Influence Of KHDRBS Family Genes On Phenotype And Androgen Receptor Of Prostate Cancer Cell

Background Prostate cancer,the most common malignancy in male genital system,like other cancer,is caused by mutiple biological events including mutation of tumor suppressor gene,inactivation of DNA repair gene and abnormal activation of oncogene,in which a variety of signal pathways are involved.Alternative splicing plays a pivotal role in prostate cancer.Through alternative splicing,AR,a nuclear receptor vital to both prostate development and prostate cancer progression,can be spliced into multiple variants,in which AR-V7 and AR-V567es have been proved implicated in the progress of castration resistant prostate cancer.The RNA binding protein KHDRBS1 was uncovered capable in assisting the spliceosome with recognizing exon 3b of AR and subsequently producing AR-V7 by inclusion of exon 3b.In addition,KHDRBS1 promotes the progression of a variety of malignant tumors,including prostate cancer,and is positively correlated with clinical stage,pathological grade and prognosis.The KHDRBS1 gene has two homologous and highly conserved subfamily members,KHDRBS2 and 3,they are consistent with KHDRBS1 in the 70%～80%amino acid sequence in the KH domain that functions in RNA binding,besides,both interact with KHDRBS1 and associated with breast cancer and renal cancer.But the relationship between KHDRBS2 and 3 and prostate cancer is unclear,therefore,in this paper,we discuss the role of KHDRBS family in PCa progression,especially the relationship between KHDRBS2 and 3 and AR,to increase the understanding of KHDRBS family and enrich the molecular mechanism of PCa progression.Objective 1)To clarify the expression level of KHDRBS family gene in prostate cancer cell lines;2)To clarify the relationship between KHDRBS2 and 3 and proliferation,migration,invasion and cell cycle of prostate cancer cell;3)To investigate mutual modification between KHDRBS 2 and 3;4.)To investigate the relationship between them and AR,AR-V7 and AR-V567es.Methods and ResultsPart Ⅰ Bioinformatics analysis of KHDRBS family in prostate cancer and verification of expression in prostate cancer cell lineMethods:1)Utilize cBioportal online tool to analyse alteration ratio,expression level of KHDRBS family genes and correlation between KHDRBS family genes and overall survival together with disease free survival in samples from 11 sequencing data sets.2)Utilize Real-time PCR to detect KHDRBS family genes in prostate cancer cell line 22RV1,LnCap,DU145 and PC3Results:1)In the 11 sequencing data sets,KHDRBS1,2 and 3 gene abnormaly alter in 4,8 and 10 sets respectively.In the corresponding set,the proportion of gene abnormal alteration of KHDRBS2 and 3,especially the latter,are mostly higher than KHDRBS 1.KHDRBS1 and 3 mRNA expression levels were significantly higher than KHDRBS2.Gene abnormal alteration of KHDRBS3 in the TCGA set affects the overall survival of patients with prostate cancer,whereas KHDRBS1 and 2 have no effect.2)KHDRBS 1 expresses in all cell lines,while KHDRBS2 only expresses in 22RV1 and KHDRBS3 mainly expresses in PC3.Part Ⅱ Roles of KHDRBS2 and 3 in prostate cancer cell linesMethods:1)Overexpress and knockdown KHDRBS2 and 3 in 22RV1 and PC3 by transient transfection and lentivirus transfection respectively,then detect cell cycle,migration,invasion,together with in vitro and in vivo proliferation by Muse cell analyzer,Transwell assay and CCK-8 assay and tumor-bearing test respectively.2)Alter the expression of KHDRBS2 and 3 in 22RV1 and PC3 with the same method,and detect corresponding changes of KHDRBS family in both cells by real-time PCR and Western-Blot.Results:1)Knocking down KHDRBS2 inhibits proliferation of 22RV1 and Knocking down KHDRBS2 inhibits proliferation of PC3,both in vivo and in vitro,respectively.2)In 22RV1 and PC3 cell,overexpression and knockdown of KHDRBS2 and 3 have no effect on the expression of KHDRBS1,but KHDRBS2 and 3 present reversal changes.Part Ⅲ The relationship between KHDRBS family and androgen receptorsMethods:1)Utilize Real-time PCR to detect KHDRBS family genes and AR,AR-V7 and AR-V567es mRNAs in prostate cancer cell line 22RV1,LnCap,DU 145 and PC3.2)Detect the expression levels of KHDRBS family genes and AR,AR-V7 and AR-V567es in 22RV1 by real-time PCR with or without the pretreatment of androgen R1881 and AR antagonist MDV3100;in same condition,detect KHDRBS family genes in PC3.3)Utilize Real-time PCR to detect mRNA level of AR,AR-V7 and AR-V567es in KHDRBS2 and 3 overexpressed 22RV1 and KHDRBS2 knocked-down 22RV1.4)Utilize Real-time PCR to detect mRNA level of KHDRB2 and 3 in KHDRBS2 and 3 overexpressed 22RV1 and KHDRB2,KHDRBS3,AR and AR-V7 in KHDRBS2 knocked-down 22RV1 with or without the pretreatment of androgen R1881.Results:1)In 22RV1 cells with high level of KHDRBS2 and low KHDRBS3,AR,AR-V7 and AR-V567es are highly expressed;and in PC3 with low level of KHDRBS2 and high KHDRBS3,AR,AR-V7 and AR-V567es barely express.2)The expression of AR,AR-V7 and AR-V567es,together with KHDRBS1 and 2 in 22RV1 cultured without androgen are significantly higher than those in normal culture medium,but no significant difference of KHDRBS3 in the same comparison is manifested.In addition,same pretreatment has no effect on KHDRBS family genes in PC3.3)Knocking-down of KHDRBS2 significantly reduced the expression of AR-V7 in 22RV1,with a P value of 0.0168.4)With or without the androgen,transition of KHDRB2 and 3 in KHDRBS2 and 3 overexpressed 22RV1 and KHDRB2,KHDRBS3,AR and AR-V7 in KHDRBS2 knocked-down 22RV1in KHDRBS2 and 3 overexpressed 22RV1 are congrugent with aforementioned tendency.Conclusion1.The analysis of prostate cancer samples by cBioportal suggests that KHDRBS2 and 3 may play an important role in the progression of PCa and CRPC.2.The expression of KHDRBS2 and 3 in 22RV1 and PC3 cells is cell-specified and may correlate to AR and its variants.3.Knocking down KHDRBS2 inhibits proliferation of 22RV1 and knocking down KHDRBS3 inhibits proliferation of PC3,both in vivo and in vitro,respectively.4.In 22RV1 and PC3,KHDRBS2 and 3 manifest mutually modulation pattern,which may facilitate delicate coordination of RNA binding proteins from same family in alternative splicing of a certain gene,so as to maintain functional stability and dynamic balance of a specific signal pathway.5.Upregulation of KHDRBS1 and 2 may be mediated by AR-FL when removal of androgen.Knockdown of KHDRBS2 inhibits the expression of AR-V7 in 22RV1 cell.KHDRBS1 and 2 may be the regulatory factors between AR-FL and AR-V7.