Inhibition Effect on Expression of RASSF1A Gene by 5HRE Combined with CEAp Element Targeted Regulation for Human Gastric Cancer SGC7901 Cells_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 ZHOUPei-hua ,  SUNXue-jun , ZHENGJian-bao , WANGWei , WANGXiao-long , CHENNan-zheng , WEIGuang-bing , FANYuan , LIXiao-bin , WANGXun-kai , LUShao-ying

Department of General Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China;

Corresponding author：

SUNXue-jun, Email: sunxy@mail.xjtu.edu.cn

Keywords：

Five copies hypoxia-responsive element; Carcinoembryonic antigen promoter; Ras association domain family 1 isoform A gene; Gastric cancer; Gene therapy

DOI：

10.7507/1007-9424.20150312

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Objective To explore the effect of five copies hypoxia-responsive element (5HRE) and carcinoembryonic antigen promoter (CEAp) element, and to explore the inhibition effect of lentiviral vectors targeted Ras association domain family 1 isoform A (RASSF1A) gene on SGC7901 human gastric cancer cells. Methods ①Expressions of carcinoembryonic antigen (CEA) mRNA and its protein, and RASSF1A protein in SGC7901, MKN28, and MCF-10A cells were detect by real time-PCR (qRT-PCR), immunocytochemistry, and Western blot, to confirm the experimental and negative control cells.②The recombinant vectors of pGL4.20-5HRE-CEAp-Luc were constructed through molecular cloning technique to transfected SGC7901, MKN28, and MCF-10A cells. Each kind of cell was divided into 2 groups:one of them didn't add CoCl₂ (normoxia group), and another group added CoCl₂ (hypoxia group). Comparison of the fold of activation was performed.③SGC7901 cells were infected by lentiviral vectors of pLV-5HRE-CEAp-RASSF1A (infection group) and negative virus (negative control group), SGC7901 cells without any treatment as blank control group. Then SGC7901 cells of 3 groups were divided into 2 groups:one of them didn't add CoCl₂ (normoxia group), and another group added CoCl₂ (hypoxia group). The expression of RASSF1A protein was tested by Western blot, and the growth inhibition rate was confirmed by cell counting kit-8 (CCK-8) assay. Comparisons of expression of RASSF1A protein and growth inhibition rate of each group were performed. Results ①Results of qRT-PCR, immunocytochemistry and Western blot showed that, SGC7901 cells showed higher expression of CEA mRNA and positive expression of RASSF1A protein than corresponding index of MKN28 cells and MCF-10A cells (P < 0.05), which were assigned as experimental cells; but MKN28 cells showed lower expression of CEA mRNA and negative expression of RASSF1A protein, which were assigned as negative control cells.②In SGC7901 and MKN28 cells transfected recombinant vectors of pGL4.20-5HRECEAp-Luc, compared with normoxia group in the same kind of cell group, the folds of activation in hypoxia group were higher (P < 0.01), but there was no significant difference between the normoxia group and hypoxia group in MCF-10A cells (P > 0.05). In the condition of with or without CoCl₂, compared with SGC7901 cells in the same condition, the folds of activation in MCF-10A and MKN28 cells were both lower (P < 0.05); compared with MKN28 cells, the fold of activation in MCF-10A cells was lower (P < 0.05).③Western blot results showed that, in the condition with and without CoCl₂, expressions of RASSF1A protein decreased in SGC7901 cells of blank control group and negative control group; weak expressions of RASSF1A protein was observed in SGC7901 cells of infection group when in condition of without CoCl₂, but increased when adding CoCl₂. But RASSF1A protein didn't expressed in MKN28 cells of blank control group, negative control group, and infection group, whether adding CoCl₂ or not. CCK-8 assay result showed that, in SGC7901 cells, the growth inhibition rate of infection group which added CoCl₂ was higher than those of other 5 groups (P < 0.05); in MKN28 cells, the growth inhibition rates of infection group and negative group were all higher than those of blank control group, whether adding CoCl₂ or not (P < 0.05), but there was no significant difference among the infection group and negative group, whether adding CoCl₂ or not (P > 0.05). Conclusions A new hypoxia inducible and cea-positive tumor-targeting transcriptional regulatory element of 5HRE-CEAp is established successfully, and lentivirus vector of pLV-5HRE-CEAp-RASSF1A can significant inhibit the growth of SGC7901 cells under hypoxia condition.

Citation： ZHOUPei-hua, SUNXue-jun, ZHENGJian-bao, WANGWei, WANGXiao-long, CHENNan-zheng, WEIGuang-bing, FANYuan, LIXiao-bin, WANGXun-kai, LUShao-ying. Inhibition Effect on Expression of RASSF1A Gene by 5HRE Combined with CEAp Element Targeted Regulation for Human Gastric Cancer SGC7901 Cells. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2015, 22(10): 1201-1208. doi: 10.7507/1007-9424.20150312 Copy

1.	Penon D, Cito L, Giordano A. Novel findings about management of gastric cancer:a summary from 10th IGCC. World J Gastroenterol, 2014, 20(27):8986-8992.
2.	Shen L, Shan YS, Hu HM, et al. Management of gastric cancer in Asia:resource-stratified guidelines. Lancet Oncol, 2013, 14(12):e535-e547.
3.	Brenner MK, Gottschalk S, Leen AM, et al. Is cancer gene therapy an empty suit?. Lancet Oncol, 2013, 14(11):e447-e456.
4.	周培华, 孙学军, 王燕, 等. 5HRE强化的肝癌靶向性自杀基因载体的构建.中国普外基础与临床杂志, 2007, 14(2):163-167.
5.	孙学军, 卢乐, 禄韶英, 等. 5HRE联合P_(CEA)调节TSST-1/CD80TM重组基因表达的逆转录病毒载体的构建.细胞与分子免疫学杂志, 2009, 25(10):863-865.
6.	药立波.医学分子生物学实验技术.第2版.北京:人民卫生出版社, 2011:353-355.
7.	Vaupel P, Mayer A. Hypoxia in cancer:significance and impact on clinical outcome. Cancer Metastasis Rev, 2007, 26(2):225-239.
8.	Keith B, Johnson RS, Simon MC. HIF1αand HIF2α:sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer, 2012, 12(1):9-22.
9.	Wenger RH, Stiehl DP, Camenisch G. Integration of oxygen signaling at the consensus HRE. Sci STKE, 2005, 2005(36):re12.
10.	Shibata T, Giaccia AJ, Brown JM. Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy. Neoplasia, 2002, 4(1):40-48.
11.	Harvey TJ, Hennig IM, Shnyder SD, et al. Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo. Cancer Gene Ther, 2011, 18(11):773-784.
12.	贺赛, 郑见宝, 孙学军, 等. 5HRE增强子和hTERT启动子联合调控CDX2基因对人结肠癌细胞系LoVo增殖的抑制.中国肿瘤生物治疗杂志, 2014, 21(3):314-319.
13.	Hammarström S. The carcinoembryonic antigen (CEA) family:structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol, 1999, 9(2):67-81.
14.	Grunnet M, Sorensen JB. Carcinoembryonic antigen (CEA) as tumor marker in lung cancer. Lung Cancer, 2012, 76(2):138-143.
15.	Chen S, Feng XY, Li YF, et al. The prognosis of gastric cancer patients with marginally elevated carcinoembryonic antigen (CEA) values after D2 radical gastrectomy. J Surg Oncol, 2013, 107(6):641-645.
16.	Qiu Y, Peng GL, Liu QC, et al. Selective killing of lung cancer cells using carcinoembryonic antigen promoter and double suicide genes, thymidine kinase and cytosine deaminase (pCEA-TK/CD). Cancer Lett, 2012, 316(1):31-38.
17.	Wang W, Sun X, Lu L, et al. Cytotoxicity of lymphocytes activated by superantigen toxic-shock-syndrome toxin-1 against colorectal cancer LoVo cells. Mol Cell Biochem, 2013, 376(1-2):1-9.
18.	Nyati MK, Sreekumar A, Li S, et al. High and selective expression of yeast cytosine deaminase under a carcinoembryonic antigen promoter-enhancer. Cancer Res, 2002, 62(8):2337-2342.
19.	Liu T, Zhang G, Chen YH, et al. Tissue specific expression of suicide genes delivered by nanoparticles inhibits gastric carcinoma growth. Cancer Biol Ther, 2006, 5(12):1683-1690.
20.	Dammann R, Li C, Yoon JH, et al. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet, 2000, 25(3):315-319.
21.	Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci, 2007, 120(Pt 18):3163-3172.
22.	Amin KS, Banerjee PP. The cellular functions of RASSF1A and its inactivation in prostate cancer. J Carcinog, 2012, 11(3):31-38.
23.	Jung HY, Jung JS, Whang YM, et al. RASSF1A suppresses cell migration through inactivation of HDAC6 and increase of acetylatedα-Tubulin. Cancer Res Treat, 2013, 45(2):134-144.
24.	De Sousa Abreu R, Penalva LO, Marcotte EM, et al. Global signatures of protein and mRNA expression levels. Mol Biosyst, 2009, 5(12):1512-1526.
25.	Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet, 2012, 13(4):227-232.
26.	Ardyanto TD, Osaki M, Nagahama Y, et al. Down-regulation of cobalt-induced HIF-1 alpha expression correlates with cell proliferation and apoptosis in human gastric carcinoma cells. Oncol Rep, 2008, 19(2):339-343.

1. Penon D, Cito L, Giordano A. Novel findings about management of gastric cancer:a summary from 10th IGCC. World J Gastroenterol, 2014, 20(27):8986-8992.
2. Shen L, Shan YS, Hu HM, et al. Management of gastric cancer in Asia:resource-stratified guidelines. Lancet Oncol, 2013, 14(12):e535-e547.
3. Brenner MK, Gottschalk S, Leen AM, et al. Is cancer gene therapy an empty suit?. Lancet Oncol, 2013, 14(11):e447-e456.
4. 周培华, 孙学军, 王燕, 等. 5HRE强化的肝癌靶向性自杀基因载体的构建.中国普外基础与临床杂志, 2007, 14(2):163-167.
5. 孙学军, 卢乐, 禄韶英, 等. 5HRE联合P_(CEA)调节TSST-1/CD80TM重组基因表达的逆转录病毒载体的构建.细胞与分子免疫学杂志, 2009, 25(10):863-865.
6. 药立波.医学分子生物学实验技术.第2版.北京:人民卫生出版社, 2011:353-355.
7. Vaupel P, Mayer A. Hypoxia in cancer:significance and impact on clinical outcome. Cancer Metastasis Rev, 2007, 26(2):225-239.
8. Keith B, Johnson RS, Simon MC. HIF1αand HIF2α:sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer, 2012, 12(1):9-22.
9. Wenger RH, Stiehl DP, Camenisch G. Integration of oxygen signaling at the consensus HRE. Sci STKE, 2005, 2005(36):re12.
10. Shibata T, Giaccia AJ, Brown JM. Hypoxia-inducible regulation of a prodrug-activating enzyme for tumor-specific gene therapy. Neoplasia, 2002, 4(1):40-48.
11. Harvey TJ, Hennig IM, Shnyder SD, et al. Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo. Cancer Gene Ther, 2011, 18(11):773-784.
12. 贺赛, 郑见宝, 孙学军, 等. 5HRE增强子和hTERT启动子联合调控CDX2基因对人结肠癌细胞系LoVo增殖的抑制.中国肿瘤生物治疗杂志, 2014, 21(3):314-319.
13. Hammarström S. The carcinoembryonic antigen (CEA) family:structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol, 1999, 9(2):67-81.
14. Grunnet M, Sorensen JB. Carcinoembryonic antigen (CEA) as tumor marker in lung cancer. Lung Cancer, 2012, 76(2):138-143.
15. Chen S, Feng XY, Li YF, et al. The prognosis of gastric cancer patients with marginally elevated carcinoembryonic antigen (CEA) values after D2 radical gastrectomy. J Surg Oncol, 2013, 107(6):641-645.
16. Qiu Y, Peng GL, Liu QC, et al. Selective killing of lung cancer cells using carcinoembryonic antigen promoter and double suicide genes, thymidine kinase and cytosine deaminase (pCEA-TK/CD). Cancer Lett, 2012, 316(1):31-38.
17. Wang W, Sun X, Lu L, et al. Cytotoxicity of lymphocytes activated by superantigen toxic-shock-syndrome toxin-1 against colorectal cancer LoVo cells. Mol Cell Biochem, 2013, 376(1-2):1-9.
18. Nyati MK, Sreekumar A, Li S, et al. High and selective expression of yeast cytosine deaminase under a carcinoembryonic antigen promoter-enhancer. Cancer Res, 2002, 62(8):2337-2342.
19. Liu T, Zhang G, Chen YH, et al. Tissue specific expression of suicide genes delivered by nanoparticles inhibits gastric carcinoma growth. Cancer Biol Ther, 2006, 5(12):1683-1690.
20. Dammann R, Li C, Yoon JH, et al. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet, 2000, 25(3):315-319.
21. Donninger H, Vos MD, Clark GJ. The RASSF1A tumor suppressor. J Cell Sci, 2007, 120(Pt 18):3163-3172.
22. Amin KS, Banerjee PP. The cellular functions of RASSF1A and its inactivation in prostate cancer. J Carcinog, 2012, 11(3):31-38.
23. Jung HY, Jung JS, Whang YM, et al. RASSF1A suppresses cell migration through inactivation of HDAC6 and increase of acetylatedα-Tubulin. Cancer Res Treat, 2013, 45(2):134-144.
24. De Sousa Abreu R, Penalva LO, Marcotte EM, et al. Global signatures of protein and mRNA expression levels. Mol Biosyst, 2009, 5(12):1512-1526.
25. Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet, 2012, 13(4):227-232.
26. Ardyanto TD, Osaki M, Nagahama Y, et al. Down-regulation of cobalt-induced HIF-1 alpha expression correlates with cell proliferation and apoptosis in human gastric carcinoma cells. Oncol Rep, 2008, 19(2):339-343.

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CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Inhibition Effect on Expression of RASSF1A Gene by 5HRE Combined with CEAp Element Targeted Regulation for Human Gastric Cancer SGC7901 Cells

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