Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 7, 2015; 21(45): 12814-12821
Published online Dec 7, 2015. doi: 10.3748/wjg.v21.i45.12814
Chemosensitization of HepG2 cells by suppression of NF-κB/p65 gene transcription with specific-siRNA
Yun Shi, Si-Ye Wang, Min Yao, Wen-Li Sai, Wei Wu, Jun-Ling Yang, Yin Cai, Wen-Jie Zheng, Deng-Fu Yao
Yun Shi, Si-Ye Wang, Min Yao, Wen-Li Sai, Wei Wu, Jun-Ling Yang, Wen-Jie Zheng, Deng-Fu Yao, Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
Min Yao, Department of Immunology, Medical School of Nantong University, Nantong 226001, Jiangsu Province, China
Yin Cai, Department of Oncology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
Author contributions: Shi Y, Wang SY, and Yao M contributed equally to this work, designed the research, analyzed the data, and wrote the first draft; Sai WL, Wu W, and Qiu LW performed quantitative real time PCR; Yang JL and Cai Y analyzed the data by Western blotting and ELISA; Zhang HJ and Zheng WJ performed immunohistochemistry, transfection, and cell proliferation, survival, and apoptosis assays; Yao DF is the guarantor and contributed to the design and interpretation of the study.
Supported by Grants from the Jiangsu Provincial Special Programs of Medical Science, BL2012053, HK201102; the Nantong Undertaking and Technological Innovation, HS2013007, BK2013048 and HS2014078; the Priority Academic Program Development of Higher Education Institution of Jiangsu Province, the National Natural Science Foundation, No. 81200634; and the international S &T Cooperation Program (2013DFA32150) of China.
Conflict-of-interest statement: The authors declare no conflict of interest.
Open-Access: This article is an open-access article which 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/
Correspondence to: Deng-Fu Yao, MD, PhD, Professor, Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, No. 20 West Temple Road, Nantong 226001, Jiangsu Province, China. yaodf@ahnmc.com
Telephone: +86-513-85052297 Fax: +86-513-85052523
Received: March 28, 2015
Peer-review started: March 29, 2015
First decision: April 24, 2015
Revised: July 4, 2015
Accepted: September 15, 2015
Article in press: September 15, 2015
Published online: December 7, 2015
Processing time: 252 Days and 21.3 Hours
Abstract

AIM: To investigate small interfering RNA (siRNA)-mediated inhibition of nuclear factor-kappa B (NF-κB) activation and multidrug-resistant (MDR) phenotype formation in human HepG2 cells.

METHODS: Total RNA was extracted from human HepG2 or LO2 cells. NF-κB/p65 mRNA was amplified by nested reverse transcription polymerase chain reaction and confirmed by sequencing. NF-κB/p65 was analyzed by immunohistochemistry. Specific-siRNA was transfected to HepG2 cells to knock down NF-κB/p65 expression. The effects on cell proliferation, survival, and apoptosis were assessed, and the level of NF-κB/p65 or P-glycoprotein (P-gp) was quantitatively analyzed by enzyme-linked immunosorbent assay.

RESULTS: HepG2 cells express NF-κB/p65 and express relatively less phosphorylated p65 (P-p65) and little P-gp. After treatment of HepG2 cells with different doses of doxorubicin, the expression of NF-κB/p65, P-p65, and especially P-gp were dose-dependently upregulated. After HepG2 cells were transfected with NF-κB/p65 siRNA (100 nmol/L), the expression of NF-κB/p65, P-p65, and P-gp were downregulated significantly and dose-dependently. The viability of HepG2 cells was decreased to 23% in the combination NF-κB/p65 siRNA (100 nmol/L) and doxorubicin (0.5 μmol/L) group and 47% in the doxorubicin (0.5 μmol/L) group (t = 7.043, P < 0.001).

CONCLUSION: Knockdown of NF-κB/p65 with siRNA is an effective strategy for inhibiting HepG2 cell growth by downregulating P-gp expression associated chemosensitization and apoptosis induction.

Keywords: Hepatocellular carcinoma; Nuclear factor-κB; Multidrug-resistant; Chemosensitization; Small interference RNA; P-glycoprotein

Core tip: Hepatic nuclear factor-kappa B (NF-κB) signaling pathway could be a potential target for designing highly effective therapeutic agents for the chemoprevention of hepatocellular carcinoma (HCC). Specific siRNAs used in combination with doxorubicin could enhance doxorubicin cytotoxicity in human HepG2 cells by downregulating NF-κB and P-glycoprotein expression. Stable NF-κB inhibition and chemosensitization could significantly inhibit tumor cell proliferation. Thus, the modulation of NF-κB might represent an advance in HCC therapy efficacy and is worthy of further research and investigation.