The Regulation Of Cell Cycle And Apoptosis Of RNF2 In Prostate Cancer Cells And Its Underlying Epigenetic Mechanism

ObjectiveThe post-transcriptional modifications of histones are important epigenetic regulations,having been proved to play vital roles in the tumorigenesis and progression of many cancer types.RNF2,also known as RING1 B or RING2,is identified as the catalytic subunit of polycomb repressive complex 1(PRC1),which can mediate the mono-ubiquitination of histone H2 A.The deregulation of RNF2 expression have exerted an oncogenic function in many cancers.However,the role and function of RNF2 in prostate cancer(PCa)have not yet been well studied and reported.In the current study,the expression status of RNF2 is firstly measured in PCa tumor tissues and benign prostatic hyperplasia(BPH)tissues.Secondly,RNF2 expression is downregulated in PCa cell lines and these RNF2 knockdown cells are subjected to cell cycle,apoptosis and cell viability examination,trying to figure out the biological functions of RNF2 in PCa.Then,next-generation sequencing is adopted to identify the genes significantly changed in expression upon RNF2 knockdown,in seeking of important downstream target genes of RNF2.Finally,ChIP assays are carried out to reveal the underlying mechanisms of how RNF2 regulates its downstream genes.Materials and methods1.Paraffn-embedded tissue microarray(TMA)sections,including PCa tumor tissues(n=81)and BPH tissues(n=71)were subjected to immunohistochemistry(IHC)staining to detect the expression level of RNF2 amd its expression level was scored using H-score method.Statistical analysis was performed to evaluate the differences of RNF2 expression in PCa tissues and BPH tissues.The correlations between RNF2 expression and Gleason score of PCa were also analyzed.2.Small interfering RNA(siRNA)duplexes specific targeting RNF2 were designed and synthesized and named as siRNF2 #1 and siRNF2 #2.siNC,which targets no specific genes,was taken as negative control.The siRNAs were transfected into DU145 and LNCaP cells and the knockdown efficiency was identified using quantitative real-time quantative PCR(RT-qPCR)and western blot(WB).Flow cytometry(FCM)analysis was used to detect the cell cycle distributions and apoptosis of different siRNAs transfected cells.Cell viability was analyzed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay(MTT).3.RNF2 specific short hairpin RNA(shRNA)and scramble shRNA were designed and cloned into lentivirus vectors,which are tagged with green fluorescence protein(GFP).Then,the shRNA expressing lentivirus vectors and packaging vectors were co-transfected into 293 T cells for lentivirus package.Supernatants containing lentivirus were harvested 3 days after transfection and named as shScr,shRNF2 #1 and shRNF2 #2.The lentiviruses were then purifed by centrifugation,and the titer was confrmed.DU145 and LNCaP cells were infected with these lentiviruses and the knockdown efficiency was identified using RT-qPCR and WB.FCM and MTT were used to detect the cell cycle distributions,apoptosis and cell viability.4.The shScr,shRNF2 #1 and shRNF2 #2 expressing lentiviruses infected DU145 cells were cultured and amplified.Then,these cells were harvested,suspended and subcutaneously injected into the back near the right thigh of nude mice to establish xenograft tumor model.Tumor growth was measured by the tumor diameter with a Vernier caliper every 5 days beginning on the 5th day of injection till the 35 th day.Tumor volume was calculated as length×width2/2,where the length and width are the longest and shortest axes in millimeters.Then xenograft tumor tissues were fxed in 4% formaldehyde immediately after resection and then paraffn-embedded.Tumor slides were subjected to IHC staining for proliferating cell nuclear antigen,(PCNA)and cleaved caspase-3(cleaved-casp3).5.siNC,siRNF2 #1 and siRNF2#2 were transfected into DU145 cells,respectively.72 hours after transfection,cells were harvested and total RNA was extracted.Total RNA was then subjected to gene expression profling,using the Affymetrix gene chip array(Biotechnology,Shanghai,China).The gene expression data were logtransformed,median centered and analyzed using the SBC Analysis System(Biotechnology,Shanghai,China).The differently expressed genes were subjected to online database,such as GeneOntology and BioCarta-ATCG,to undergo Gene Ontology(GO)analysis.Further more,the differently expressed genes were analyzed and testified by RT-qPCR and WB,seeking the potential downstream target genes of RNF2.The above mentioned xenograft tumor tisssue slides were also subjected to IHC staining for RNF2 and its potential target genes.6.Specific siRNA and shRNA targeting thioredoxin interacting protein(TXNIP),the potential target gene of RNF2 screened above,were designed and named as siTXNIP and shTXNIP,respectively.The shTXNIP was cloned into lentivirus vectors,which are tagged with GFP and co-transfected with packaging vectors into 293 T cells for lentivirus package.The lentiviruses were then purifed by centrifugation,and the titer was confrmed.The knockdown efficiency was tested using RT-qPCR and WB.Then,siTXNIP and shTXNIP were used to downregulate TXNIP expression to see if TXNIP knockdown could abrogate the biological changes exerted by RNF2 knockdown.FCM and MTT methods were used to detect the cell cycle distributions,apoptosis and cell viability.7.PCR primers were designed based on the region within 1000 bp before the transcription start site(TSS)of TXNIP.Then,chromatin immunoprecipitation(ChIP)assays were used to exam the enrichment of RNF2 to TXNIP promotor region and the mono-ubiquitination level of histone H2 A in 119 lysine residue(H2AK119Ub).Then,siRNA was used to knockdown RNF2 expression and ChIP assay was adopted to exam the enrichment fold change of RNF2 to the promoter region of TXNIP and the change of H2AK119 Ub.Results1.The results of IHC staining of TMA and H-Score assessment showed RNF2 expression in PCa tissues is much higher than BPH tissues,with the H-Score of 117.53±62.34 and 90.14±63.66,respectively.Statistical analysis showed that RNF2 expression is higher in PCa tissues with Gleason>7(H-Score: 150.50±62.03)than those with Gleason?7(H-Score: 97.10±53.63).2.The results of RT-qPCR and WB showed that siRNF2 #1 and siRNF2 #2 have good RNF2 knockdown efficiency,both in mRNA level and protein level.FCM analysis showed that the percentages of G1 phase cells and apoptotic cells significantly increased in RNF2 knockdown DU145 and LNCaP cells compared with negative control cells.MTT assay showed decreased viability in RNF2 knockdown cells.3.We succeed in getting shRNA expressing lentiviruses with good infection efficiency.RT-qPCR and WB showed good knockdown efficiency of RNF2.The results of FCM analysis and MTT assay showed that shRNF2 #1 and shRNF2 #2 induced notable G1 pahase arrest,apoptosis and impaired cell viability in DU145 and LNCaP cells,when compared with shScr infected cells.4.We successfully developed nude mice xenograft models using shSCr?shRNF2 #1 and shRNF2 #2 infected DU145 cells.Results showed that the tumor growth in RNF2 knockdown group(shRNF2 #1 and shRNF2 #2)was dramatically inhibited in comparision with the control group(shScr),with the final tumor volume of 304.33±63.72 mm3,331.93±84.42 mm3 and 846.80±55.29 mm3,respectively.IHC staining of the xenograft tumor tissues showed RNF2 knockdown tumor tissues had decreased PCNA and increased cleaved-casp3 when compared with the control group.5.We performed gene microarray analyses using the mRNA from RNF2 knockdown and control DU145 cells.Using a 1.5 fold cutoff,we found that 632 genes were differentially expressed in RNF2 knockdown cells.Of these genes,144 were upregulated and 488 were downregulated.Results of GO analysis showed that many of the most signifcantly changed genes participate in the regulation of cell cycle,mitotic cell division,apoptosis and proliferation.Considering the transcriptional repressive function of PcGs,we mainly focused on the tumor suppressor genes increased in RNF2 knockdown cells,among which,TXNIP is one of the most signifcantly increased genes.We further comfirmed the increased expression of TXNIP in RNF2 knockdown DU145 and LNCaP cells in both mRNA and protein levels,using RT-qPCR and WB.The results of IHC staining showed increased TXNIP expression in RNF2 knockdown DU145 xenograft tumor tissues than negative control group tumor tissues.6.The results of RT-qPCR and WB showed good TXNIP knockdown efficiency of both siTXNIP and shTXNIP.FCM assay showed that simultaneous knockdown of RNF2 and TXNIP can effectively abrogate the G1 phase arrest and increased apoptosis in RNF2 single knockdown cells.MTT assay showed that simultaneously knockdown of RNF2 and TXNIP can rescue the inhibited cell viability in RNF2 single knockdown cells.7.Results of ChIP assay showed that RNF2 can specifcally enriched to the TXNIP promoter region and the H2AK119 Ub level was higher when compared with control group.When RNF2 was knocked down,the enrichment of RNF2 was dramatically decreased,as well as the level of H2AK119 Ub.Conclusions1.RNF2 expression in PCa is significantly higher than that in BPH and the expression level of RNF2 is positively correlated with Gleason score,suggesting an oncogenic function of RNF2 in PCa.2.RNF2 plays a vital role in the regulation of the cell cycle,apoptosis and cell viability in PCa cells.Specific knockdown of RNF2 can induces cell cycle arrest,increasing apoptosis,inhibition of cell viability,as well as significant growth inhibition of nude mice tumor xenograft.3.RNF2 knockdown can change the expreission of large amounts of downstream genes,among which,TXNIP is a vital functional target gene.The biological functions of RNF2 are at least partly depending on the trancriptional regulation of TXNIP.4.RNF2 may regulate the expression of TXNIP through the enrichment to TXNIP promotor,as well as increase the level of H2AK119 Ub.5.RNF2 is a potential biomarker and therapeutic target of PCa.