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Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells

  • Research Article
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Tumor Biology

Abstract

Abnormal clusterin (CLU) expression is associated with multidrug resistance (MDR) of hepatocellular carcinoma (HCC). In the present study, the CLU expression was analyzed in human hepatoma cells and chemoresistant counterpart HepG2/ADM cells. Compared with L02 cells, the overexpression of cellular CLU was identified in HepG2, HepG2/ADM, SMMC7721, Hep3B ,and PLC cells and relatively lower expression in Bel-7404, SNU-739, and MHCC97H cells. Specific short hairpin RNAs (shRNAs) to silence CLU gene transcription were designed, and the most effective sequences were screened. After the HepG2/ADM cells transfected with shRNA-1, the inhibition of CLU expression was 73.68 % at messenger RNA (mRNA) level by real-time quantitative RT-PCR with obvious enhancement in cell chemosensitivity, increasing apoptosis induced by doxorubicin using fluorescence kit, and Rh-123 retention qualified with flow cytometry. Knockdown CLU also significantly decreased the drug efflux pump activity through the depression of MDR1/P-glycoprotein (q = 11.739, P < 0.001). Moreover, silencing CLU led to downregulation of β-catenin (q = 13.544, P = 0.001), suggesting that downregulation of CLU might be a key point to reverse multidrug resistance of HepG2/ADM cells.

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Abbreviations

A:

Absorbance

ABC:

ATP-binding cassette

CLU:

Clusterin

HCC:

Hepatocellular carcinoma

MDR:

Multidrug resistance

nCLU:

Nuclear clusterin

PBS:

Phosphate buffered saline

RT-PCR:

Reverse transcriptase-PCR

sCLU:

Secretory clusterin

shRNA:

Short hairpin RNA

Wnt:

Wnt/β-catenin signaling pathway

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Acknowledgments

This work was supported by the Grants from the National Natural Science Foundation (81200634), the Projects of Jiangsu Medical Science (PADA, 2013-WSW-011, 2014-YY-028, HK201102, and BL2012053) and the Qinglan Project of Jiangsu Higher Education, and the International S&T Cooperation Program (2013DFA32150) of China

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    Authors and Affiliations

    1. Research Center of Clinical Medicine, Affiliated Hospital of Nantong University, 20 West Temple Road, Nantong, 226001, Jiangsu Province, China

      Wenjie Zheng, Wenli Sai & Dengfu Yao

    2. Medical School of Nantong University, Nantong, 226001, China

      Min Yao

    3. The Hospital of Nantong Maternal and Childcare Service, Nantong, 226001, China

      Hongbin Gu & Yao Yao

    4. Center of Oncology, Affiliated Hospital of Nantong University, Nantong, 226001, China

      Qi Qian

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    1. Wenjie Zheng
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    Correspondence to Dengfu Yao.

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    Wenjie Zheng and Wenli Sai contributed equally to this work.

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    Zheng, W., Sai, W., Yao, M. et al. Silencing clusterin gene transcription on effects of multidrug resistance reversing of human hepatoma HepG2/ADM cells. Tumor Biol. 36, 3995–4003 (2015). https://doi.org/10.1007/s13277-015-3043-9

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    • DOI: https://doi.org/10.1007/s13277-015-3043-9

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