Autophagic degradation of SQSTM1 inhibits ovarian cancer motility by decreasing DICER1 and AGO2 to induce MIRLET7A-3P.

The relationship between macroautophagy/autophagy and miRNA in regulating cancer cell motility is not clearly delineated. Here, we found that induction of BECN1-dependent or -independent autophagy decreased ubiquitin-binding proteins SQSTM1/p62 and CALCOCO2/NDP52. Downregulation of SQSTM1 (but not CALCOCO2) led to a decrease of the miRNA-processing enzyme DICER1 and the miRNA effector AGO2. The autophagy-mediated reduction of levels of SQSTM1, DICER1 or AGO2 resulted in increased MIRLET7A-3P (but not MIRLET7A-5P or PRE-MIRLET7A miRNA) and suppressed ovarian cancer motility. The investigation of the MIRLET7A effects on cancer cell motility showed that synthetic MIRLET7A-3P (3 nM) inhibited, whereas MIRLET7A-5P (100 nM) increased cancer cell motility. Moreover, downregulation of MIRLET7A-3P with antisense of MIRLET7A-3P miRNA (MIRLET7A-3P inhibitor; 3 nM) reversed the nutrient depletion- and rVP1-mediated suppression of ovarian cancer cell motility. In addition, restoring SQSTM1, DICER1 and AGO2 with inhibition of autophagic degradation or overexpression of DICER1 and AGO2 reversed the autophagy-associated enhancement of MIRLET7A-3P and inhibition of motility. Examination of ovarian cancer tissue microarray further showed that the levels of SQSTM1, DICER1 and AGO2 in the tumor were higher than those in the non-tumor cells and negatively correlated with the levels of autophagy and MIRLET7A-3P. Our results demonstrated that induction of autophagy to decrease SQSTM1, DICER1 and AGO2 and increase MIRLET7A-3P is a potential therapeutic strategy for suppressing ovarian cancer cell motility. Abbreviations: ACTB: actin beta; AGO2: argonaute 2, RISC catalytic component; ATG: autophagy related; BCIP/NBT: 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium; BECN1: beclin 1, autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CQ: chloroquine; DICER1: dicer 1, ribonuclease III; EBSS: Earle balanced salt solution; FBS: fetal bovine serum; HGF: hepatocyte growth factor; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MIRLET7A: microRNA LET-7A: MIR16: microRNA 16; MIR29C: microRNA 29C; miRNA: microRNA; MMP: matrix metallopeptidase; PRE-MIRNA: precursor microRNA; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; RISC: RNA-induced silencing complex; rVP1: recombinant foot-and-mouth disease virus capsid protein VP1; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; WIPI: WD repeat domain, phosphoinositide interacting.