Basic Study
Copyright ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 14, 2019; 25(22): 2752-2762
Published online Jun 14, 2019. doi: 10.3748/wjg.v25.i22.2752
MiR-34a overexpression enhances the inhibitory effect of doxorubicin on HepG2 cells
Shun-Zhen Zheng, Ping Sun, Jian-Ping Wang, Yong Liu, Wei Gong, Jun Liu
Shun-Zhen Zheng, Jian-Ping Wang, Yong Liu, Wei Gong, Jun Liu, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan 250021, Shandong Province, China
Ping Sun, Department of Clinical Laboratory, Blood Station of Jinan, Jinan 250021, Shandong Province, China
Author contributions: Zheng SZ and Li J designed the research; Zheng SZ, Sun P, Wang JP, Liu Y, and Gong W performed the research; Sun P and Wang JP analyzed the data; Zheng SZ wrote the paper.
Supported by: the National Natural Science Foundation of China, No. 81302124, Shandong Key R and D Program No. 2017GSF218038 and Shandong Provincial Natural Science Foundation, No. ZR2014HP065.
Institutional review board statement: This study was reviewed and approved by the Medical Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University.
Institutional animal care and use committee statement: No animal models were used in this study.
Conflict-of-interest statement: None of the authors has any conflicts of interest to declare.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: No animal models were used in this study.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Jun Liu, MD, Professor, Surgeon, Department of Liver Transplantation and Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, No. 324 Jingwu Road, Jinan 250021, Shandong Province, China. dr_liujun1967@126.com
Telephone: +86-531-68776932 Fax: +86-531-87925615
Received: March 14, 2019
Peer-review started: March 14, 2019
First decision: April 4, 2019
Revised: April 13, 2019
Accepted: April 29, 2019
Article in press: April 29, 2019
Published online: June 14, 2019
Processing time: 95 Days and 3.2 Hours
Abstract
BACKGROUND

Hepatocellular carcinoma (HCC) is the third leading cause of death from malignant tumors worldwide. More than 50% of HCC cases occur in China. The prognosis remains poor and overall efficacy is still unsatisfactory. Chemotherapy resistance is the most important reason for the poor outcome. Much progress has been made in the study of chemotherapy resistance of HCC; however, the specific mechanisms of progression of HCC have still only been partially established. Therefore, the mechanism of chemotherapy resistance in HCC requires more research.

AIM

To investigate the effect of miR-34a expression on the growth inhibition of HepG2 cells by doxorubicin.

METHODS

A recombinant lentiviral vector containing miR-34a was constructed and transfected into HepG2 cells. The expression of miR-34a was detected by reverse transcription-polymerase chain reaction (commonly known as RT-PCR) before and after transfection. Cells were exposed to 2 μM doxorubicin or phosphate-buffered saline before and after transfection. Cell viability in each group was detected by MTT assay, and cell cycle and apoptosis were detected by flow cytometry. Changes in expression levels of phospho (p)-p53, sirtuin (SIRT) 1, cyclin D1, cyclin-dependent kinase (CDK) 4, CDK6, BCL-2, multidrug resistance protein (MDR) 1/P glycoprotein (P-gp), and AXL were detected by Western blotting.

RESULTS

Recombinant lentiviral vector LV-hsa-mir-34a was successfully constructed by restriction endonuclease digestion and sequencing. RT-PCR showed that expression of miR-34a in HepG2 cells was significantly upregulated after transfection (P < 0.01). MTT assay showed that growth of HepG2 cells was inhibited after upregulation of miR-34a, and viability was significantly decreased after combined treatment with doxorubicin (P < 0.01). Flow cytometry showed that the number of HepG2 cells in G1 phase increased, and G1 phase arrest was more obvious after intervention with doxorubicin (P < 0.01). The apoptosis rate of HepG2 cells was increased after upregulation of miR-34a, and became more obvious after intervention with doxorubicin (P < 0.01). Western blotting showed that upregulation of miR-34a combined with treatment with doxorubicin caused significant changes in the expression levels of p-p53, SIRT1, cyclin D1, CDK4, CDK6, BCL-2, MDR1/P-gp and AXL proteins (P < 0.01).

CONCLUSION

MiR-34a may enhance the inhibitory effect of doxorubicin by downregulating MDR1/P-gp and AXL, which may be related to p53 expression.

Keywords: miR-34a; Doxorubicin; Hepatocellular carcinoma; HepG2 cells; Growth inhibition

Core tip: Hepatocellular carcinoma (HCC) is the third leading cause of death from malignant tumors worldwide. The prognosis remains poor, and the overall efficacy is still unsatisfactory. Chemotherapy resistance is the most significant cause of poor HCC outcome. We found that upregulation of miR-34a can increase the chemosensitivity of HCC cells to doxorubicin. MiR-34a may enhance the inhibitory effect of doxorubicin by downregulating multidrug resistance protein 1/P glycoprotein and AXL, which may be related to p53 expression.