诱导性表达人巨细胞病毒UL138蛋白的胃癌细胞株构建及其效应评价

摘要
图/表
参考文献
相关文章 (0)

全文: PDF (1837 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要目的：构建可诱导表达人巨细胞病毒（HCMV）UL138蛋白的稳定遗传胃癌细胞系，并评价UL138对胃癌细胞的生物学效应。方法：将pCMV-tet3G质粒转染BGC-823胃癌细胞，通过G418及荧光素酶活性检测筛选BGCTet-on细胞株。再将含有UL138外源基因的pTRE3G-UL138质粒转染稳定BGCTet-on细胞株，通过G418及潮霉素双抗筛选，获得诱导性表达UL138基因的胃癌细胞（BGC-UL138Tet-on）。强力霉素（DOX）诱导后，采用Western blot验证UL138蛋白表达，采用流式细胞术及CCK8试剂盒检测UL138蛋白表达对胃癌细胞的生长周期及增殖的影响。构建荷瘤裸鼠模型，体内验证UL138蛋白表达对胃癌细胞增殖的影响。结果：成功构建了DOX诱导性表达的、携带UL138基因的BGC-UL138Tet-on稳转细胞株。UL138蛋白表达可显著抑制胃癌细胞增殖；流式细胞术检测显示UL138蛋白表达阻滞BGC-823细胞周期在G1期。荷瘤裸鼠模型体内实验发现，DOX腹腔注射诱导UL138蛋白表达后，BGC-UL138Tet-on移植瘤体积缩小甚至消失。结论：成功构建可诱导表达UL138蛋白的胃癌细胞株，进一步证实HCMV基因UL138的表达可在体内和体外抑制胃癌细胞的增殖，细胞实验提示细胞周期阻断在G1期。

	服务
	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	张良
	陈文静
	郭刚强
	孙祥威
	叶璐璐
	胡畅远
	金劲激
	沈贤
	薛向阳

关键词 ：巨细胞病毒；UL138；Tet-on系统；胃肿瘤；基因, 肿瘤抑制

Abstract：Objective: To establish a stably inherited gastric cell line with inducible expression of human cytomegalovirus UL138 protein and then to evaluate the biological effect of UL138 protein on gastric cancers (GCs), which will offer a convincing tool for researching the specific function of UL138 protein impact on GCs. Methods: Transfect pCMV-tet3G plasmids into BGC-823 and then screen for stablely transfected clones BGCTet-on by G418 and luciferase assay. Then transfect the recombinant plasmid pTRE3G-UL138 into the selected clone and screen for stably transfected clones BGC-UL138Tet-on by G418 and hygromycin. Validate the expression of UL138 induced by doxycycline (DOX) by western blot. Besides, detect the growth impact of UL138 protein on GCs by flow cytometry and CCK8 test. Establish GC xenograft nude mice models and verify the GC inhibition function of UL138 protein in vivo. Results: Gastric cancer cell line BGC-UL138Tet-on with DOX inducible expression of human cytomegalovirus UL138 protein was successfully established. Expression of UL138 protein inhibited proliferation of GC cells distinctly. Flow cytometry test indicated UL138 protein could block GC cell cycle at G1 phase. The data of GC xenograft nude mice models further demonstrated that the BGC-UL138Tet-on xenografts were decreased, and even disappeared, when UL138 expression was induced by DOX intraperitoneal injection. Conclusion: We successfully established a specific GC cell line with inducible expression of human cytomegalovirus UL138 protein and then we found HCMV UL138 gene could inhibit proliferation of GC cells in vitro and in vivo and block its cell cycle.

Key words： cytomegalovirus UL138 Tet-on system gastric neoplasms genes, tumor suppressor

收稿日期: 2016-05-31

基金资助:国家自然科学基金资助项目（81472308，81001343，31470891）；温州市科技局科研基金资助项目（Y2015 0054，Y20150057）

通讯作者: 薛向阳，副教授，Email：wzxxy001@163.com

作者简介: 张良（1991-），男，浙江温州人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2017/V47/I2/79

[1] SODERBERG-NAUCLER C. Does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer?[J]. J Intern Med, 2006, 259(3): 219-246.
[2] FANNIN RIDER P J, DUNN W, YANG E, et al. Human cytomegalovirus microRNAs[J]. Curr Top Microbiol Immunol, 2008, 325: 21-39.
[3] DUNN W, TRANG P, ZHONG Q, et al. Human cytomegalovirus expresses novel microRNAs during productive viral  infection[J]. Cell Microbiol, 2005, 7(11): 1684-1695.
[4] PFEFFER S, SEWER A, LAGOS-QUINTANA M, et al.Identification of microRNAs of the herpesvirus family[J]. Nat Methods, 2005, 2(4): 269-276.
[5] STARK T J, ARNOLD J D, SPECTOR D H, et al. High-resolution profiling and analysis of viral and host small RNAs during human cytomegalovirus infection[J]. J Virol, 2012,  86(1):226-235.
[6] GATHERER D, SEIRAFIAN S, CUNNINGHAM C, et al.High-resolution human cytomegalovirus transcriptome[J]. Proc Natl Acad Sci U S A, 2011, 108(49): 19755-19760.
[7] PETRUCELLI A, RAK M, GRAINGER L, et al. Characterization of a novel Golgi apparatus-localized latency determinant encoded by human cytomegalovirus[J]. J Virol, 2009, 83(11):5615-5629.
[8] 金劲激, 涂建欣, 吴明, 等. 胃癌组织中人巨细胞病毒感染及其临床意义[J]. 温州医科大学学报, 2014, 44(10): 703-707.
[9] Chen W, Lin K, Zhang L, et al. The cytomegalovirus protein UL138 induces apoptosis of gastric cancer cells by binding to heat shock protein 70[J]. Oncotarget, 2016, 7(5): 5630-5645.
[10] 陈文静, 黄崇安, 陈静, 等. 潜伏相关抗原UL138蛋白的制备及其用于人巨细胞病毒感染血清学检测的评价[J]. 温州医科大学学报, 2015, 45(10): 703-708.
[11] CRAIG J M, MACAULEY J C, WELLER T H, et al. Isolation of intranuclear inclusion producing agents from infants
with illnesses resembling cytomegalic inclusion disease[J].Proc Soc Exp Biol Med, 1957, 94(1): 4-12.
[12] HO M. The history of cytomegalovirus and its diseases[J]. Med Microbiol Immunol, 2008, 197(2): 65-73.
[13] 陈静, 朱小春, 张丽芳, 等. 巨细胞病毒UL133-UL138基因表达与系统性红斑狼疮的相关性研究[C]. 二零一四年浙江省风湿病学学术年会论文汇编, 2014: 14-15.
[14] 陈静, 章慧娣, 楼威洋, 等. 人巨细胞病毒感染与系统性红斑狼疮的相关性[J]. 温州医科大学学报, 2014, 44(5): 318-323.
[15] SOROCEANU L, MATLAF L, KHAN S, et al. Cytomegalovirus immediate-early proteins promote stemness properties in glioblastoma[J]. Cancer Res, 2015, 75(15): 3065-
3076.
[16] BISHOP R K, VALLE OSEGUERA C A, SPENCER J V. Human Cytomegalovirus interleukin-10 promotes proliferation and migration of MCF-7 breast cancer cells[J]. Cancer
Cell Microenviron, 2015, 2(1): e678.
[17] GEDER L, SANFORD E J, ROHNER T J, et al. Cytomegalovirus and cancer of the prostate: in vitro transformation of human cells[J]. Cancer Treat Rep, 1977, 61(2):139-146.
[18] TAFVIZI F, FARD Z T. Detection of human cytomegalovirus in patients with colorectal cancer by nested-PCR[J]. Asian Pac J Cancer Prev, 2014, 15(3):1453-1457.
[19] JIN J, HU C, WANG P, et al. Latent infection of human cytomegalovirus is associated with the development of gas tric cancer[J]. Oncol Lett, 2014, 8(2):898-904.
[20] QI Y, HE R, MA Y P, et al. Human cytomegalovirus UL138 open reading frame is highly conserved in clinical strains[J]. Chin Med Sci J, 2009, 24(2): 107-111.
[21] LEE S H, ALBRIGHT E R, LEE J H, et al. Cellular defense against latent colonization foiled by human cytomegalovirus UL138 protein[J]. Sci Adv, 2015, 1(10): e1501164.
[22] GRAINGER L, CICCHINI L, RAK M, et al. Stress-inducible alternative translation initiation of human cytomegalo virus latency protein pUL138[J]. J Virol, 2010, 84(18):9472-9486.
[23] PETRUCELLI A, UMASHANKAR M, ZAGALLO P, et al.  Interactions between proteins encoded within the human cytomegalovirus UL133-UL138 locus[J]. J Virol, 2012, 86(16): 8653-8662.
[24] MONTAG C, WAGNER J A, GRUSKA I, et al. The laten-cy-associated UL138 gene product of human cytomegalovirus sensitizes cells to tumor necrosis factor alpha (TNF-
alpha) signaling by upregulating TNF-alpha receptor 1 cell surface expression[J]. J Virol, 2011, 85(21): 11409-11421.
[25] WEEKES M P, TAN S Y, POOLE E, et al. Latency-associated degradation of the MRP1 drug transporter during latent human cytomegalovirus infection[J]. Science, 2013, 340(6129): 199-202.
[26] KONOPKA W, DUNIEC K, MIODUSZEWSKA B, et al. hCMV and Tet promoters for inducible gene expression in rat neurons in vitro and in vivo[J]. Neurobiol Dis, 2005, 19(1-2): 283-292.
[27] 白志强, 王斌, 刘志军, 等. 人巨细胞病毒即刻早期蛋白IE2在Tet-On系统调控下的表达及其抗凋亡作用[J]. 病毒学报, 2009, 25(3): 190-195.
[28] YAO F, WALKER P D, MACKENZIE R G. A Tet-on system for DRD1-expressing cells[J]. PLoS One, 2013, 8(8):e72681.
[29] RUSSELL S J, PENG K W, BELL J C. Oncolytic virotherapy[J]. Nat Biotechnol, 2012, 30(7): 658-670.
[30] YOON S S, NAKAMURA H, CARROLL N M, et al. Anoncolytic herpes simplex virus type 1 selectively destroys diffuse liver metastases from colon carcinoma[J]. FASEB J, 2000, 14(2): 301-311.