Study The Function Of BAF155 And MED19 In Human Prostate Cancer PC3 Cells

Prostate cancer(PCa)is one of the most common malignancies and the sixth leading cause of cancer-related death in men.According to WHO global epidemiological statistics,the global ranking of cancer incidence among men,PCa is second only to lung cancer,ranking second.The incidence of PCa in developed countries is much higher than in developing countries.In China,the prevalence of PCa has rapidly increased year by year as a result of the changes in lifestyle,dietary structure,and aging of the population.At present,PCa is clinically divided into early stage(?,?)localized in prostate cancer and feasible radical prostatectomy;late stage(?,?)has adjacent organ invasion or peritoneal lymph node,distant metastasis,endocrine secretion Treatment-based,feasible orchiectomy,with hormone blocking treatment.The median remission period is 18-24 months,and more progresses to Androgen-Independent Prostate Cancer(AIPC).It has high proliferative and high invasiveness.The current clinical treatment methods are not ideal.Therefore,the mechanism and treatment of AIPC have become the focus of current research.Tumor cells are able to develop adaptive resistance to virtually all targeted therapies,posing continuing challenges to the medical fields.This is true for the metastatic prostate cancer(mPCa)that is currently incurable and lethal.Since the first demonstration of clinical benefits of surgical or medical castration by Huggins and Hodges in 19411,androgen deprivation therapy(ADT)has remained a mainstay in the treatment of mPCa.ADT is effective by limiting availability of androgen,which controls the nuclear translocation and action of the androgen receptor(AR)as a transcription factor by binding to its ligand-binding domain(LBD).Although more than 80%of patients with mPCa initially respond to ADT,they eventually develop castration-resistant prostate cancer(CRPC).At this stage,the patient is considered depleted of androgen and thus the PCa presumably grew independently of AR signaling.Chromosome instability is one of the markers of cancer,which is closely related to cancer metastasis.SWI/SNF(Switch in mating type/sucrose non-fermentation)complex is one of the chromatin remodeling complexes.It consists of BRG1/BRM,INI1(SMARCB1,BAF47,SNF5),BAF155,BAF170,BAF180 and BAF250A subunits.Chromatin remodeling SWI/SNF complex is an important regulator of developmental gene expression.The Switch in mating type/sucrose non fermentation(SWI/SNF)yeast complex is highly conserved in yeast,fruit flies and mammals.The SWI/SNF complex plays an important role in various cell biological processes such as cell cycle,tumor formation and apoptosis.The mechanism of cancer development involved in the SWI/SNF complex is unclear and has become a research hotspot.In particular,the development of cancer induced by SWI/SNF loss may be related to DNA repair,transcription,cell cycle regulation,and nucleosome location.The BRG1-associated factor 155(BAF155)is a member of the SWI/SNF complex,BAF155 is found in the cyclin E complex,and cyclin E-related kinases can cause BAF155 to occur Phosphorylation.The role of BAF155 in tumorigenesis is unclear,but because BAF155 is located on chromosome 3p221.31,which includes some suspected tumor suppressor genes such as FUS1 and SEM3B,the absence of BAF155 may contribute to the development of the tumor.Med 19 is an important member of Mediator family.It plays an important role in gene transcription.Knocking out Medl9 subunit reduces the stability of Mediator complex and inhibits the phosphorylation of RNA polymerase ? terminal,thus lowering the transcription level.Silencing its expression can inhibit the growth and spread of tumors.Previous studies have shown that Med 19 is involved in the progression of various tumors.Med 19 promotes the proliferation of breast cancer cells by regulating the expression of CBFA2T3/HEB.Medl9 can promote the proliferation and migration of non-small cell lung cancer cells,and also enhance the sensitivity of the chemotherapeutic drug Cisplatin.Inhibiting Med 19 enhances the chemical sensitivity of adriamycin by down-regulating HMGB1 signal transduction in human breast cancer cells.Med19 is targeted by microRNA-101-3p/microRNA-422a,and promotes the progression of breast cancer by regulating EGFR/MEK/ERK signaling pathway1In many cancers,the overexpression of Mediator Complex Subunit 19(Med 19)plays a promotive role.Tumor-host interaction at the invasive front of cancer represents a critical interface encompassing a dynamic process of de-differentiation of carcinoma cells known as epithelial mesenchymal transition(EMT).EMT can be identified histologically by the presence of "tumor budding",a feature which can be highly specific for tumors showing an infiltrating tumor growth pattern.Importantly,tumor budding and tumor border configuration have generated considerable interest as additional prognostic factors and are also recognized as such by the International Union Against Cancer.Tumor metastasis initially requires cell invasion,which is initiated by EMT.However,the role of Med19 in prostate cancer remains unclear.Therefore,this study attempts to investigate how Med 19 regulates the proliferation,migration and apoptosis of prostate cancer cells initiated by EMT in prostate cancer.Therefore,this study attempts to investigate how Med 19 regulates the proliferation,migration and apoptosis of prostate cancer cells initiated by EMT in prostate cancer.The function of BAF155 in human prostate cancer PC3 cells has not yet been fully defined.The biological effects and molecular mechanisms of BAF155 and Medl9 in human prostate cancer cells were studied in this paper.1.To explore the role of BAF155 in proliferation,differentiation and metastasis of prostate cancer cells,the expression of BAF155 in PC3 was analyzed by Western blotting.2.Screening of cell lines with high expression of BAF155 in PC3,Hela and SNUC2B cells.Overexpression of BAF155 by lentivirus BAF155-shRNA and BAF155-siRNA and interference analysis of BAF155 after interference were carried out.Proliferation,invasiveness and apoptosis of cell lines were examined to verify the growth of prostate cancer cells.Whether the biological behavior is regulated by BAF155 or not lays a foundation for the study of the molecular mechanism of prostate cancer formation and follow-up treatment.3.In PC3 cells,we studied the knockout efficiency of Med19-siRNA knockout,how Med19 regulates the proliferation and migration of prostate cancer cells,and how Med 19 affects the initiation of EMT to inhibit the proliferation and migration of cancer cells.The research contents of doctor students are divided into the following two parts:Part IStudy the function of BAF155 in human prostate cancer PC3 cells Research Objective1.The expression of BAF155 in PC3 cells was detected by Western blotting.2.The full-length coding sequence of wild-type BAF155 was subcloned into vector pcDNA3.1(+),and the super expression vector of BAF155(pc-BAF155)was constructed and sequenced.3.pcDNA3.1,pc-BAF 155 and pc-BAF 155+si-BAF155 were transfected into cells respectively.Quantitative reverse transcription-polymerase chain reaction(QRT-PCR)was used to detect the expression level of BAF155 and immunoblotting was used to detect the efficiency of BAF155 transfection.4.PC3 cells transfected with BAF 155 were tested for viability,apoptosis and migration by MTT assay,apoptosis detection and migration assay,respectively.5.The expression levels of p16,phosphatidylinositol 3-hydroxy kinase(PI3K)/protein kinase B(AKT)and WNT/beta-catenin pathway related proteins were detected by Western blot.6.Statistical analysis of the data shows that all the experiments in this study were repeated three times.The experimental data are expressed by mean(+standard deviation).There is statistical significance when P<0.05.Research results1.It was found that BAF 155 was overexpressed in Hela cells and BRG1 was low expression;BAF155 was deleted and BRG1 was low expression in PC3 cells;BRG1 was overexpressed in SNUC2B cells,but BAF 155 was not.2.In PC3 cells,overexpression of BAF 155 can increase the transcriptional overexpression of BAF 155,but after the deletion of BAF 155,the expression of BAF 155 decreases.3.Overexpression of BAF 155 significantly inhibited PC3 cell proliferation(inhibition rate was 50%)and migration ability(inhibition rate was 56%)and promoted PC3 cell apoptosis(apoptosis rate was 28%)(P<0.05).After transfection of si-BAF155 into cells,cell migration ability increased again.4.Further studies on the expression level of p16 protein showed that the expression level of p16 was significantly increased after transfection of pc-BAF155(2.3 fold increase)(P<0.05).Overexpression of BAF155 could significantly inhibit the expression levels of p/t-PI3K,p/t-AKT,Wnt3a,beta-catenin and Wnt5a(P<0.05)(inhibition multiples were 0.78?0.45?0.67?0.63?0.520),thus inhibiting the proliferation of PC3 cells and promoting apoptosis.After transfection of siRNA-BAF155 into cells,the expression of these proteins returned to normalResearch ConclusionsOver-expression of BAF155 could inhibit proliferation and migration while inducing apoptosis of PC3 cells through up-regulating p16 and inactivating PI3K/AKT and WNT/?-catenin pathways.Research MeaningThis study was the first to study the anti-cancer effect of BAF 155 in prostate cancer cells.It was found that BAF155 could up-regulate p16 and down-regulate PI3K/AKT levels in PC3 cells,inactivate WNT/beta-catenin pathway,and ultimately induce PC3 cell apoptosis.Part ?Basic study on the silencing of med19 gene in human prostate cancer PC3 cellsResearch BackgroundMediator Complex Subunit 19(Med 19)is overexpressed and plays promotional roles in many cancers.However,the roles of Med 19 in PCa are still obscure.Research ObjectiveTo observe the effect of lentivirus-mediated inhibition of Med 19 on cell migration and invasion in the PC3 cells and explore the mechanism of epithelial-mesenchymal transition transformation.Research Methods1.PC3 cells were infected with siRNA lentivirus vector targeting Med 19.2.Quantitative real-time fluorescence PCR(QRT-PCR)and Western blot were used to detect the transcription and translation of Med 19-siRNA infection group(siRNA)and Med19 gene in the blank group(scRNA).3.Boyden chamber invasion test and scratch test were used to detect the changes of invasion and migration ability of PC3 cells after infection.4.QRT-PCR was used to detect the expression of E-Cadherin,N-Cadherin,Vimentin,ZEB2,Snail-1 and Snail-2 in PC3 cells after infection.Research results1.Establishment and characterization of PC3-Med19-si/sc cells.After lentivirus infection and FACS classification,GFP expression was determined by fluorescence microscopy to confirm its transduction efficiency.The positive rate of GFP in all cells was more than 95%.2.Med 19-siRNA lentivirus infected PC3 cells,the expression level of Med19 gene in the experimental group was significantly lower than that in the blank group(18%lower gene transcription rate),the difference was statistically significant(P<0.01).3.The expression of Med L9 protein in PC3 cells infected with Med19-siRNA lentivirus was significantly lower in the experimental group than in the no-load group(P<0.01).4.Boyden chamber invasion test and scratch test showed that the invasion and migration ability of cells in the infected group were significantly reduced(P<0.01).The invasion rate of PC3-Med 19-si cells was(19.71±4.00%)while that of control PC3-Med 19-sc cells was 32.27±3.72%(*P<0.05).The percentage of scratch repair area in PC3-Med 19-si cells was significantly lower than that in control cells(70.83±9.37%)v.s.(87.33±4.63%),(**P<0.01).5.The expression of N-Cadherin,Vimentin,ZEB2,Snail-1 and Snail-2 in infected group decreased,while the expression of E-Cadherin increased.Compared with PC3-Med19-sc group and PC3-Medl9-si group,the relative gray values of the following proteins were:N-Cadherin:(1 ± 0.12756),(0.45586 ± 0.05091);E-Cadherin:(1±0.07178);(1.78180±0.14847);Vimentin:(1±0.07177);(0.46652±0.05091);ZEB2:(1±0.04083),(0.43528+0.04862);Snail-1:(1 ±0.12756);(0.58777±0.02400);Snail-2:(1±0.04830),(0.57435±0.04786).The results of factor variance analysis showed that P<0.05.Research ConclusionMed 19 inhibition can reduce migration ability of prostate cancer PC3 cells by epithelial-mesenchymal transition.Research InnovationThis study is the first to report that Med 19 inhibits the expression of EMT and related genes,thus inhibiting the proliferation and migration of prostate cancer cells.Med 19 plays an important role in prostate cancer.