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

Autophagy. 2021 Apr;17(4):840-854. doi: 10.1080/15548627.2020.1733262. Epub 2020 Mar 1.

Abstract

Although MIR516A 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 MIR516A was markedly upregulated in human BC tissues and cell lines, while inhibition of MIR516A expression attenuated BC cell monolayer growth in vitro and xenograft tumor growth in vivo, accompanied with increased expression of PHLPP2. Further studies showed that MIR516A was able to directly bind to the 3'-untranslated region of PHLPP2 mRNA, which was essential for its attenuating PHLPP2 expression. The knockdown of PHLPP2 expression in MIR516A-inhibited cells could reverse BC cell growth, suggesting that PHLPP2 is a MIR516A downstream mediator responsible for MIR516A 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 MIR516A resulted in a dramatic inhibition of xenograft tumor formation in vivo. Collectively, our results reveal that MIR516A has a novel oncogenic function in BC growth by directing binding to PHLPP2 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 MIR516A identified in other cancers.Abbreviation: 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; MIR516A: microRNA 516a (microRNA 516a1 and microRNA 516a2); MS: mass spectrometry; PHLPP2: PH domain and leucine rich repeat protein phosphatase.

Keywords: Autophagy; BECN1; MIR516A; PHLPP2; bladder cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions / genetics
  • Autophagy / genetics*
  • Base Sequence
  • Beclin-1 / metabolism*
  • Carcinogenesis / genetics*
  • Carcinogenesis / pathology
  • Cell Line, Tumor
  • Cell Proliferation / genetics
  • Cullin Proteins / metabolism
  • Down-Regulation / genetics
  • Gene Expression Regulation, Neoplastic*
  • Humans
  • Ki-67 Antigen / genetics
  • Ki-67 Antigen / metabolism
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Phosphoprotein Phosphatases / genetics*
  • Phosphoprotein Phosphatases / metabolism
  • Protein Binding
  • Proteolysis
  • Urinary Bladder Neoplasms / genetics*
  • Urinary Bladder Neoplasms / pathology
  • Urinary Bladder Neoplasms / ultrastructure
  • Xenograft Model Antitumor Assays

Substances

  • 3' Untranslated Regions
  • Beclin-1
  • CUL4A protein, human
  • Cullin Proteins
  • Ki-67 Antigen
  • MIRN516 microRNA, human
  • MKI67 protein, human
  • MicroRNAs
  • PHLPP2 protein, human
  • Phosphoprotein Phosphatases

Grants and funding

This work was partially supported by the Natural Science Foundation of China NSFC81702530, NSFC81872587 and NSFC31970696]; Wenzhou Science and Technology Bureau [Y20170028 and Y20190065], Wenzhou Medical University [89216021]; Key Project of Science and Technology Innovation Team of Zhejiang Province [2013TD10], Key Discipline of Zhejiang Province in Medical Technology (First Class, Category A) and Xinmiao Talent Program of Zhejiang Province [2017R413064].