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MiR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy.

ABSTRACT: Multi-drug resistance is a major challenge to hepatocellular carcinoma (HCC) treatment, and the over-expression or deletion of microRNA (miRNA) expression is closely related to the drug-resistant properties of various cell lines. However, the underlying molecular mechanisms remain unclear. CCK-8, EdU, flow cytometry, and transmission electron microscopy were performed to determine cell viability, proliferation, apoptosis, autophagic flow, and nanoparticle characterization, respectively. In this study, the results showed that the expression of miR-26b was downregulated following doxorubicin treatment in human HCC tissues. An miR-26b mimic enhanced HCC cell doxorubicin sensitivity, except in the absence of p53 in Hep3B cells. Delivery of the proteasome inhibitor, MG132, reversed the inhibitory effect of miR-26b on the level of p53 following doxorubicin treatment. Tenovin-1 (an MDM2 inhibitor) protected p53 from ubiquitination-mediated degradation only in HepG2 cells with wild type p53. Tenovin-1 pretreatment enhanced HCC cell resistance to doxorubicin when transfected with an miR-26b mimic. Moreover, the miR-26b mimic inhibited doxorubicin-induced autophagy and the autophagy inducer, rapamycin, eliminated the differences in the drug sensitivity effect of miR-26b. In vivo, treatment with sp94dr/miR-26b mimic nanoparticles plus doxorubicin inhibited tumor growth. Our current data indicate that miR-26b enhances HCC cell sensitivity to doxorubicin through diminishing USP9X-mediated p53 de-ubiquitination caused by DNA damaging drugs and autophagy regulation. This miRNA-mediated pathway that modulates HCC will help develop novel therapeutic strategies.

SUBMITTER: Chen E

PROVIDER: S-EPMC7975695 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Metastasis is responsible for the rapid recurrence and poor survival of malignancies. Epithelial-mesenchymal transition (EMT) has a critical role in metastasis. Increasing evidence indicates that EMT can be regulated by microRNAs (miRNAs). The aim of this study was to investigate the role of miR-26b in modulating epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma (HCC), as well as to identify its underlying mechanism of action.The expression level of miR-26b was assessed in multiple HCC cell lines (HepG2, MHCC97H, Hep3B, MHCC97L, HCCC9810, BEL-7402, Huh7 and QGY-7703), as well as in liver tissue from patients with HCC. Follow-up studies examined the effects of a miR-26b mimic (increased expression) and a miR-26b inhibitor (decreased expression) on markers of EMT, wound healing and cell migration. The molecular target of miR-26b was also identified using a computer algorithm and confirmed experimentally.MiR-26b expression was decreased in HCC cell lines and was inversely correlated with the grade of HCC. Increased expression of miR-26b inhibited the migration and invasiveness of HCC cell lines, which was accompanied by decreased expression of the epithelial marker E-cadherin and increased expression of the mesenchymal marker vimentin, at both the mRNA and protein expression levels. A binding site for miR-26b was theoretically identified in the 3'UTR of USP9X. Further studies revealed that overexpression of miR-26b repressed the endogenous level of USP9X protein expression. Overexpression of miR-26b also repressed Smad4 expression, whereas its inhibition elevated Smad4 expression.Taken together, our results indicate that miR-26b were inhibited in HCC. In HCC cell lines, miR-26b targeted the 3'UTR of USP9X, which in turn affects EMT through Smad4 and the TGF-? signaling pathway. Our analysis of clinical HCC samples verifies that miR-26b also targets USP9X expression to inhibit the EMT of hepatocytes. Thus, miR-26b may have some effects on the EMT of HCC cells.

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MiR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy.

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