The Anti-tumor Role And Mechanism Of CTFT Trascriptionally Inhibiting Mir-34a-5p In Bladder Cancer

Bladder cancer(BCa)is the most common cancer in urology of China,which seizes the seventh common cancer in men and below fifteenth common cancer in wemen.Derived from urinary transitional epithelium cells,approximately one third of diagnosed BCa patients already develop locally invasive or metastatie disease.The standard care for invasive BCa remains radical cystectomy for now,and the 5-year survival rate is still lower than 62%.The data support continuing efforts to study the carcinogenic mechanisms and develop novel and effective therapeutic targets of BCa.MicroRNAs(miRNAs)are small(19-22 nucleotides)non-coding RNAs that suppress gene expression after transcription.Lots of microRNA show specific expression pattern in different tissue or bioprocess,and in tumors,changed regulators lead to a tumor specific microRNA expression pattern.Our study aimed to explore the inhibition mechanism of miR-34a-5p during transcription in p53-mutated BCa cells and clarify the inhibition effect and molecular mechanism of miR-34a-5p in BCa.The main investigations and results are as follows:1)miR-34a-5p was downregulated in bladder cancer.To evaluate miR-34a-5p expression in bladder cancer,we performed quantitative real-time PCR(qRT-PCR)in T24,UM-UC-3,5637 and J82 cell lines.The expression level was generally lower in 4 BCa cell lines compared to the SV-HUC-1 cell line(p<0.05).Lower miR-34a-5p level indicated lower OS(p<0.05).BSP indicated higher methylation levels in upstream of the TSS of miR34aHG in T24 and UM-UC-3 cell lines.5-Aza-2’-deoxycytidine(5-Aza)treatment significantly upregulated the expression of miR-34a-5p.2)The expression level of CTCF and miR-34a-5p in 4 BCa cell lines were negatively correlated.Knockdown of CTCF and inhibition of HDAC1 all leaded to higher m:iR-34a-5p and miR34aHG expression level.ChIP and ChIP/Re-ChIP demonstrated CTCF and HDAC1 bound together at the 500-700bp of downstream of TSS.3)Additionally,we found that knockdown of CTCF induced the lower methylation levels of the CpG island nearest TSS.4)Overexpression of miR-34a-5p,Knockdown of CTCF and inhibition of HDAC1 inhibited proliferation of BCa cells through G1 phase arrest.Western Blot and luciferase reporter assays were used to manifest CDK6 mRNA was the direct target of miR-34a-5p.Knockdown and rescue experiments of CDK6 affirmed its critical role for G1 progression in BCa.5)CTCF transcriptionally inhibited p15/p16 expression in BCa.Knockdown and overexpression of CTCF,together with ChIP,clarified that CTCF transcriptionally inhibited p 15/p 16 expression in BCa.6)Knockdown of CTCF,together with inhibition of HDAC1,could downregulated.CDK6 expression,which at least in part due to transcriptional activated miR-34a-5p.7)Contrast to shCTCF+vehicle group of UM-UC-3 xnograft mouse model,shCTCF+Entinostat group possessed smaller tumor,higher miR-34a-5p and lower CDK6 expression level.8)Online Kaplan-Meier survival analysis manifested that higher expression level of CDK6 associated with poor survival rate in BCa patients(p=0.048),nevertheless,expression level of CTCF did not significantly affect the overall survival rate(p=0.197).In conclusion,the study indicated that miR-34a-5p was transcriptionally inhibited in BCa by CTCF/HDAC1-DNA methylation’ combined regulatory factors.CDK6 was the direct target of miR-34a-5p and play critical role in G1 progression of BCa.CTCF could also transcriptionally inhibited p15/p16 expression and then activated CDK6 through this bypass.