CN105251019A - Inhibition of invasion and transferring of prostatic cancer by utilization of targeting polar molecule Par3 - Google Patents

Abstract

The invention relates to inhibition of invasion and transferring of prostatic cancer by utilization of a targeting polar molecule Par3. A prostate cancer cell subcloning cell line of the stable knock-down Par3 is constructed, a corresponding nude mouse prostate in-situ transplanting tumor model is established, and the effects of the stable knock-down Par3 on inhibition of invasion and transferring of the prostatic cancer are verified in vitro and in vivo. After the stable knock-down Par3 is discovered, through damage of formation of a Par3-aPKC-KIBRA ternary complex, phosphorylation of an important member MST1/2 and Lats1 in a Hippo passage is enhanced, therefore the Hippo passage is activated, a protooncogene YAP at the downstream is phosphorylated, thus the cytoplasm ofthe YAP protein is retarded, nuclear import of the non-phosphorylated YAP protein is reduced, and finally interaction of YAP and TEAD transcription factors in cell nuclei is weakened, and transcription of a series of invasion/ transferring promoting genes is inhibited. The fact that the stable knock-down Par3 has functions of inhibiting invasion and transferring of prostatic cancer cells is verified in vitro, in vivo, on phenotypes and in molecular mechanism.

Description

Targeting the polar molecule Par3 inhibits the invasion and metastasis of prostate cancer

technical field

The invention belongs to the field of biomedicine, and in particular relates to a polar molecule partitioningdefective3homolog (Par3) expressed by eukaryotic cells. Using specific shRNA to stably target and knock down Par3 in prostate cancer cells can effectively inhibit the invasion and metastasis of prostate cancer, suggesting that Par3 as a potential drug target can be applied to the invasion and metastasis of prostate cancer in targeted gene therapy.

Background technique

Prostate cancer is a common malignant tumor in elderly men, and its incidence rate ranks second among male tumors worldwide. In my country, the incidence of prostate cancer increases significantly after the age of 60 and reaches a peak at the age of 75-79 [1]. Data in recent years have shown that the 5-year survival rate of patients with localized prostate cancer is almost 100%, while the 5-year survival rate of patients with distant metastasis is only 29% [2], revealing that the metastasis of prostate cancer seriously threatens patients survival prognosis. Therefore, systematically and exhaustively studying the mechanisms related to prostate cancer metastasis, screening and identifying effective drug targets, will provide positive guidance for the clinical diagnosis, treatment and prognosis evaluation of prostate cancer metastasis.

Polarity is one of the main characteristics of epithelial cells, which refers to the unequal gradient distribution of the cytoplasmic components of cells in a certain spatial order. The establishment and maintenance of epithelial top-bottom polarity mainly depends on three evolutionarily conserved complexes "PAR complex (Par3/PAR6/aPKC)", "crumbs complex (CRB/MPP5/PATJ)" and "SCRIB complex ( scribble/Lgl/Dlg)" synergistic or antagonistic interaction [3]. Normal polarity distribution is crucial for epithelial cells to perform physiological functions such as secretion, absorption, material transport, and barrier. At the same time, since most human cancers originate from epithelial tissue, the abnormality of epithelial polarity (including abnormal expression and distribution of polar molecules) is also closely related to the occurrence and development of tumors[4,5].

Recent studies have shown that, as a key molecule in regulating polarity, the loss of Par3 molecule in the PAR complex is closely related to the occurrence of breast cancer and skin cancer [4,5]. McCaffrey et al. found that knocking out Par3 in mammary epithelial cells could promote the occurrence of mammary epithelial tumors in the context of Notch and H-Ras overexpression through mouse xenograft tumor experiments [4]. Iden et al. first constructed mice that specifically knocked out Par3 in skin tissue. In vivo experiments showed that tissue-specific knockout of Par3 inhibited the progression of epidermis-derived cutaneous papilloma but promoted the progression of dermis-derived keratoacanthoma, suggesting that polar molecules regulate the complexity and organization of tumor development. specificity [5]. Clinical case analysis showed that the expression level of Par3 was negatively correlated with the prognosis of pancreatic cancer patients and lymph node metastasis of esophageal squamous cell carcinoma [6, 7], and positively correlated with the prognosis of renal clear cell carcinoma [8]. The above animal experiment results and clinical data show that the downregulation of Par3 expression has a tissue-specific regulatory effect on the occurrence and development of tumors from different tissue sources. As a prostate cancer that is also derived from epithelial tissue, so far, the correlation between the invasion/metastasis of the tumor and the expression and distribution of Par3, as well as the corresponding molecular mechanism, have not been systematically reported.

The Hippo-YAP pathway was first discovered in Drosophila as a signaling pathway closely related to the individual development process [9]; in mammals, this pathway is highly homologous to the pathway in Drosophila, mainly composed of two parts: Hippo complex Substances (MST1/2, Lats1/2) and downstream effector molecule YAP[10]. Under normal physiological conditions, when the Hippo pathway is activated by extracellular signals by phosphorylating MST1/2, it further phosphorylates Lats1/2 and finally phosphorylates YAP. Phosphorylated YAP binds to 14-3-3 and α-catenin and is retained in the cytoplasm to prevent it from entering the nucleus and exerting transcriptional regulation functions. Conversely, when the Hippo pathway is at rest, YAP in the cytoplasm is in a non-phosphorylated state and enters the nucleus, where it interacts with the TEAD family of transcription factors to initiate the transcription of various cell proliferation-related genes [11]. Recent studies have shown that in addition to regulating individual development, the Hippo-YAP pathway also plays an important role in tumor development, invasion/metastasis, and drug resistance [12]. Although the molecular mechanism of the Hippo-YAP pathway itself has been basically clarified, the upstream signal that activates the pathway and the regulatory mechanism of signal transduction are still unclear. Not only that, but also the specific mechanism of the pathway's regulation of prostate cancer metastasis has not yet been clarified. However, relevant studies on embryonic development of Drosophila and nematodes have suggested that cell polarity changes seem to be a possible upstream signal source of this pathway, but no relevant reports have been reported in human prostate cancer cells.

[1] RenSC, ChenR, SunYH. Prostate cancer research in China. AsianJAndrol2013; 15(3): 350-353.

[2] ZhangBY, LiYF, LaiY, LiYS, ChenZJ. Effect of compound Chinese traditional medicine PC-SPES II in inhibiting proliferation of human prostate cancer cell LNCa Pandon expressions of AR and PSA. ZhongguoZhongYaoZaZhi2015; 40(5): 950-956.

[3] Martin Belmonte F, Perez Moreno M. Epithelial cell polarity, stem cells and cancer. Nat Rev Cancer 2011; 12(1): 23-38.

[4] McCaffreyLM, MontalbanoJ, MihaiC, MacaraIG.

[5] IdenS, van RielWE, R, SongJY, HiroseT, OhnoS, CollardJG.

[6] GuoX, WangM, ZhaoY, WangX, ShenM, ZhuF, et al. Par3 regulates invasion of pancreatic cancer cells via interaction with Tiam1. ClinExpMed2015Jun18. [Epubaheadofprint]

[7] ZenK, YasuiK, GenY, DohiO, WakabayashiN, MitsufujiS, et al. Defective expression of polarity protein PAR-3gene (PARD3) inesophageal squamous cell carcinoma. Oncogene2009; 28 (32): 2910-2918.

[8] DugayF, LeGoffX, Rioux-LeclerqN, ChesnelF, JouanF, HenryC, et al.

[9]TumanengK, RussellRC, GuanKL.OrgansizecontrolbyHippoandTORpathways.CurrBiol2012;22(9):R368-R379.

[10] HuangJ, WuS, BarreraJ, MatthewsK, PanD. The Hipposignaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, theDrosophila Homolog of YAP. Cell2005; 122(3): 421-434.

[11] ZengQ, HongW.Theemergingroleofthehippathwayincellcontactinhibition,organsizecontrol, andcancerdevelopmentinmammals.CancerCell2008;13(3):188-192.

[12] ChenD, SunY, WeiY, ZhangP, RezaeianAH, etal.LIFRisabreastcancermetastasissuppressorupstreamoftheHippo-YAPpathwayandaprognosticmarker.NatMed2012;18(10):1511-1517.

Contents of the invention

The purpose of the present invention is to provide a target for inhibiting the metastasis of prostate cancer.

Technical scheme of the present invention is as follows:

The use of Par3 as a drug target in the preparation of drugs for inhibiting prostate cancer metastasis.

The application of Par3 as a drug target in the preparation of drugs for inhibiting the invasion of prostate cancer.

The drug for inhibiting prostate cancer metastasis can activate the expression of the tumor suppressor signaling pathway Hippo-YAP by targeting and knocking down Par3, and finally inhibit the entry of the proto-oncogene YAP into the nucleus, thereby blocking the transcription of downstream pro-metastasis genes and inhibiting the expression of prostate cancer. cancer metastasis.

The drug for inhibiting the invasion of prostate cancer can target the knockdown of Par3, activate the expression of the tumor suppressor signaling pathway Hippo-YAP, and finally inhibit the proto-oncogene YAP from entering the nucleus, thereby blocking the transcription of downstream pro-invasion genes , Inhibit the invasion of prostate cancer.

The present invention is the first to explore its regulatory effect on prostate cancer metastasis from the perspective of polar molecule expression and localization changes. In the present invention, for the first time, the influence of the expression change of Par3 on the metastasis of prostate cancer is discussed. The present invention finds for the first time that down-regulating the expression of Par3 can effectively inhibit the invasion/metastasis of prostate cancer. Through molecular mechanism research, the present invention finds for the first time that Par3 is a possible upstream of Hippo-YAP, an important signaling pathway related to metastasis. One of the significance of the present invention is that it is verified for the first time in human prostate cancer cells that the polar molecule Par3 is one of the upstream molecules regulating the Hippo-YAP signaling pathway, and participates in the regulation of prostate cancer invasion/metastasis through this pathway .

The present invention evaluates the inhibitory effect of knocking down Par3 on the invasion/metastasis of prostate cancer in vivo and in vitro by constructing a prostate cancer subclone cell line stably knocking down Par3 and combining it with a prostate cancer orthotopic tumor implantation model. The present invention found that stably knocking down Par3 can activate the expression of the tumor suppressor signaling pathway Hippo-YAP, resulting in the failure of the proto-oncogene YAP to enter the nucleus, thereby inhibiting the transcription of various downstream pro-metastasis genes, and realizing the inhibition of prostate cancer invasion/metastasis . The present invention found that stably knocking down Par3 can effectively inhibit the invasion and metastasis of prostate cancer by regulating the Hippo-YAP signaling pathway, suggesting that Par3 as a potential drug target can be applied to target the invasion and metastasis of prostate cancer into gene therapy. The invention provides positive reference significance for the diagnosis and prognosis of prostate cancer. The research method involved in the invention also provides methodological guidance for other related studies on tumor invasion/metastasis.

The present invention evaluates the feasibility of using Par3 as a potential therapeutic target in targeted gene therapy for prostate cancer invasion/metastasis, and lays the foundation for further clinical application. Specifically, the subclone cell line PC3- shPar3 (corresponding control is PC3-con). In vitro, the ability of stably knocking down Par3 to inhibit the migration of prostate cancer cells was evaluated by transwell assay (see Example 2 for details) and scratch test (see Example 3 for details). The experiment found that after stably knocking down Par3 in vitro, the invasive ability of tumor cells (number of invasive cells/field of view) decreased to 80.5% of that of the control (Figure 2); the ability of migration (number of migrated cells/field of view) decreased to 66.6% of that of the control. % (accompanying drawing 2); at the same time, compared with the control, the scratch healing ability of prostate cancer cells (reflecting the ability of cell adhesion and migration) was significantly decreased after stably knocking down Par3 (accompanying drawing 3). In vivo, through orthotopic implantation of prostate cancer cell lines, it was found that the growth of tumor cells in situ in the prostate was significantly reduced after stably knocking down Par3, and the invasion of paraprostatic lymph nodes and metastasis to distant organs were significantly reduced (attached Figure 4).

The present invention first discovers and identifies Par3 in a prostate cancer cell line as an upstream regulatory molecule of a known important tumor suppressor signaling pathway Hippo-YAP pathway. Through co-immunoprecipitation (coIP), real-time quantitative PCR (qRT-PCR), western blotting (WesternBlot) experiments and immunofluorescence staining (IF) experiments (Fig. 5), it was found that after specific and stable knockdown of Par3, the inhibitory The formation of the Par3-aPKC-KIBRA ternary complex promoted the expression of tumor suppressor genes MST1/2 and Lats1/2 in the Hippo-YAP pathway significantly increased, and the phosphorylation level of Lats1 protein was significantly increased, suggesting that after specific knockdown of Par3 The activity of Lats1 protein was restored and the tumor suppressor signaling pathway was activated.

For the first time, the present invention found that stably knocking down Par3 in a prostate cancer cell line can lead to the phosphorylation of the downstream proto-oncogene YAP by activating the Hippo-YAP pathway, thereby retaining it in the cytoplasm [14] and preventing the protein from entering the nucleus. Through qRT-PCR, Western Blot and immunofluorescence (IF) experiments after nuclear-cytoplasmic separation (Fig. 6), it was found that after specific and stable knockdown of Par3, the phosphorylation level of YAP in the cytoplasm was significantly increased, and it was non-phosphorylated in the nucleus The protein level of YAP decreased significantly, suggesting that the activity of the proto-oncogene YAP was inhibited after the specific and stable knockdown of Par3, preventing it from transmitting downstream signals that promote metastasis.

The present invention identifies and verifies for the first time that MMP1, MMP9, Zeb1, Snail1 and Twist1 are invasion/transfer genes regulated by YAP protein in prostate cancer cell lines. After chromatin immunoprecipitation (ChIp), qRT-PCR, and Western Blot (Fig. 7), it was found that non-phosphorylated YAP protein interacted with known TEAD transcription factor families (mainly TEAD1, TEAD2, TEAD4), It can be combined with the promoter region of the above-mentioned genes to activate the transcription of the above-mentioned genes. When Par3 was specifically and stably knocked down, the nuclear import of non-phosphorylated YAP protein was blocked, the transcription of the above genes was inhibited, and the invasion/metastasis of prostate cancer cells was inhibited at the molecular level (Fig. 8).

Compared with prior art, the beneficial effect of the present invention is:

In the present invention, by constructing a prostate cancer cell subcloning cell line stably knocking down Par3, and establishing a corresponding nude mouse prostate orthotopic tumor implantation animal model, the ability of stably knocking down Par3 to inhibit the invasion and metastasis of prostate cancer is verified in vitro and in vivo effect. The present invention found that after stably knocking down Par3, by destroying the formation of the Par3-aPKC-KIBRA ternary complex, the phosphorylation of important members MST1/2 and Lats1 in the Hippo pathway was enhanced, thereby activating the Hippo pathway and phosphorylating its downstream The proto-oncogene YAP leads to the cytoplasmic retention of YAP protein and reduces the nuclear import of non-phosphorylated YAP protein, which eventually weakens the interaction between YAP and TEAD transcription factors in the nucleus and inhibits the transcription of a series of pro-invasion/metastasis genes. The present invention verifies that stably knocking down the polar molecule Par3 has the effect of inhibiting the invasion and metastasis of prostate cancer cells in vivo and in vitro, phenotype and molecular mechanism. It suggested that the polar molecule Par3, as a new potential therapeutic target, has a broad prospect in the clinical treatment of prostate cancer.

Description of drawings

Figure 1: Construction and identification results of prostate cancer subclonal cell lines stably knocking down Par3. The sorted EGFP-positive cells were lysed with Trizol and total RNA was extracted respectively. Total RNA was reverse transcribed using the mRNA reverse transcription kit from TAKARA. The expression level of Par3 mRNA was detected by qRT-PCR experiment using TOYOBO's SYBR-Green reagent. The results showed that the stable expression of shPar3#1 could knock down the expression level of Par3 to 32% of the control, and the stable expression of shPar3#2 could knock down the expression level of Par3 to 40% of the control. The results of detecting the expression level of Par3 protein by Western Blot were consistent with the experimental results of qRT-PCR, indicating that the prostate cancer subclone cell line stably knocking down Par3 required for subsequent experiments has been successfully established. We used the subcloned cell line obtained by stably expressing shPar3#1 and named it PC3-shPar3 cells, and the corresponding control named PC3-con for subsequent experiments. **: p<0.01

Figure 2: Diagram of the results of in vitro migration and invasion experiments. Tumor cell migration (without matrigel coating) and invasion (with matrigel coating) experiments were performed on cells in PC3-con and PC3-shPar3 groups. Through fixation, staining, microphotography and statistical counting, it was found that after the stable knockdown of Par3, the migration and invasion abilities of PC3 cells were down-regulated, suggesting that the stable knockdown of Par3 in vitro had an inhibitory effect on the migration and invasion of prostate cancer cells. Microscopic magnification is 200 times, **: p<0.01.

Figure 3: Diagram of the results of the scratch test in vitro. PC3-con and PC3-shPar3 cells were routinely digested and seeded in 6-well plates for scratch experiments. Photomicrographs were taken after scratching for 0 hour, 24 hours and 48 hours after incubation in medium containing 2% serum. The results showed that the scratch healing speed of PC3-con cells was significantly greater than that of PC3-shPar3 cells after cultured in low serum for 24 hours and 48 hours, suggesting that stably knocking down Par3 in vitro inhibited the ability of prostate cancer cell lines to adhere to and migrate. The microscopic magnification is 100 times.

Figure 4: Diagram of orthotopic transplantation of prostate in vivo. (a) Experimental strategy of orthotopic prostate tumor implantation in nude mice. First, PC3-con and PC3-shPar3 cells were subcutaneously implanted into tumors, and the tumor tissues were removed after the tumors grew to a more suitable volume (≈1 cm ³ , about 4 weeks). The tumor tissue was digested, and the human prostate cancer subclone cells were inoculated in the left anterior ventral lobe of the prostate in nude mice. (b) The mice were sacrificed 8 weeks after the orthotopic tumor implantation, and relevant organs were observed and removed. The results showed that after the stable knockdown of Par3, the in situ growth of human prostate cancer cells in other prostate lobes, the invasion/metastasis in organs, the proximal metastasis of paraprostatic lymph nodes and the distant metastasis of liver were all significantly decreased. It suggested that the stable knockdown of Par3 could effectively inhibit the metastasis of human prostate cancer cells. Black solid arrows indicate orthotopic growth of prostate tumor; black dotted arrows indicate proximal metastasis of tumor in paraprostatic lymph nodes; white arrows indicate bladder; white triangles indicate large metastatic nodules of tumor in liver site. There are a large number of small metastatic nodules within the range of the white dotted line.

Figure 5: Diagram identifying Par3 as an upstream regulator of the Hippo-YAP pathway. (a) Using PC3 cell lysates for co-IP experiments, it was found that both Par3 and aPKC could bind to KIBRA, and KIBRA protein could also bind to MST1 and Lats1 proteins. (b) After the stable knockdown of Par3, the expression level of phosphorylated aPKC was significantly up-regulated, and the expression of KIBRA was significantly down-regulated, suggesting that the stable knockdown of Par3 in vitro may disrupt the formation of the Par3-aPKC-KIBRA complex. (c) Total RNA was extracted from PC3-con and PC3-shPar3 cells using Trizol conventional methods, and after conventional mRNA reverse transcription reactions were performed, qRT-PCR experiments were performed using SYBR-Green reagents. The results showed that the expressions of MST1/2 and Lats1 were significantly upregulated after the stable knockdown of Par3. Nuclear and cytoplasmic proteins were extracted using nuclear plasma separation technology for Western Blot experiments. It was found that the total protein level and phosphorylated MST1/2 and Lats1 protein levels were significantly up-regulated after the stable knockdown of Par3. It is suggested that after stably knocking down Par3, the Hippo pathway may be activated by increasing the phosphorylation of MST1/2 and Lats1 by destroying the Par3-aPKC-KIBRA complex. **: p<0.01.

Figure 6: Stable knockdown of Par3 enhances YAP phosphorylation and reduces nuclear import of non-phosphorylated YAP. (a) Total RNA was extracted from PC3-con and PC3-shPar3 cells using Trizol conventional methods, and after conventional mRNA reverse transcription reactions were performed, qRT-PCR experiments were performed using SYBR-Green reagents. The results showed that the level of YAP mRNA was significantly down-regulated after stably knocking down Par3. After the nuclear and cytoplasmic proteins were isolated, Western Blot experiments were performed, and it was found that the expression level of phosphorylated YAP in the cytoplasm was significantly up-regulated after the stable knockdown of Par3, while the expression level of non-phosphorylated YAP in the nucleus was significantly down-regulated. (b) Immunofluorescence staining revealed that after stably knocking down Par3, YAP was expressed diffusely in the nucleus and cytoplasm rather than concentrated in the nucleus. It is suggested that stably knocking down Par3 in prostate cancer cell lines can activate the Hippo-YAP pathway, leading to enhanced phosphorylation of the downstream proto-oncogene YAP and retaining it in the cytoplasm, thereby reducing the nuclear import of non-phosphorylated YAP protein. Photomicrography magnification 400X, scale bar length represents 20 microns. **: p<0.01

Figure 7: Non-phosphorylated YAP proteins interact with known TEAD family of transcription factors and initiate transcriptional expression of pro-invasion/translocation factors. (a) The promoter regions of invasion/metastasis-related genes MMP1, MMP9, Zeb1, Snail1 and Twist1 were all predicted to contain TEAD family binding sites. (b) By combining ChIP with qRT-PCR, it was confirmed that non-phosphorylated YAP interacted with TEAD1 in PC3 cells and could be enriched at these binding sites to varying degrees. (c) Through Western Blot experiments, it was found that stably knocking down Par3 in vitro can effectively inhibit the expression of the above-mentioned pro-invasion/transfer genes. It is suggested that MMP1, MMP9, Zeb1, Snail1 and Twist1 are invasion/transfer genes regulated by Hippo-YAP pathway. Stable knockdown of Par3 in the prostate cancer cell line PC3 can weaken the transcription of the above genes, and play a role in inhibiting the invasion and metastasis of prostate cancer cells at the molecular level.

Figure 8: Molecular mechanism of stably knocking down Par3 to inhibit prostate cancer metastasis. Stable knockdown of Par3 in prostate cancer cell lines activates the Hippo pathway and blocks nuclear import of the proto-oncogene YAP protein by disrupting the formation of the Par3-aPKC-KIBRA ternary complex, ultimately leading to pro-invasion/metastasis The transcription of the gene is weakened, and the function of inhibiting the invasion and metastasis of prostate cancer cells is realized at the molecular level. The black solid arrow indicates that it has a promoting effect on the downstream; the ⊥ symbol indicates that it has an inhibitory effect on the downstream; the black solid double arrow indicates the existence of interaction; the black dashed downward arrow indicates the down-regulation of expression; the black dotted upward arrow indicates the up-regulation of expression. p indicates phosphorylation.

detailed description

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

A subcloned cell line PC3-shPar3 stably knocking down Par3 and a control PC3-con were constructed and identified in the prostate cancer cell line PC3 (the human prostate cancer cell line PC3 is used here as an example, but not limited thereto).

Prostate cancer cell line PC3 was purchased from the Cell Bank of the Chinese Academy of Sciences, and Par3 interference plasmids (pshPar3#1, pshPar3#2) and control plasmids were purchased from Origen Company (Cat. The designed shRNA against Par3 (i.e. shPar3#1 and shPar3#2), also contains a control backbone plasmid). 1×10 ⁶ PC3 cells were seeded in a 6-well plate, and 24 hours later, the Par3 interference plasmid and the control plasmid were transfected with lipofectarmine2000 transfection reagent (purchased from LifeTechnology Company). 72 hours after transfection, the cells were transferred to a culture dish with a diameter of 10 cm by a conventional adherent cell subculture method and expanded to culture until the cell density reached 70%-80%. Puromycin (purchased from sigma-Aldrich, USA) was added to a final concentration of 0.5 μg/ml, the medium was changed every 3 days, and puromycin was given continuously for 10 days, and puromycin selection was stopped after observing that the cells no longer continued to die. Surviving cells were collected, and EGFP-positive cells were sorted out by conventional flow cytometry methods (the fusion protein of puromycin resistance gene and EGFP was contained in the plasmid backbone, and the fusion protein shared the same promoter with Par3shRNA).

EGFP-positive cells were lysed using Trizol (purchased from LifeTechnology Company) and total RNA was extracted. Total RNA was reverse-transcribed using the mRNA reverse transcription kit from TAKARA, and the expression levels of Par3 mRNA in the Par3 stable knockdown and control subcloned cell lines were detected by qRT-PCR using the SYBR-Green reagent from TOYOBO. The results showed that the stable expression of shPar3#1 could knock down the expression level of Par3 to 32% of the control, and the stable expression of shPar3#2 could knock down the expression level of Par3 to 40% of the control. The result of using WesternBlot to detect the expression level of Par3 protein was consistent with the experimental result of qRT-PCR (accompanying drawing 1, qRT-PCR primer sequences are shown in Table 1). The above experimental results show that this method can be used to obtain a subclonal cell line that stably knocks down Par3 in the prostate cancer cell line PC3. At the same time, due to the stable expression of shPar3#1, a subcloned cell line with a lower Par3 expression level can be obtained, so the subcloned cell line obtained by stably expressing shPar3#1 is used in subsequent examples, and it is named PC3-shPar3 cells, and the corresponding control is named for PC3-con.

Example 2

In vitro transwell experiments (using PC3-shPar3 and control PC3-con cells as examples, but not limited thereto).

BD company's transwell cell culture plate (the size of the mesh at the bottom of the upper chamber with a pore size of 8 microns) was used to conduct tumor cell migration (without matrigel coating) and invasion (with matrigel coating) experiments. Adherently cultured PC3-con and PC3-shPar3 cells were serum-starved for 24 hours, digested and prepared a serum-free cell suspension with a density of 5x10 ⁵ /ml. 100 microliters of cell suspension was added to the upper chamber, 700 microliters of medium containing 10% FBS was added to the lower chamber, and the cell culture plate was routinely cultured in a cell culture incubator. For the migration experiment, after 4 hours of conventional culture, the upper chamber was taken out, and the residual cell suspension inside the chamber and on the screen was sucked off using a micropipette. The upper chamber was soaked twice in PBS, fixed with 4% paraformaldehyde for 10 minutes and stained with 0.2% crystal violet for 3 minutes. The upper chamber was soaked twice in PBS again and gently wiped with a cotton swab to remove the residual crystal violet at the lower part of the screen, and the upper chamber was dried naturally in a ventilated place before photomicrographing. For the invasion experiment, after 8 hours of conventional culture, the same procedure was used for the experiment. A phase contrast microscope (200 times magnification) was used to photograph 5 to 8 randomly distributed fields of view under bright field and count the cells positive for crystal violet staining in the fields of view (that is, cells that had invaded and migrated, Figure 2). The results showed that after stably knocking down Par3, the migration and invasion abilities of PC3-shPar3 cells were inhibited, and the average number of positive cells stained with crystal violet in each visual field was 80.5%±0.97% of the control cells (70±4.2 cells/field than 87±6.5 cells/field) and 66.6%±1.14% (56±4.5 cells/field than 84±5.1 cells/field), suggesting that stably knocking down Par3 can inhibit the invasion/metastasis of prostate cancer cells (attached figure 2).

Example 3

Scratch test (using PC3-shPar3 and control PC3-con cells as an example, but not limited thereto).

PC3-con and PC3-shPar3 cells that were conventionally adhered to the wall were digested with trypsin and seeded in a 6-well plate at a seeding density of 70%-80%. Routine culture in the cell incubator for 24 hours after inoculation to ensure that the cells completely cover the bottom of the culture plate. Use a 1ml micropipette tip perpendicular to the orifice plate and use a scale to assist in scratching, try to ensure that the width of each scratch is consistent. After scratching, the cell culture medium was sucked off, and the plate was washed twice with PBS to wash away the cell debris produced by the scratching. Add DMEM containing 2% serum for routine culture in a cell culture incubator. Three time points of 0 hour, 24 hours and 48 hours after the scratch were selected, and microscopic photography was performed under bright field using a phase contrast microscope (magnification 100 times). The results showed that the scratch width of PC3-con cells was significantly reduced at 24 hours and 48 hours after scratching, and the scratch healing speed of PC3-con cells was significantly greater than that of PC3-shPar3 cells (Figure 3), suggesting that after stably knocking down Par3 Inhibits the adherent migration ability of prostate cancer cell lines.

Example 4

Prostate cancer cell line nude mouse prostate orthotopic tumor implantation model (PC3-shPar3 and control PC3-con cells are used as examples, but not limited thereto).

In order to more effectively observe the effect of knocking down Par3 on the metastatic ability of prostate cancer cell lines, in this example, a subcutaneous heterotopic tumor transplantation model of prostate cancer cell lines in nude mice was first constructed to enhance the expression of humanized tumor cell lines in mice. Metastatic capacity in vivo. After the formation of subcutaneous tumors, human-derived prostate cancer cells were screened out from the tumor tissues and then implanted in the prostate of nude mice in situ (Fig. 4a). The entire experimental operation, postoperative care and feeding of mice were strictly implemented in accordance with the relevant operation manual, which complied with the ethical norms of animal experiments.

First, mix ^1x106 PC3-con or PC3-shPar3 cells (this is an example, but not limited to this) with an equal volume of matrigel (50% final concentration, BD company) on ice, and mix them on ice at the age of 6 weeks. Tumors were subcutaneously planted on the outer left and right thighs of male nude mice, and the tumor mass was removed after the tumors grew to a suitable volume (≈1 cm ³ , about 4 weeks). Use tissue scissors in an ultra-clean bench to cut the tumor block into pieces, add 4 ml of type IV collagenase at a final concentration of 1 mg/ml (purchased from Life Technology Company), digest it in a water shaker at 37°C for 20 minutes, and then add 1 ml 0.05% trypsin (containing 0.02% EDTA) continued to digest for 5 minutes. After terminating digestion with DMEM complete medium, centrifuge at 1000 rpm for 5 minutes using a horizontal centrifuge. The collected cell pellet was resuspended with 10 ml of PBS, and centrifuged again under the same conditions to remove residual enzymes and medium components. The cell pellet was resuspended with 1 ml of PBS again, and the adipose tissue and connective tissue components were removed by cell sieve with a pore size of 40 microns, and the human-derived prostate cancer subcloning cell lines PC3-con and PC3-shPar3 cells constructed in Example 1 were , in a 37°C incubator for 24 hours. The above cells that had been cultured for 24 hours were routinely digested to prepare a cell suspension with a final concentration of 2.5× ^{10 7} cells/ml. Surgical mice were anesthetized using a mixture of isoflurane and air and fixed on a small animal surgical board. Before the operation, sterilize the abdomen of 8-week-old male nude mice (sexually mature) with iodine and alcohol to sterilize the skin respectively, use tissue scissors to cut the skin and muscle at the midline of the lower abdomen of the mouse about 1 cm, and use ophthalmic forceps Mouse seminal vesicles were carefully isolated and exposed in vitro. Add 8.6 microliters of matrigel (30% final concentration) to 20 microliters of cell suspension (about 0.5×10 ⁶ cells) on ice and mix thoroughly. Insulin syringes with 31G needles (purchased from BD Company) were used to inoculate the cells in the left anterior ventral lobe of the prostate (the transparent sac-shaped organ immediately below the left seminal vesicle), and inject slowly at a uniform speed to prevent extravasation of the cells into the abdominal cavity. 1 to 2 minutes after injection, lightly touch the injection site with ophthalmic tweezers, confirm that the cell-matrigel mixture has solidified, and then push the seminal vesicle back into the abdominal cavity for reset. Use No. 4 sutures to close the abdominal muscles and No. 6 sutures to close the skin. Add 1/1000 broad-spectrum antibiotics to drinking water to prevent wound infection when the mice are reared after operation. The mice were sacrificed at 8 weeks after operation, and relevant organs were observed and removed. The results showed that after stably knocking down Par3, the in situ growth of human prostate cancer cells in other prostate lobes, intra-organ invasion/metastasis, proximal metastasis of paraprostatic lymph nodes, and distant metastasis of liver were all significantly reduced (attached Figure 4b), suggesting that stably knocking down Par3 can effectively inhibit the metastasis of human prostate cancer cells.

Example 5

Par3 was identified as an upstream regulatory molecule of the Hippo-YAP pathway (taking PC3 cells, PC3-shPar3 and control PC3-con cells as examples, but not limited thereto).

The adherent cultured PC3 cells (about 1×10 ⁷ cells) were washed twice with PBS after removing the medium. After removing the PBS, place the culture dish on ice and add 5 ml of RIPA lysate (purchased from Beyotime Biotechnology Co., Ltd.) to lyse for 10 minutes, and collect the cell lysate in a centrifuge tube. Add 50 microliters of protein Gagarose microspheres (purchased from Thermofisher) to every 1 milliliter of cell lysate, and shake on a horizontal shaker at 4° C. for 1 hour. After thorough mixing, the mixture was centrifuged and 1/10 of the volume of the supernatant was taken as input, and the remaining part was used with Par3 antibody (purchased from Millipore Company), aPKC antibody (purchased from Santa Cruz Company), MST1 antibody and 1 microgram each of Lats1 antibody (both purchased from Cell Signaling Technology Company) was used as bait for coIP, and the obtained protein complex was tested using KIBRA antibody (purchased from Santa Cruz Company). On the other hand, coIP was performed using the KIBRA antibody as a bait, and the obtained protein complexes were tested separately using antibodies to the above four proteins. The results showed that Par3, aPKC and KIBRA could interact with each other, and KIBRA could also interact with MST1 and Lats1 (Fig. 5a). It is suggested that the change of expression and (or) localization of Par3 transmits the signal to the Hippo pathway through the Par3-aPKC-KIBRA complex. 1x10 ⁶ PC3-con, PC3-shPar3 cells were subjected to Western Blot experiments, and it was found that stably knocking down Par3 up-regulated the expression level of phosphorylated aPKC, and at the same time down-regulated the expression of KIBRA, suggesting that stably knocking down Par3 in vitro can destroy Par3-aPKC - Formation of the KIBRA complex (Fig. 5b). Total RNA was extracted from 1x10 ⁶ PC3-con and PC3-shPar3 cells using Trizol routine methods, and after conventional mRNA reverse transcription reaction, qRT-PCR experiments were performed using TOYOBO's SYBR-Green reagent (see Table 1 for primer sequences). The results showed that the mRNA expression levels of MST1/2 and Latsl were significantly increased after stably knocking down Par3. Nuclear and cytoplasmic proteins were extracted using a nucleoplasmic separation kit (purchased from Thermofisher) for Western Blot experiments of corresponding proteins. The results showed that after stably knocking down Par3, the total protein expression levels of MST1/2 and Lats1 were significantly up-regulated, and the expression levels of phosphorylated MST1/2 and phosphorylated Lats1 were also up-regulated (Fig. 5c). It is suggested that stably knocking down Par3 can effectively activate Hippo pathway through the mediation of Par3-aPKC-KIBRA complex.

Example 6

Stable knockdown of Par3 inhibits the activity of YAP (take PC3 cells, PC3-shPar3 and control PC3-con cells as examples, but not limited thereto).

RNA was extracted from PC3-con and PC3-shPar3 cells using Trizol conventional methods, and conventional mRNA reverse transcription was performed, followed by qRT-PCR experiments using TOYOBO's SYBR-Green reagent (see Table 1 for primer sequences). The results showed that the mRNA expression level of YAP was down-regulated after stably knocking down Par3 (Fig. 6a). Nuclear and cytoplasmic proteins were extracted using a nucleoplasmic separation kit (purchased from Thermofisher) for Western Blot experiments of corresponding proteins. The results showed that after stably knocking down Par3, the level of phosphorylated YAP protein in the cytoplasm was significantly up-regulated, while the level of non-phosphorylated YAP protein in the nucleus was significantly down-regulated (Fig. 6a). IF experiments found that the expression of nuclear YAP was significantly down-regulated after stably knocking down Par3, while the expression of YAP (co-stained with Tublin) in the cytoplasm was significantly increased (Fig. 6b). These results suggest that stable knockdown of Par3 in prostate cancer cell lines can lead to enhanced phosphorylation of the downstream proto-oncogene YAP and its retention in the cytoplasm by activating the Hippo-YAP pathway, thereby reducing the nuclear import of non-phosphorylated YAP proteins.

Example 7

Stable knockdown of Par3 inhibits the transcription of metastasis-related genes (example, but not limited to, PC3-shPar3 and control PC3-con cells).

In the present invention, the PROMO software ( http://algeen.lsi.upc.es , see website description for details on how to use the software) was first used to predict the role of TEAD transcription factors in the invasion/transfer genes MMP1, MMP9, and Zeb1 by bioinformatics methods. Possible binding sites in the promoter regions of , Snail1 and Twist1 (Fig. 7a). Using conventional ChIP (the kit was purchased from CellSignalingTechnology, please refer to the kit manual for details of the experimental method, see Table 2 for the primer sequence) combined with qRT-PCR and sequencing, it was confirmed that the TEAD transcription factor (TEAD1 is used as an example) in the promoter of related genes Region enrichment (Fig. 7b). By Western Blot experiment, it was found that the protein expression levels of the above-mentioned transfer-promoting genes were significantly down-regulated after stably knocking down Par3 (Fig. 7c). The results of this experiment suggest that the stable knockdown of Par3 reduces the entry of non-phosphorylated YAP into the nucleus. Thus inhibiting its interaction with the known TEAD transcription factor family (mainly TEAD1, TEAD2, TEAD4), and finally inhibiting the transcriptional expression of pro-invasion/transferring genes.

These results above indicated that stably knocking down Par3 in prostate cancer cell lines activated the Hippo pathway and blocked the nuclear import of the proto-oncogene YAP protein by disrupting the formation of the Par3-aPKC-KIBRA ternary complex, eventually leading to The transcription of the pro-invasion/metastasis gene was weakened, and the function of inhibiting the invasion and metastasis of prostate cancer cells was realized at the molecular level (Fig. 8).

Table 1: qRT-PCR primer sequences

Table 2: ChIP related primers

Claims (2)

1. The application of Par3 as a drug target in the preparation of drugs for inhibiting prostate cancer metastasis. 2. The application of Par3 as a drug target in the preparation of drugs for inhibiting the invasion of prostate cancer.