Effect of Silencing of Hypoxia Inducible Factor-1 Alpha Gene on Chemosensitivity of Human Hepatocellular Carcinoma Cells and Correlative Mechanisms_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

LIU Liping , CHEN Xiaoping , YANG Shengli , ZHANG Wanguang , XU Tao , JIN Weidong , LIANG Huifang

Hepatic Surgery Center, Tongji Hospita1, Huazhong University of Science and Technology, Wuhan 430030, China;

Corresponding author：

Keywords：

Hepatocellular carcinoma; Hypoxia inducible factor-1 alpha; RNA interference; Chemosensitivity

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【 Abstract 】 Objective To observe the effect of disruption of hypoxia inducible factor-1 α (HIF-1 α ) pathway by small hairpin RNA (shRNA) on chemosensitivity of human hepatocellular carcinoma (HCC) cells and to reveal the correlative mechanisms. Methods Plasmid of pshRNA-HIF-1α was transfected into HepG2 cells by lipofectamine. HepG2/pshRNA-HIF-1α (HepG2/pshRNA) cell lines were obtained by selection of HepG2 cells in G418. Meanwhile, plasmid of empty vector (pHK) was transfected as a control (HepG2/pHK). The mRNA and protein expression levels of HIF-1α and mdr1 were investigated by RT-PCR and Western blot respectively. Using CoCl2 to simulate the hypoxia condition, growth inhibition and apoptosis rates of HepG2 cells under different dosages of chemotherapeutic agents (adriamycin) were measured by MTT assay and flow cytometry (FCM) ． Results Compared with HepG2/pHK cells, the mRNA and protein expression levels of HIF-1αand mdr1 were obviously down-regulated in HepG2/pshRNA cells. At the same time, the proliferation inhibition and apoptosis rates were evidently increased after transfection with pshRNA-HIF-1α(P＜0.05)，which decreased the expression of HIF-1αto 82.18% at mRNA level and 75.51% at protein level. There was no significant effect of transfection pHK (P gt;0.05). Conclusion These data demonstrates that HIF-1α interference by shRNA increased the sensitivity of HCC chemotherapy and the reversal of multidrug resistance, which may be done by down-regulating the transcription of mdr1 and the translation of P-gp. Blocking HIF-1αin HCC cells may offer an new avenue for gene therapy．

Citation： LIU Liping,CHEN Xiaoping,YANG Shengli,ZHANG Wanguang,XU Tao,JIN Weidong,LIANG Huifang. Effect of Silencing of Hypoxia Inducible Factor-1 Alpha Gene on Chemosensitivity of Human Hepatocellular Carcinoma Cells and Correlative Mechanisms. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2008, 15(4): 234-239. doi: Copy

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1. 　Xu RH, Pelicano H, Zhou Y, et al. Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia [J]. Cancer Res, 2005; 65(2)∶613.
2. 　Wouters BG, Weppler SA, Koritzinsky M, et al. Hypoxia as a target for combined modality treatments [J]. Eur J Cancer, 2002; 38(2)∶240.
3. 　Chen XP, Wang Q, Guan J, et al. Reversing multidrug resistance by RNA interference through the suppression of MDR1 gene in human hepatoma cells [J]. World J Gastroenterol, 2006; 12(21)∶3332.
4. 　罗华友, 严律南, 杨家印, 等. ASODN 逆转耐药肝癌细胞多药耐药基因mdr1 的体内实验研究 [J]. 中国普外基础与临床杂志, 2004; 11(2)∶142.
5. 　朱虹, 陈孝平, 罗顺峰, 等. 缺氧诱导因子1α依赖性缺氧诱导人肝癌细胞多药耐药相关基因的表达及意义 [J]. 中华外科杂志, 2005; 43(5)∶277.
6. 　Comerford KM, Cummins EP, Taylor CT. c-Jun NH2-terminal kinase activation contributes to hypoxia-inducible factor 1alpha-dependent P-glycoprotein expression in hypoxia [J]. Cancer Res，2004; 64(24)∶9057.
7. 　Semenza GL. HIF-1 and human disease: one highly involved factor [J]. Genes Dev, 2000; 14(16)∶1983.
8. 　Jaakkola P, Mole DR, Tian YM, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation [J]. Science, 2001; 292(5516)∶468.
9. 　Wartenberg M, Ling FC, Müschen M, et al. Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species [J]. FASEB J, 2003; 17(3)∶503.
10. 　Nordsmark M, Bentzen SM, Rudat V, et al. Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study [J]. Radiother Oncol, 2005; 77(1)∶18.
11. 　Eschmann SM, Paulsen F, Reimold M, et al. Prognostic impact of hypoxia imaging with 18F-misonidazole PET in non-small cell lung cancer and head and neck cancer before radiotherapy [J]. J Nucl Med, 2005; 46(2)∶253.
12. 　权毅, 严律南. 多药耐药基因mdr1 在肝细胞性肝癌中的表达及其意义 [J]. 中国普外基础与临床杂志, 2003; 10(4)∶370.
13. 　Kamiyama N, Takagi S, Yamamoto C, et al. Expression of ABC transporters in human hepatocyte carcinoma cells with cross-resistance to epirubicin and mitoxantrone [J]. Anticancer Res, 2006; 26(2A)∶885.
14. 　　Britz-Cunningham SH, Adelstein SJ. Molecular targeting with radionuclides: state of the science [J]. J Nucl Med, 2003; 44(12)∶1945.
15. 　Ambudkar SV, Kimchi-Sarfaty C, Sauna ZE, et al. P-glycoprotein: from genomics to mechanism [J]. Oncogene, 2003; 22(47)∶7468.
16. 　Clayton J. RNA interference: the silent treatment [J]. Nature, 2004; 431(7008)∶599.
17. 　Dykxhoorn DM, Palliser D, Lieberman J. The silent treatment: siRNAs as small molecule drugs [J]. Gene Ther, 2006; 13(6)∶541.
18. 　Yuan Y, Hilliard G, Ferguson T, et al. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha [J]. J Biol Chem, 2003; 278(18)∶15911.
19. 　Bos R, van der Groep P, Greijer AE, et al. Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma [J]. Cancer, 2003; 97(6)∶1573.
20. 　 Dales JP, Garcia S, Meunier-Carpentier S, et al. Overexpression of hypoxia-inducible factor HIF-1alpha predicts early relapse in breast cancer: retrospective study in a series of 745 patients [ J ] . Int J Cancer, 2005; 116(5) ∶ 734.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Effect of Silencing of Hypoxia Inducible Factor-1 Alpha Gene on Chemosensitivity of Human Hepatocellular Carcinoma Cells and Correlative Mechanisms

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