PRPF6 Up-regulates AR-mediated Gene Transcription And Its Effect On Hepatocellular Carcinoma Growth

Objective: The androgen receptor(AR),a member of the nuclear receptor superfamily,is a transcription factor activated by ligand-dependent and ligand-independent mechanisms.After binding to the ligand(such as androgen or DHT),AR activates into the nucleus and binds to the androgen response elements(AREs)of the target gene promoter,and induces a series of target gene transcription to exert its biological roles.Hepatocellular carcinoma(HCC)is the most common primary malignant tumor of the liver.The incidence is mainly in males,and AR is the main factor causing this gender difference.In this process,the transcriptional activity of AR is regulated by a variety of co-regulators.In recent years,more and more studies have shown that the regulation of co-regulators on AR gene expression play a crucial role in the development of HCC.Therefore,it is of great significance to study the regulatory mechanism of AR co-regulators in AR-mediated gene transcription and their roles in HCC.PRPF6,a new AR coactivator,was isolated and identified by yeast two-hybrid technique using AR-AF1 as a bait by our research team in 2002.PRPF6 is a splicing factor for mRNA precursors,which plays an important role in the formation of splicesome.Studies have shown that the loss of PRPF6 function not only leads to the accumulation of mRNA precursors,but also to the cell cycle arrest phenotype.Recently,it has been found that PRPF6 can promote the proliferation of colon cancer cells.However,so far,there are few studies on AR coactivator PRPF6 in liver cancer.Whether PRPF6 is involved in the regulation of AR activity in HCC and what role it plays in the occurrence and development of HCC has not been reported.In the present study,we investigate the roles of PRPF6 in liver cancer,and to determine whether PRPF6 participates in and regulates AR-mediated gene transcription.In order to provide a new target for the treatment of liver cancer,the mechanism of PRPF6 regulating AR-mediated gene transcription was further discussed.Methods: 1.To determine the expression of PRPF6 in clinical liver cancer tissues: Firstly,the expression pattern of PRPF6 gene in clinical liver cancer samples and its relationship with the prognosis of patients were analyzed by UALCAN database.Next,the expression level of PRPF6 protein in adjacent noncancerous tissues and liver cancer tissues were detected by immunohistochemistry(IHC).2.To determine whether there is an interaction between PRPF6 and AR in HCC cells: The interaction between PRPF6 and AR in LM3 cells was detected by immunocoprecipitation assay(CoIP);The colocalization between PRPF6 and AR in LM3 cells was detected by immunofluorescence(IF)assay;Five plasmids expressing truncated mutants of PRPF6 were constructed and their interaction and localization with AR were detected.3.To determine whether PRPF6 can up-regulate AR-mediated gene transcription in HCC cells: The regulation of PRPF6 or five truncated mutants on AR-mediated gene transcription in LM3 cells were detected by dual luciferase reporter assay;The regulation of PRPF6 on AF1 or AF2,two important regulatory regions of AR,mediated gene transcription also were detected;The regulation of PRPF6 knockdown on AR target gene mRNA level were detected by Real-time PCR;The regulation of knockdown or overexpression of PRPF6 on AR target gene protein level were detected by Western blot;The AR recruitment in the ARE of CCRK promoter was detected by chromatin immunocoprecipitation assay(ChIP).4.To explore the mechanism of PRPF6 regulating AR itself gene transcription in HCC cells: Real-time PCR or Western blot were used to detect AR gene or protein expression with the treatment of DHT;ChIP were performed to detect whether PRPF6 or AR was recruited in the AREs region of AR gene and the effect of PRPF6 on AR recruitment and histone modification here.5.To clarify the function of PRPF6 in HCC cells: Firstly,the LM3 and PLC5-AR cell lines with stable PRPF6 knockdown were constructed;Then,the effect of PRPF6 on the proliferation of HCC cells was detected by MTS and cell clone formation assay,the effect of PRPF6 on the migration of HCC cells was detected by scratch and Transwell experiments,and the effect of PRPF6 on the cell cycle of HCC cells was detected by the flow cytometry.5.The effect of PRPF6 on the tumor formation and growth of liver cancer cells in vivo were tested by tumor-bearing mice.Results: 1.The expression of PRPF6 in clinical liver cancer tissues: The analysis of UALCAN database showed that the mRNA expression of PRPF6 in clinical liver cancer tissues was significantly higher than that in normal liver tissues,and the expression level increased with the increase of tumor pathological grades and clinical grades.In addition,high level of PRPF6 was associated with shorter overall survival rates.Next,we detected the expression of PRPF6 protein in 75 cases of clinical liver cancer tissues and 33 cases of adjacent noncancerous liver tissues by IHC.Hscore scores analysis showed that the expression of PRPF6 in liver cancer tissues was significantly higher than that in adjacent noncancerous liver tissues,and the expression level increased with the increase of pathological grades.We further analyzed the relationship between PRPF6 protein expression and clinical features of HCC in the high and low PRPF6 expression groups based on the results of IHC analysis.The data shown that PRPF6 expression was positively correlated with pathological grades,and there was no significant association with the other clinical features.2.PRPF6 interacts with AR in HCC cells: CoIP results demonstrated that PRPF6 interacted with both endogenous or exogenous AR in LM3 or PLC5-AR cells overexpressing AR in DHT-independent manner,and the treatment of DHT enhanced their interaction;The plasmids expressing AR or different truncated mutants of PRPF6 were co-transfected in HEK293 cells,CoIP results showed that except for PRPF6-N,all of them could interact with AR.IF results showed that endogenous PRPF6 and AR colocalized in the nucleus without the treatment of DHT in LM3 cells,,while in HEK293 cells overexpressing AR and different truncated mutants of PRPF6,PRPF6-FL or truncated mutants N,5TPR,10 TPR,15TPR localized in the nucleus,PRPF6-C was localized in the cytoplasm and nucleus without or with DHT.While AR was mainly localized in the cytoplasm,and co-localized in the nucleus with PRPF6-FL or its truncated proteins with the treatment of DHT.3.PRPF6 up-regulates AR-mediated gene transcription in HCC cells: Dual luciferase reporter assays demonstrated that PRPF6-FL or its truncated mutants,including 5TPR,10 TPR and 15 TPR,enhanced ARinduced transcriptional activity without DHT,and the up-regulation effect was more significant in the presence of DHT.While truncated PRPF6-N or PRPF6-C had no obvious effect on it.The transcriptional activity of AF1,a ligand-independentent region of AR,was regulated by PRPF6,while that of AF2,a ligand-dependent region of AR,was not affected by PRPF6.Real-time RCR detected some AR target genes in HCC cells,the results showed that PRPF6 knockdown decreased the mRNA expression levels of AR and its target genes,including CCRK,in the presence and absence of DHT,while VEGFA and FKBP5 mRNA expression level did not change.Western blot results showed that PRPF6 knockdown decreased the protein levels of AR and its target gene CCRK in LM3 cells,while the expression of FKBP5 protein remained unchanged.Similar results were obtained in PLC5-AR cells.In addition,our data demonstrated that ectopic expression of PRPF6 enhanced AR and CCRK protein expression in LM3 or PLC5-AR cells.ChIP results showed that PRPF6 could be recruit to the ARE of AR target gene CCRK,and the recruitment of AR reduced by PRPF6 knockdown.4.The mechanism of PRPF6 up-regulating AR itself gene transcription in HCC cells: Real-time PCR and Western blot results showed that compared with the control group,the expression levels of AR gene and protein increased with the stimulation of DHT,suggesting that AR transcription is induced by its own ligand(DHT)in HCC cells.ChIP results showed that compared with the control group,the recruitment of AR in its own AREs regions increased under DHT stimulation,and PRPF6 was also recruited to the ARE I site.Knockdown of PRPF6 reduced AR recruitment and the modification level of histone H3K36me3 at ARE I region of AR gene.5.Knockdown of PRPF6 inhibits the growth of HCC cells in vitro: MTS and cell clone formation experiments demonstrated that PRPF6 knockdown inhibited the proliferation of HCC cells.Scratch and Transwell experiments showed that PRPF6 knockdown had no effect on the migration of HCC cells.Flow cytometry analysis results showed that PRPF6 knockdown significantly reduced the S phase of HCC cells.6.Knockdown of PRPF6 inhibits the growth of HCC cells in vivo: The results of tumor-bearing mice experiment showed that the tumors formed by LM3 cells carrying shPRPF6 subcutaneously in male BALB/c nude mices were smaller than the control group,and the growth rate of tumors was slower.The results of IHC showed that the expression level of the cell proliferation antigen Ki-67 in PRPF6 silenced tumor tissues also decreased accordingly.PRPF6 knockdown also decreased the protein level of CCRK in tumor tissues of mice.Conclusion: 1.PRPF6 is associated with the clinical stage of liver cancer and the prognosis of patients.2.PRPF6 promotes the S phase and promotes the growth of HCC cells.3.In HCC cells,AR is mainly localized in the nucleus.PRPF6 interacts with AR and can be recruited in the promoter region of AR target genes to up-regulate AR-mediated gene transcription.4.PRPF6 participates in up-regulating AR itself gene transcription induced by androgen in HCC cells.PRPF6 facilitates the recruitment of AR to ARE region of AR gene,subsequently increasing the level of H3K36me3 modification and promoting transcription of AR gene,thereby further enhancing the regulation of PRPF6 on AR-mediated gene transcription.