The influence of silencing HIF-1α gene on expressions of VEGF gene and MMP-2gene in SMMC-7721 cell line_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

HOU Lizhao ^1,2 , HUANG Yayun ³ , LU Yueqing ³ , Yangdancairang ^1,2 , REN Li ^1,2 , ZHOU Ying ^1,2 , WANG Haijiu ^1,2 ,  FAN Haining ^1,2

1. Department of Hepatopancreatobiliary Surgery, Affiliated Hospital of Qinghai University, Xining 810001, P. R. China;
2. Qinghai Province Key Laboratory of Hydatid Disease Researsh, Xining 810001, P. R. China;
3. Medical College of Qinghai University, Xining 810001, P. R. China;

Corresponding author：

FAN Haining, Email: fanhaining@medmail.com.cn

Keywords：

hypoxia inducible factor-1α; vascular endothelial growth factor; matrix metalloproteinase-2; RNA interference; SMMC-7721 cell line

DOI：

10.7507/1007-9424.201704081

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Objective To explore the influence on the expressions of vascular endothelial growth factor (VEGF) gene and matrix metalloproteinase-2 (MMP-2) gene in hepatocellular carcinoma of SMMC-7721 cells with RNA interference (RNAi) silencing the expression of hypoxia inducible factor-1α (HIF-1α) gene. Methods Firstly, constructed short hairpin RNA (shRNA) targeting for HIF-1α gene, and then transfected it to SMMC-7721 cells after combining with plasmid. The SMMC-7721 cells were divided into three groups, silencing group, negative control group, and blank control group, which were transfected with HIF-1α-shRNA-pGenesil-1 recombinant vector, shRNA-HK-pGenesil-1 recombinant vector, and pGenesil-1 vector respectively. Transfection cells were screened by the concentration of 500 μg/mL G418, and then positive and negative cell clones with transfection recombination carrier were obtained. Detected the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups with real time PCR (RT-PCR) technology, under the condition of hypoxic training 6 h, 12 h, and 24 h, as well as conventional oxygen training. Results There was no expression of HIF-1α mRNA at conventional oxygen condition in the 3 groups, and there was no significant difference in expressions of VEGF mRNA and MMP-2 mRNA among the 3 groups (P>0.05) at the condition of conventional oxygen training. The expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the silencing group, compared with the the negative control group and the blank control group, were obviously decreased (P<0.05) under the condition of hypoxic training (6, 12, and 24 h), while there was no significant difference between the negative control group and the blank control group at each time point (P>0.05), but the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups under every condition of hypoxic training were all higher than those of conventional oxygen condition (P<0.05). Under the condition of hypoxic training, the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups decreased over time, and there was significant difference between any 2 time points in each group (P<0.05). Conclusion RNAi technique can effectively silence the expression of HIF-1α mRNA of SMMC-7721 cells, and then silence the expressions of VEGF and MMP-2 mRNA, to inhibit the invasion and metastasis of hepatocellular carcinoma.

Citation： HOU Lizhao, HUANG Yayun, LU Yueqing, Yangdancairang, REN Li, ZHOU Ying, WANG Haijiu, FAN Haining. The influence of silencing HIF-1α gene on expressions of VEGF gene and MMP-2gene in SMMC-7721 cell line. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2017, 24(10): 1206-1212. doi: 10.7507/1007-9424.201704081 Copy

1.	付艳, 邢卉春. 原发性肝癌的流行状况及危险因素分析. 中国肝脏病杂志(电子版), 2014, 6(2): 87-90.0.
2.	陈琳, 张志伟, 陈义发, 等. 293 例原发性肝癌术后复发的综合治疗效果分析. 腹部外科, 2017, 30(2): 89-92, 96.0.
3.	Hockel M, Schlenger K, Aral B, et al. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res, 1996, 56(19): 4509-4515.
4.	郑怀, 石学林, 黄广升. HIF-1α、ToPo-Ⅱ、Ki-67 在乳腺癌组织中的表达研究. 白求恩医学杂志, 2016, 14(2): 139-141.0.
5.	Sundfør K, Lyng H, Rofstad EK. Tumour hypoxia and vascular density as predictors of metastasis in squamous cell carcinoma of the uterine cervix. Br J Cancer, 1998, 78(6): 822-827.
6.	Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res, 1996, 56(5): 941-943.
7.	Ogiso Y, Tomida A, Lei S, et al. Proteasome inhibition circumvents solid tumor resistance to topoisomeraseⅡ-directed drugs. Cancer Res, 2000, 60(9): 2429-2434.
8.	Beavon IR. Regulation of E-cadherin: does hypoxia initiate the metastatic cascade? Mol Pathol, 1999, 52(4): 179-188.
9.	Dachs GU, Tozer GM. Hypoxia modulated gene expression: angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer, 2000, 36(13 Spec No): 1649-1660.
10.	Jögi A, Øra I, Nilsson H, et al. Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype. Proc Natl Acad Sci U S A, 2002, 99(10): 7021-7026.
11.	黄耿文, 杨连粤. 缺氧致肿瘤恶性转化的分子机制. 世界华人消化杂志, 2001, 9(11): 1300-1304.0.
12.	Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol, 1992, 12(12): 5447-5454.
13.	Schmaltz C, Hardenbergh PH, Wells A, et al. Regulation of proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol, 1998, 18(5): 2845-2854.
14.	Zhao B, Ma A, Cai J, et al. VEGF-A regulates the expression of VEGF-C in human retinal pigment epithelial cells. Br J Ophthalmol, 2006, 90(8): 1052-1059.
15.	Neagoe PE, Lemieux C, Sirois MG. Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and -2 heterodimer. J Biol Chem, 2005, 280(11): 9904-9912.
16.	Veikkola T, Karkkainen M, Claesson-Welsh L, et al. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res, 2000, 60(2): 203-212.
17.	Inda AM, Andrini LB, García MN, et al. Evaluation of angiogenesis with the expression of VEGF and CD34 in human non-small cell lung cancer. J Exp Clin Cancer Res, 2007, 26(3): 375-378.
18.	Clauss M, Schaper W. Vascular endothelial growth factor: a jack-of-all-trades or a nonspecific stress gene? Circ Res, 2000, 86(3): 251-252.
19.	Jacquemier J, Mathoulin-Portier MP, Valtola R, et al. Prognosis of breast-carcinoma lymphagenesis evaluated by immunohistochemical investigation of vascular-endothelial-growth-factor receptor 3. Int J Cancer, 2000, 89(1): 69-73.
20.	Rak J, Mitsuhashi Y, Sheehan C, et al. Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts. Cancer Res, 2000, 60(2): 490-498.
21.	Van Valckenborgh E, Croucher PI, De Raeve H, et al. Multifunctional role of matrix metalloproteinases in multiple myeloma: a study in the 5T2MM mouse model. Am J Pathol, 2004, 165(3): 869-878.
22.	颜子颖, 王海林. 精编分子生物学实验指南. 北京: 科学出版社, 1998: 286-287.
23.	Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer, 2003, 3(10): 721-732.
24.	Kleiner DE, Stetler-Stevenson WG. Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol, 1999, 43 Suppl: S42-S51.
25.	McLaughlin SH, Smith HW, Jackson SE. Stimulation of the weak ATPase activity of human hsp90 by a client protein. J Mol Biol, 2002, 315(4): 787-798.
26.	经庆玲. MMP-2、MMP-9、VEGF 在人非小细胞肺癌中的表达及其与临床病理特征的相关性分析. 南宁: 广西医科大学, 2014.
27.	王阿曼, 蔡欣, 周涛, 等. MMP-2、VEGF、CD105 和 Ki-67 在小细胞肺癌中的表达及预后相关性研究. 临床肿瘤学杂志, 2012, 17(11): 988-993.0.

1. 付艳, 邢卉春. 原发性肝癌的流行状况及危险因素分析. 中国肝脏病杂志(电子版), 2014, 6(2): 87-90.0.
2. 陈琳, 张志伟, 陈义发, 等. 293 例原发性肝癌术后复发的综合治疗效果分析. 腹部外科, 2017, 30(2): 89-92, 96.0.
3. Hockel M, Schlenger K, Aral B, et al. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res, 1996, 56(19): 4509-4515.
4. 郑怀, 石学林, 黄广升. HIF-1α、ToPo-Ⅱ、Ki-67 在乳腺癌组织中的表达研究. 白求恩医学杂志, 2016, 14(2): 139-141.0.
5. Sundfør K, Lyng H, Rofstad EK. Tumour hypoxia and vascular density as predictors of metastasis in squamous cell carcinoma of the uterine cervix. Br J Cancer, 1998, 78(6): 822-827.
6. Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res, 1996, 56(5): 941-943.
7. Ogiso Y, Tomida A, Lei S, et al. Proteasome inhibition circumvents solid tumor resistance to topoisomeraseⅡ-directed drugs. Cancer Res, 2000, 60(9): 2429-2434.
8. Beavon IR. Regulation of E-cadherin: does hypoxia initiate the metastatic cascade? Mol Pathol, 1999, 52(4): 179-188.
9. Dachs GU, Tozer GM. Hypoxia modulated gene expression: angiogenesis, metastasis and therapeutic exploitation. Eur J Cancer, 2000, 36(13 Spec No): 1649-1660.
10. Jögi A, Øra I, Nilsson H, et al. Hypoxia alters gene expression in human neuroblastoma cells toward an immature and neural crest-like phenotype. Proc Natl Acad Sci U S A, 2002, 99(10): 7021-7026.
11. 黄耿文, 杨连粤. 缺氧致肿瘤恶性转化的分子机制. 世界华人消化杂志, 2001, 9(11): 1300-1304.0.
12. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol, 1992, 12(12): 5447-5454.
13. Schmaltz C, Hardenbergh PH, Wells A, et al. Regulation of proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol, 1998, 18(5): 2845-2854.
14. Zhao B, Ma A, Cai J, et al. VEGF-A regulates the expression of VEGF-C in human retinal pigment epithelial cells. Br J Ophthalmol, 2006, 90(8): 1052-1059.
15. Neagoe PE, Lemieux C, Sirois MG. Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and -2 heterodimer. J Biol Chem, 2005, 280(11): 9904-9912.
16. Veikkola T, Karkkainen M, Claesson-Welsh L, et al. Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res, 2000, 60(2): 203-212.
17. Inda AM, Andrini LB, García MN, et al. Evaluation of angiogenesis with the expression of VEGF and CD34 in human non-small cell lung cancer. J Exp Clin Cancer Res, 2007, 26(3): 375-378.
18. Clauss M, Schaper W. Vascular endothelial growth factor: a jack-of-all-trades or a nonspecific stress gene? Circ Res, 2000, 86(3): 251-252.
19. Jacquemier J, Mathoulin-Portier MP, Valtola R, et al. Prognosis of breast-carcinoma lymphagenesis evaluated by immunohistochemical investigation of vascular-endothelial-growth-factor receptor 3. Int J Cancer, 2000, 89(1): 69-73.
20. Rak J, Mitsuhashi Y, Sheehan C, et al. Oncogenes and tumor angiogenesis: differential modes of vascular endothelial growth factor up-regulation in ras-transformed epithelial cells and fibroblasts. Cancer Res, 2000, 60(2): 490-498.
21. Van Valckenborgh E, Croucher PI, De Raeve H, et al. Multifunctional role of matrix metalloproteinases in multiple myeloma: a study in the 5T2MM mouse model. Am J Pathol, 2004, 165(3): 869-878.
22. 颜子颖, 王海林. 精编分子生物学实验指南. 北京: 科学出版社, 1998: 286-287.
23. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer, 2003, 3(10): 721-732.
24. Kleiner DE, Stetler-Stevenson WG. Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol, 1999, 43 Suppl: S42-S51.
25. McLaughlin SH, Smith HW, Jackson SE. Stimulation of the weak ATPase activity of human hsp90 by a client protein. J Mol Biol, 2002, 315(4): 787-798.
26. 经庆玲. MMP-2、MMP-9、VEGF 在人非小细胞肺癌中的表达及其与临床病理特征的相关性分析. 南宁: 广西医科大学, 2014.
27. 王阿曼, 蔡欣, 周涛, 等. MMP-2、VEGF、CD105 和 Ki-67 在小细胞肺癌中的表达及预后相关性研究. 临床肿瘤学杂志, 2012, 17(11): 988-993.0.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

The influence of silencing HIF-1α gene on expressions of VEGF gene and MMP-2gene in SMMC-7721 cell line

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