MiR-26a-5p Inhibits Cell Proliferation and Enhances Doxorubicin Sensitivity in HCC Cells via Targeting AURKA

Abstract

Objective: To investigate the role of miR-26a-5p in cell proliferation and doxorubicin sensitivity in hepatocellular carcinoma.

Methods: We evaluated miR-26a-5p expression in hepatocellular carcinoma tissues and cell lines by reverse transcription polymerase chain reaction. Cell Counting Kit-8 was used to examine cell proliferation. Relationship between miR-26a-5p and aurora kinase A was evaluated by luciferase report system. Western blot was used to detect expression of aurora kinase A.

Results: In this study, we observed miR-26a-5p was downregulated in hepatocellular carcinoma tissues and cell lines. Gain-of-function experiments showed that proliferation rate of hepatocellular carcinoma cells decreased under condition of miR-26a-5p mimics. We found miR-26a-5p mimics could enhance doxorubicin sensitivity of hepatocellular carcinoma cells. Further study showed that aurora kinase A was target gene of miR-26a-5p. Suppression of aurora kinase A could lead to lower cell proliferation and higher doxorubicin sensitivity of hepatocellular carcinoma cells.

Conclusion: Our study found that miR-26a-5p could inhibit cell proliferation and enhance doxorubicin sensitivity in hepatocellular carcinoma cells by targeting aurora kinase A.

Keywords: MiR-26a-5p; aurora kinase A (AURKA); doxorubicin sensitivity; hepatocellular carcinoma (HCC).

Figure 1.

miR-26a-5p is downregulated in hepatocellular carcinoma (HCC) tissues and cell lines. A, Expression level of miR-26a-5p is detected in 35 paired HCC tissues and neighbor tissues, and the result is analyzed by the Student t test. P < .05 means statistical significance. B, Expression level of miR-26a-5p is detected in HCC cell lines and normal immortalized hepatocyte.

Figure 2.

miR-26a-5p inhibits proliferation and enhances doxorubicin sensitivity in hepatocellular carcinoma (HCC) cells. A, Cell viability of Huh7 and SMMC-7721 at 48 and 72 hours was compared between miR-26a-5p mimics and negative control; the result was analyzed by the Student t test, ***P < .0005. B, Cell viability of Huh7 and SMMC-7721 was detected under different concentrations (0, 0.0625, 0.125, 0.25, 0.5, and 1.0 µg/mL) of doxorubicin. And the results were analyzed by analysis of variance (ANOVA),***P < .0005,** P < .005.

Figure 3.

Aurora kinase A (AURKA) is a target gene regulated by miR-26a-5p. A, TargetScanHuman 7.1 showed AURKA 3′ untranslated region (3′UTR) had complementary pairing with miR-26a-5p, and we constructed the wild-type and mutant-type AURKA 3′UTR. B, The results of luciferase activity experiment in Huh7 and SMMC-7721,** P < .005,***P < .0005. C, Protein level of AURKA when treated with miR-26a-5p mimics or miR-26a-5p inhibitor; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as an internal control.

Figure 4.

miR-26a-5p regulates proliferation and doxorubicin sensitivity via targeting aurora kinase A (AURKA). A, Protein level of AURKA in hepatocellular carcinoma (HCC) cells is detected among groups of negative control, AURKA siRNA, miR-26a-5p mimics + AURKA siRNA, and miR-26a-5p inhibitor + AURKA siRNA. B and C, Cell viability of HCC cells is detected among groups of negative control, AURKA siRNA, miR-26a-5p mimics + AURKA siRNA, and miR-26a-5p inhibitor + AURKA siRNA, under different times (24, 48, and 72 hours) or different concentrations of doxorubicin (0, 0.0625, 0.125, 0.25, 0.5, and 1.0 µg/mL). Analysis of variance (ANOVA) was used to analyze statistical difference. ***P < .0005, **P < .005.

Figure 5.

Overexpression of aurora kinase A (AURKA) can reverse the effect of miR-26a-5p in hepatocellular carcinoma (HCC) cells. A, Protein level of AURKA in HCC cells is detected among groups of negative control, miR-26a-5p mimics, and miR-26a-5p mimics + AURKA overexpression (OE). B and C, Cell viability of HCC cells is detected among groups of negative control, OE control, miR-26a-5p mimics, and miR-26a-5p mimics + AURKA OE under different times (24, 48, and 72 hours) or different concentration of doxorubicin (0, 0.0625, 0.125, 0.25, 0.5, and 1.0 µg/mL), Analysis of variance (ANOVA) was used to analyze statistical difference. *P < .05, **P < .005,***P < .0005.