X. CUI1,2, Q. Du1, D. Zhou2, D. Geller1 1University Of Pittsburg,Department Of Surgery,Pittsburgh, PA, USA 2The second hospital of Anhui University,Department Of General Surgery,Hefei, ANHUI, China
Introduction:
The inflammatory reaction in hepatocellular carcinoma (HCC) tumor microenvironment correlates with recurrence and poor prognosis after liver resection surgery. MicroRNA-200a (miR-200a) negatively regulates epithelial-mesenchymal transition (EMT) and promotes immunomodulatory function in tumors. However, the effects of miR-200a in response to HCC tumors undergoing chemotherapy are unknown. Here, we investigated the effects of miR-200a on cell growth and autophagy in HCC tumor cells treated with doxorubicin.
Methods:
qPCR for miR-200a was performed in tumor and adjacent non-tumor tissues from 30 patients with HCC. Proliferation assay and half-inhibitory concentration (IC 50) were employed in Huh-7 liver tumor cells with/without doxorubicin. We stably expressed miR-200a in Huh-7 cells using lentivirus transduction, and down-regulated endogenous miR200a using shRNA. Effect of miR-200a on cell growth and autophagy with/without doxorubicin treatment was determined. Informed consent was obtained from the patients involved in this research.
Results:
MiR-200a level in HCC tumors was lower than the adjacent non-tumor tissue (Fig. 1A, p<0.01). Huh-7 human liver tumor cells were successfully transfected with Lv-miR-200a, and shRNA targeting miR-200a (Lv-anti-miR-200a) silenced miR-200a expression (Fig.1B). MiR-200a expression was negatively associated with tumor differentiation and liver fibrosis (p=0.030; p=0.032, data not shown). Lentiviral miR-200a over-expression significantly inhibited HCC cell proliferation (Fig. 1C) and reduced the IC50 value of doxorubicin (Fig.1D), while inhibition of endogenous miR200a had the opposite effect. Autophagy with autolysosomes (red dots) and autophagosomes (yellow dots) was increased in HCC cells overexpressing miR-200a, while silencing of endogenous miR-200a decreased basal autophagy (Fig.1E, 1F). Rapamycin was used as positive control to induce autophagy, and the miR-200a – induced autophagy was abrogated with the autophagy inhibitor 3-methyladenine (3-MA).
Conclusion:
These data show a novel role of miR-200a in HCC tumor biology. Endogenous miR-200a expression is down-regulated in human HCC tissues, while overexpression of miR-200a exerts anti-tumor effects, increases autophagy, and enhances doxorubicin effects.
