Oncogenic role of <i>MIR516A</i> in human bladder cancer was mediated by its attenuating PHLPP2 expression and BECN1-dependent autophagy

Abstract

Although <i>MIR516A</i> has been reported to be downregulated and act as a tumor suppressor in multiple cancers, its expression and potential contribution to human bladder cancer (BC) remain unexplored. Unexpectedly, we showed here that <i>MIR516A</i> was markedly upregulated in human BC tissues and cell lines, while inhibition of <i>MIR516A</i> expression attenuated BC cell monolayer growth <i>in vitro</i> and xenograft tumor growth <i>in vivo</i>, accompanied with increased expression of PHLPP2. Further studies showed that <i>MIR516A</i> was able to directly bind to the 3'-untranslated region of <i>PHLPP2</i> mRNA, which was essential for its attenuating PHLPP2 expression. The knockdown of <i>PHLPP2</i> expression in <i>MIR516A</i>-inhibited cells could reverse BC cell growth, suggesting that PHLPP2 is a <i>MIR516A</i> downstream mediator responsible for <i>MIR516A</i> oncogenic effect. PHLPP2 was able to mediate BECN1/Beclin1 stabilization indirectly, therefore promoting BECN1-dependent macroautophagy/autophagy, and inhibiting BC tumor cell growth. In addition, our results indicated that the increased autophagy by attenuating <i>MIR516A</i> resulted in a dramatic inhibition of xenograft tumor formation <i>in vivo</i>. Collectively, our results reveal that <i>MIR516A</i> has a novel oncogenic function in BC growth by directing binding to <i>PHLPP2</i> 3'-UTR and inhibiting PHLPP2 expression, in turn at least partly promoting CUL4A-mediated BECN1 protein degradation, thereby attenuating autophagy and promoting BC growth, which is a distinct function of <i>MIR516A</i> identified in other cancers.<b>Abbreviation:</b> ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; BAF: bafilomycin A₁; BC: bladder cancer; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CUL3: cullin 3; CUL4A: cullin 4A; CUL4B: cullin 4B; IF: immunofluorescence: IHC-p: immunohistochemistry-paraffin; <i>MIR516A</i>: microRNA 516a (microRNA 516a1 and microRNA 516a2); MS: mass spectrometry; PHLPP2: PH domain and leucine rich repeat protein phosphatase.