miR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy - PubMed (original) (raw)

. 2021 Feb 8;17(3):781-795.

doi: 10.7150/ijbs.52517. eCollection 2021.

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

Enjiang Chen et al. Int J Biol Sci. 2021.

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.

Keywords: Doxorubicin; USP9X; hepatocellular carcinoma; microRNA-26b; p53..

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1

Figure 1

miR-26b enhances HCC cell sensitivity to doxorubicin. A. QRT-PCR analysis of the changes in miRNA after treatment with doxorubicin in SNU449 and SNU387 cells. B. The level of miR-26b was determined following treatment with or without Doxorubicin by qRT-PCR in HCC cells. **P < 0.01. C. A CCK-8 assay analysis showed that treatment with an miR-26b mimic can enhance the sensitivity of HCC cells to doxorubicin, with the exception of Hep3B cells. D and E. An EdU incorporation assay of cellular proliferation in different treatment groups. **P < 0.01. F and G. The apoptosis ratio was determined by flow cytometry. *P < 0.05; **P < 0.01. H. QRT-PCR used to determine miR-26b expression in adjacent cancer and adjacent tissues. I. We used StarBase v 3.0 project to analyze the level of miR-26b in normal and cancer tissues.

Figure 2

Figure 2

p53 was related to the sensitivity of HCC cells to doxorubicin. A. P53 protein expression was detected by Western blot. B - E. The cell viability was examined in doxorubicin, or miR-26b mimic plus doxorubicin treated cells following transfection with p53 siRNA.

Figure 3

Figure 3

miR-26b enhances HCC cell sensitivity to doxorubicin via USP9X-dependent p53 degradation. A. Western blot indicating the expression of p53 after treated with doxorubicin, doxorubicin plus miR-26 mimic, or MG132. **P < 0.01, ***P < 0.001. B. Tenovin-1 combined with an miR-26b mimic could enhance doxorubicin sensitivity in HepG2 cells, except other HCC cells. C. The predicted miR-26b binding site in the USP9X 3′ UTR and dual fluorescence reporter gene experiments verify that miR-26b binds to the promoter region of USP9X. *P < 0.05, **P < 0.01, ***P < 0.001. D. The expression of USP9X was up-regulated following treatment with the miR-26b mimic. *P < 0.05, **P < 0.01. E. P53 protein expression was determined by Western blot. F. The expression of UXP9X was determined by Western Blot.

Figure 4

Figure 4

miR-26b enhances doxorubicin sensitivity via autophagy. A. The expression of proteins in HCC cells in different treatment groups detected by Western blot. _*_P < 0.05, _**_P < 0.01.B-D. Confocal microscopy analysis of LC3 double fluorescent cells. *P < 0.05; **P < 0.01; ***P < 0.001. E-F. Electron microscopy detected autophagic flow in SNU449 and HepG2 cells. **P < 0.01 G. CCK-8 detected the sensitivity to doxorubicin in HCC cells after treatment with different conditions (transfected with miR-26b mimics, miR-26b mimic interference, and treated with rapamycin). *P < 0.05 vs Negative Control.

Figure 5

Figure 5

Treatment with an miR-26b mimic inhibits tumor growth in vivo. A. Growth curves of xenograft tumors treated with the control, doxorubicin, sp-94dr/miR-26b mimic, or doxorubicin plus with sp-94dr/miR-26b mimic (n = 6 per group). B. The mice were weighed on days 0-15 following treatment with either the control, doxorubicin, sp-94dr/miR-26b mimic, or doxorubicin plus with sp-94dr/miR-26b mimic. C. The morphology of the subcutaneous xenograft tumors in each of the different treatment groups. D. The tumor inhibition rate was determined following treatment with doxorubicin, sp-94dr/miR-26b mimic, or doxorubicin plus with sp-94dr/miR-26b mimic. *P < 0.05. E. Ki-67 staining was used to analyze the rate of Ki-67-positive staining in each of the treatment groups (40× magnification). *P < 0.05; **P < 0.01 F. The presence of apoptotic cells was detected in each of the treatment groups by a TUNEL assay. *P < 0.05; ***P < 0.001**.**

Figure 6

Figure 6

Treatment with Sp-94dr/miR-26b mimic combined with doxorubicin down-regulates p53 and USP9X expression in vivo. A and B. Immunohistochemistry-positive (IHC-P) cells were examined for the expression of USP9X and p53. *P < 0.05; **P < 0.01; ***P < 0.001. C. QRT-PCR analysis the expression of miR-26b, USP9X, and p53 in control, doxorubicin, sp-94dr/miR-26b mimic, and doxorubicin plus with sp-94dr/miR-26b mimic-treated cells. *P < 0.05; **P < 0.01; ***P < 0.001. D. USP9X, p53, and p62 protein expression were evaluated using a Western blot. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Figure 7

Schematic diagram of the regulatory mechanism of the miR-26b/USP9X/p53 axis in regulating HCC sensitivity to doxorubicin.

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