The Anti-tumor Role And Mechanism Of MiR-148a-3p In Bladder Cancer

Bladder cancer is one of the most common cancers in the world,and approximately one-third of bladder cancer patients develop locally advanced and metastatic disease.The 5-year survival rate is lower than 62%.Therefore,it is of great importance to understand the carcinogenic mechanisms and develop novel therapeutic targets of bladder cancer.MicroRNAs(miRNAs)are small(20?23 nucleotides)non-coding RNAs that comprise a novel class of target gene regulators,and they act by accelerating the degradation and/or blocking the translation of their target mRNAs.In our previous studies,we identified a series of miRNAs involved in bladder cancer proliferation,migration and invasion,including miR-26a,miR-101,miR-124-3p,miR-320c,miR-409-3p,miR-490-5p,miR-433 and miR-576-3p.However,the biological function of miRNAs in bladder cancer is still unclear.Our Study aimed to explore the suppression effect,progression effect and molecular mechanism of miR-148a-3p in bladder cancer.The main investigations and results are as follows:1)miR-148a-3p was downregulated in bladder cancer.ISH analysis demonstrated that miR-148a-3p expression was significantly lower in bladder cancer tissues compared to adjacent non-tumor tissues,and that miR-148a-3p localized to the cytoplasm.To further evaluate miR-148a-3p expression in bladder cancer,we performed quantitative real-time PCR(qRT-PCR)in T24,UM-UC-3,and J82 cell lines.miR-148a-3p expression was significantly downregulated in all three different bladder cancer cell lines compared to the SV-HUC-1 cell line2)The results of BSP indicated that methylation levels of miR-148a-3p were significantly higher in bladder cancer cell lines(T24 and UM-UC-3).5-Aza-2'-deoxycytidine(5-Aza)significantly upregulated the expression of miR-148a-3p.3)miR-148a-3p overexpression inhibited cell proliferation in vivo and in vitro.Besides,miR,148a-3p suppressed the cell motility and EMT phenotype of T24 and UM-UC-3 cells by regulating AKT2/Snail signaling.4)ERBB3 and AKT2 were direct miR-148a-3p targets.We used bioinformatics prediction and gene expression microarray analysis to predict the downstream target of miR-148a-3p.qRT-PCR,Western Blot,IHC and luciferase reporter assays were used to verify that ERBB3 and AKT2 were direct miR-148a-3p targets.5)Silencing ERBB3 inhibited cell motility and EMT phenotype of T24 and UM-UC-3 cells by regulating AKT2/Snail signaling.Silencing ERBB3 and AKT2 inhibited cell proliferation by cell cycle arrest.We further compared the expression of miR-148a-3p,DNMT1 and ERBB3 in bladder cancer tissues.miR-148a-3p was significantly negatively correlated with ERBB3(r=0.149,P=0.017)and DNMT1(r=0.108,P=0.041)expression.Moreover,we observed significant positive correlation between ERBB3 and DNMT1(r=0.116,P=0.042).Kaplan-Meier survival curves indicated that the protein ERBB3 expression was significantly associated with a poor overall survival rate of bladder cancer patients(P=0.026).Finally,in a multivariate Cox model including age,gender,tumor grade,lymph node invasion,ERBB3 expression,DNMT1 expression and miR-148a-3p expression,we found that ERBB3 overexpression was a poor independent prognostic factor for the overall survival in patients with bladder cancer(P=0.044).6)miR-148a-3p/ERBB3/AKT2/c-myc established a positive feedback loop to regulate bladder cancer.In conclusion,this study indicated that miR-148a-3p was a novel suppressor in bladder cancer.miR-148a-3p inhibited bladder cancer cell proliferation and EMT by regulating ERBB3/AKT2/c-myc and ERBB3/AKT2/Snail signaling,and miR-148a-3p/ERBB3/AKT2/c-myc established a positive feedback loop to regulate bladder cancer.