内质网蛋白29过表达慢病毒载体的构建及其对胃癌细胞生物学行为的影响

doi:10.13481/j.1671-587x.20190206

吉林大学学报(医学版) ›› 2019, Vol. 45 ›› Issue (02): 244-250.doi: 10.13481/j.1671-587x.20190206

• 基础研究 • 上一篇

内质网蛋白29过表达慢病毒载体的构建及其对胃癌细胞生物学行为的影响

朱广伟^1,2, 黄金胜^1,2, 黄永建¹, 郑炜¹, 杨树钢¹, 林春霖¹, 陈恒恺¹, 叶建新^1,2

1. 福建医科大学附属第一医院胃肠外科二区, 福建福州 350005;
2. 福建医科大学消化道恶性肿瘤教育部重点实验室, 福建福州 350005

收稿日期:2018-06-08 发布日期:2019-03-29
通讯作者: 叶建新,教授,硕生研究生导师(Tel:0591-87982082,E-mail:yjx0201@vip.sina.com) E-mail:yjx0201@vip.sina.com
作者简介:朱广伟(1983-),男,河南省郑州市人,主治医师,医学博士,主要从事消化道恶性肿瘤转移分子机制方面的研究。
基金资助:
国家自然科学基金资助课题（81702424，81872364）；国家卫计委临床重点专科建设项目（普通外科）资助课题（2013）；福建省卫计委青年课题项目资助课题（2017-1-51）；福建省科技厅自然科学基金资助课题（2017J01279）

Construction of lentiviral vector with overexpression of ERp29 and its effect on biological behavior of gastric cancer cells

ZHU Guangwei^1,2, HUANG Jinsheng^1,2, HUANG Yongjian¹, ZHENG Wei¹, YANG Shugang¹, LIN Chunlin¹, CHEN Hengkai¹, YE Jianxin^1,2

1. Department of Gastrointestinal Surgery, First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China;
2. Key Laboratory of Gastrointestinal Cancer, Ministry of Education, Fujian Medical University, Fuzhou 350005, China

Received:2018-06-08 Published:2019-03-29

摘要/Abstract

摘要： 目的：构建内质网蛋白29（ERp29）慢病毒过表达载体，探讨ERp29过表达对胃癌MGC803和SGC7901细胞增殖、迁移和侵袭的影响。方法：构建带有红色荧光蛋白标签的ERp29基因表达质粒的慢病毒（pCDH-ERp29）和对照质粒（pCDH-Vector），分别感染MGC803和SGC7901细胞；荧光显微镜观察细胞感染情况；CCK-8和平板克隆实验检测稳定感染ERp29基因的胃癌MGC803和SGC7901细胞的增殖能力；Transwell小室实验分别检测稳定感染ERp29基因的胃癌MGC803和SGC7901细胞的迁移和侵袭能力；应用Real-time PCR和Western blotting技术分别检测稳定感染ERp29基因的胃癌MGC803和SGC7901细胞中ERp29 mRNA和蛋白表达水平。结果：酶切鉴定显示成功构建pCDH-ERp29过表达载体，荧光显微镜下观察到成功感染胃癌细胞。CCK-8实验和平板克隆实验检测，与pCDH-Vector组比较，pCDH-ERp29组MGC803和SGC7901细胞增殖能力无明显变化（P>0.05）；Transwell小室实验，与pCDH-Vector组比较，pCDH-ERp29组MGC803和SGC7901细胞的迁移和侵袭细胞数明显减少（P<0.05）。pCDH-ERp29组细胞中ERp29 mRNA和蛋白表达水平明显高于pCDH-Vector组（P<0.05）。结论：成功构建过表达ERp29基因的慢病毒载体，过表达ERp29基因可明显抑制胃癌MGC803和SGC7901细胞的迁移和侵袭。

关键词: 胃肿瘤, 内质网蛋白29, 慢病毒载体, 细胞增殖, 细胞迁移, 细胞侵袭

Abstract: Objective: To construct a lentiviral vector with overexpression of endoplasmic reticulum protein 29 (ERp29), and to explore the effects of ERp29 overexpression on the proliferation, migration and invasion of gastric cancer MGC803 and SGC7901 cells.Methods: The lentiviral plasmid with ERp29 (pCDH-ERp29) tagged with red fluorescentce protein and control plasmid (pCDH-Vector) were constructed, and then the MGC803 and SGC9901 cells were infected with these lentivital vectors. Under fluorescence microscope, the infection of these cells were observed. CCK-8 assay and clone formation assay were performed to test the proliferation abilities of MGC803 and SGC7901 cells infected with ERp29. Transwell chamber assay was used to test the abilities of migration and invasion of MGC803 and SGC7901 cells infected with ERp29. Real-time PCR and Western blotting methods were used to detect the expression levels of ERp29 mRNA and protein in MGC803 and SGC7901 cells which were stably infected by ERp29, respectively.Results: The enzyme digestion identification result showed that the pCDH-ER29 overexpression vector was successfully constructed. The fluorescence microscope result showed the successful infection in the gastric cancer cells. The CCK-8 assay and clone formation assay results showed that compared with pCDH-Vector group, the proliferation ability of the cells in pCDH-ERp29 group had no marked changes(P>0.05). Transwell chamber assay revealed that compared with pCDH-Vector group, the number of migration and invasion cells in the MGC803 and SGC7901 cells in pCDH-ERp29 group were significantlydecreased(P<0.05). The expression levels of ERp29 mRNA and protein in the cells in pCDH-ERp29 group were significantly increased compared with pCDH-Vector group(P<0.05).Conclusion: The lentiviral vector with overexpression of ERp29 is constructed successfully, and the overexpression of ERp29 in MGC803 and SGC7901 cells can significantly inhibit their abilities of migration and invasion.

Key words: stomach neoplasms, endoplasmic reticulum protein 29, lentiviral vector, cell proliferation, cell migration, cell invasion

中图分类号:

R735.2

朱广伟, 黄金胜, 黄永建, 郑炜, 杨树钢, 林春霖, 陈恒恺, 叶建新. 内质网蛋白29过表达慢病毒载体的构建及其对胃癌细胞生物学行为的影响[J]. 吉林大学学报(医学版), 2019, 45(02): 244-250.

ZHU Guangwei, HUANG Jinsheng, HUANG Yongjian, ZHENG Wei, YANG Shugang, LIN Chunlin, CHEN Hengkai, YE Jianxin. Construction of lentiviral vector with overexpression of ERp29 and its effect on biological behavior of gastric cancer cells[J]. Journal of Jilin University(Medicine Edition), 2019, 45(02): 244-250.

参考文献

[1] CHEN W Q, ZHENG R S, BAADE P D, et al. Cancer statistics in China, 2015[J]. CA:Cancer J Clin, 2016, 66(2):115-132.
[2] 王锦州.胃腺癌组织差异表达蛋白质的分离与鉴定[D].福州:福建医科大学,2013.
[3] DEMMER J, ZHOU C, HUBBARD M J. Molecular cloning of ERp29, a novel and widely expressed resident of the endoplasmic reticulum[J]. FEBS Lett, 1997, 402(2/3):145-150.
[4] MKRTCHIAN S, SANDALOVA T. ERp29, an unusual redox-inactive member of the thioredoxin family[J]. Antioxid Redox Signal, 2006, 8(3/4):325-337.
[5] LIEPINSH E, BARYSHEV M, SHARIPO A, et al. Thioredoxin fold as homodimerization module in the putative chaperone ERp29:NMR structures of the domains and experimental model of the 51 kDa dimer[J]. Structure, 2001, 9(6):457-471.
[6] CHERETIS C, DIETRICH F, CHATZISTAMOU I, et al. Expression of ERp29, an endoplasmic reticulum secretion factor in basal-cell carcinoma[J]. Am J Dermatopathol, 2006, 28(5):410-412.
[7] FAN N J, GAO J L, LIU Y, et al. Label-free quantitative mass spectrometry reveals a panel of differentially expressed proteins in colorectal cancer[J]. Biomed Res Int, 2015,2015:365068.
[8] DENG Y J, TANG N, LIU C, et al. CLIC4, ERp29, and Smac/DIABLO derived from metastatic cancer stem-like cells stratify prognostic risks of colorectal cancer[J]. Clin Cancer Res, 2014, 20(14):3809-3817.
[9] SHNYDER S D, MANGUM J E, HUBBARD M J. Triplex profiling of functionally distinct chaperones (ERp29/PDI/BiP) reveals marked heterogeneity of the endoplasmic reticulum proteome in cancer[J]. J Proteome Res, 2008, 7(8):3364-3372.
[10] ZHANG K, YAO H P, YANG Z, et al. Comparison of ILK and ERP29 expressions in benign and malignant pancreatic lesions and their clinicopathological significances in pancreatic ductal adenocarcinomas[J]. Clin Transl Oncol, 2016, 18(4):352-359.
[11] BAMBANG I F, LU D, LI H P, et al. Cytokeratin 19 regulates endoplasmic reticulum stress and inhibits ERp29 expression via p38 MAPK/XBP-1 signaling in breast cancer cells[J]. Exp Cell Res, 2009, 315(11):1964-1974.
[12] YUAN L W, LIU D C, YANG Z L. Correlation of S1P1 and ERp29 expression to progression, metastasis, and poor prognosis of gallbladder adenocarcinoma[J]. HBPD Int, 2013, 12(2):189-195.
[13] BAMBANG I F, XU S C, ZHOU J B, et al. Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells[J]. Lab Invest, 2009, 89(11):1229-1242.
[14] WU J, YANG Y Y, GAO S S, et al. ERp29 inhibits tumorigenicity by suppressing epithelial mesenchymal transition in gastric cancer[J]. Oncotarget, 2017, 8(45):78757-78766.
[15] ZHANG Y, HU Y, WANG J L, et al. Proteomic identification of ERP29 as a key chemoresistant factor activated by the aggregating p53 mutant Arg282Trp[J]. Oncogene, 2017, 36(39):5473-5483.
[16] 钟文进.组织芯片研究ERp29在胃癌中的表达及意义[D].福州:福建医科大学,2014.

内质网蛋白29过表达慢病毒载体的构建及其对胃癌细胞生物学行为的影响

Construction of lentiviral vector with overexpression of ERp29 and its effect on biological behavior of gastric cancer cells

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