synthesis was performed by PrimeScript RT Kit (TaKaRa, Dalian, China). Real-time polymerase chain reaction (qPCR) was performed using SYBR GreenPremix Ex Taq (TaKaRa, Dalian, China).

5. Construction of stable transgenic plants

The overexpression and knockdown viruses of C7orf50 were synthesized by Jikai Genomics Co., Ltd., and then transfected with liver cancer cell lines and screened with puromycin.

6. Cell Proliferation, Migration and Invasion Assays

(1) Cell proliferation experiment: using the CCK-8 kit, according to the instructions, the cells were placed in a 96-well plate, respectively, on the 0th day, the 1st day, the 2nd day, the 3rd day, the 4th day, and the 5th day Add CCK-8 solution every day, observe and record the OD value, and make the growth curve.

(2) Cell clone formation experiment: cells were seeded in six-well plates and cultured in a cell incubator. After 14 days, they were fixed with paraformaldehyde and stained with crystal violet staining solution, and photographed and statistical data were taken.

(3) Transwell chamber migration and invasion experiment: according to the operation of the Transwell filter chamber (BD Biosciences, USA), place or not place Matrigel glue in the upper chamber, and then take 600 μL of complete medium and place it in a twenty-four-well plate (i.e., the lower chamber). room), put 200 μL of serum-free cells in the upper room, and culture them in a conventional incubator. After 48 hours, take out the small room, rinse with clean water, and then place them in paraformaldehyde for fixation and crystal violet staining. After staining in the liquid, carefully wipe the adherent cells in the upper chamber with a cotton swab and rinse with water, then observe the cells passing through the lower chamber under a microscope, count the cells and take pictures.

(4) Scratch test: cells were inoculated into six-well plates, cultured in a cell incubator, scratched after overgrown, photographed at 0h, 24h, and 48h, and the cell growth conditions were recorded. .

7. Animal models

Four to six-week-old male nude mice (BALB/c nu/nu) were purchased from Shanghai Institute of Physical Medicine, Chinese Academy of Sciences, and all animals were maintained under specific pathogen-free (SPF) conditions. Mice were subcutaneously injected with 3×10 ⁶ liver cancer cells. Tumor volumes were measured at indicated time intervals. Mice bearing tumors were sacrificed 30 days after inoculation and tumors were removed for further study. All experiments were subject to approval by the Animal Care and Use Committee of Shanghai Cancer Institute.

Example 1

Verify that C7orf50 gene or protein is related to liver tumor metastasis ability and prognosis:

1.1 Extract the expression of C7orf50 gene in liver cancer tissue and paracancerous tissue through TCGA database, as shown in Figure 1A, the expression of C7orf50 gene in cancer tissue is higher than that in paracancerous tissue; and extract the expression of C7orf50 gene and the patient The relationship between prognosis, as shown in Figure 1B, the expression level of the surface C7orf50 gene is closely related to the prognosis of patients. 20 cases were randomly selected from previously collected liver cancer samples to detect the expression level of C7orf50 protein. As shown in Figure 1C, C7orf50 protein was highly expressed in most samples (16/20).

1.2 RT-qPCR was used to detect the expression of C7orf50 mRNA in liver cancer cell lines with different metastatic potentials. As shown in Figure 1D, C7orf50 mRNA was relatively highly expressed in high metastatic potential cell lines (HCCLM3, MHCC97H and MHCC97L). Relatively low expression in low metastatic potential cell lines (HepG2 and PLC/PRF/5), and lowest expression in normal liver cell line L0-2; immunoblotting was used to further detect the expression of C7orf50 protein in liver cancer cell lines with different metastatic potential As shown in Figure 1E, C7orf50 protein was relatively highly expressed in high metastatic potential cell lines (HCCLM3, MHCC97H and MHCC97L), relatively low in low metastatic potential cell lines (HepG2 and PLC/PRF/5), and relatively low in The normal liver cell line L0-2 showed the lowest expression. It shows that C7orf50 gene or protein is related to liver tumor metastasis ability.

Example 2

RNAi knockdown of C7orf50 gene can inhibit the proliferation, invasion and migration of liver cancer cells, and can inhibit lung metastasis of liver cancer:

2.1 The knockdown of C7orf50 gene was constructed in HCCLM3 by using lentivirus, and the stable transfection cell line was constructed after knockdown, and the stable transfection cell line with overexpression of C7orf50 gene was constructed in PLC/PRF/5 by lentivirus, and the stable transduction was detected The expression level of C7orf50 mRNA in the cell line is shown in Figure 2A, and the expression level of C7orf50 protein in the stable cell line was detected, as shown in Figure 2B.

2.2 Use cell counting kit 8 (CCK-8) (Dojindo, Japan) to measure the in vitro proliferation ability of HCCLM3 and PLC/PRF/5 cell stable transfection lines, operate according to the instructions of CCK-8, and the experimental results are shown in HCCLM3 cells Knockdown of C7orf50 gene reduces the proliferation ability of hepatocellular carcinoma cells, but the proliferation ability of HCCLM3 cells that restores and supplements C7orf50 gene after knockdown is significantly restored. Overexpression of C7orf50 gene in PLC/PRF/5 cells The proliferative ability of PLC/PRF/5 cells with the gene was also significantly enhanced, as shown in Figure 2C.

2.3 The cell colony formation experiment can detect cell population dependence and proliferation ability. Take the same number of logarithmic growth phase cells in each group as shown in Figure 2D in a culture dish, fix the cells after 2 weeks, and take pictures with crystal violet staining to record the number of clones. The results showed that knocking down the C7orf50 gene could effectively inhibit the proliferation of liver cancer cells, as shown in Figure 2D.

2.4 Cell migration and invasion assay (transwell filter chamber, BD Biosciences, USA) and scratch test can detect the invasion and migration ability of liver cancer cells. The results show that knocking down C7orf50 gene can inhibit the invasion and migration ability of liver cancer cells, while overexpression of C7orf50 will inhibit the invasion and migration ability of liver cancer cells. Enhance the invasion and migration ability of liver cancer cells, the results of cell migration and invasion experiments are shown in Figure 2E, and the results of scratch experiments are shown in Figure 2F.

Example 3

RNAi knockdown of C7orf50 gene can effectively inhibit the growth of liver tumors and lung metastasis of liver cancer:

3.1 The knockdown of C7orf50 gene was constructed in HCCLM3 by using lentivirus, and the stable transfection cell line was constructed after knockdown, and the stable transfection cell line with overexpression of C7orf50 gene was constructed in PLC/PRF/5 by lentivirus, and then the constructed Knockdown of the C7orf50 gene, and the stably transfected cell line that was replenished after the knockdown were injected into mice subcutaneously. The normal HCCLM3 cell line was injected as a control group, and the constructed stable cell line overexpressing the C7orf50 gene was injected subcutaneously into mice. , normal PLC/PRF/5 cell line was injected as a control group, and after tumor formation, a small piece of subcutaneous tumor was orthotopically transplanted into the liver of nude mice (mouse orthotopic tumor model). The in vivo imaging results of the mouse orthotopic tumor model are shown in Figure 3A. The tumor volume of the HCCLM3 cell line with the knockdown of the C7orf50 gene was significantly smaller than that of the control HCCLM3 cell line, and the tumor volume of the C7orf50 genome after knockdown was larger than that of the C7orf50 gene group. The knockdown group increased, and the tumor volume of the PLC/PRF/5 cell line overexpressing the C7orf50 gene was significantly larger than that of the control PLC/PRF/5 cell line, indicating that RNAi knockdown of C7orf50 can inhibit the growth of liver tumors. The fluorescence quantitative results of C7orf50 gene knockdown or overexpression liver tumor in situ are shown in Figure 3C, and the weight quantitative results of C7orf50 gene knockdown or overexpression liver tumor in situ are shown in Figure 3E. The weight of hepatic tumor in situ was significantly lower than that of the control group and the replenishment group, further indicating that RNAi knockdown of C7orf50 can inhibit the proliferation of liver cancer and the growth of liver tumors.

3.2 The lung tissues of liver cancer mice were taken for HE staining. As shown in Figure 3B, the lung metastasis ability of the tumor in situ in the C7orf50 gene knockout group was significantly smaller than that in the control group, while the tumor in situ with C7orf50 gene knockout had Compared with the C7orf50 knockdown group, the ability of lung metastasis was increased, and the orthotopic tumors overexpressing the C7orf50 gene had obvious lung metastasis compared with the control group. The results of fluorescence quantification of lung metastases in C7orf50 gene knockdown or overexpression are shown in Figure 3D, and the quantitative results of lung metastatic nodules in C7orf50 gene knockdown or overexpression are shown in Figure 3F. The number of knots was significantly less than that in the control group and the replenishment group, further indicating that knockdown of C7orf50 by RNAi can effectively inhibit lung metastasis of liver cancer.

The foregoing is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any form and in essence. Several improvements and supplements are made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims (2)

1. Use of an inhibitor of a C7orf50 gene or protein for the preparation of a medicament for treating liver cancer, wherein the inhibitor of a C7orf50 gene or protein comprises RNAi specific for a C7orf50 gene, microRNA specific for a C7orf50 gene, shRNA of a C7orf50 gene, siRNA of a C7orf50 gene, an antibody to a C7orf50 protein, or an inhibitor of activity of a C7orf50 protein.

2. Use of an inhibitor of the C7orf50 gene or protein as in claim 1, wherein the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient that is an inhibitor of the C7orf50 gene or protein.