hsa-miR-100-3p抑制剂慢病毒表达载体稳定感染BEAS-2B细胞株的建立

董伟; 刘媛媛; 陈志军; 姜玉; 朱其苹; 汪思应

doi:10.16462/j.cnki.zhjbkz.2016.02.021

hsa-miR-100-3p抑制剂慢病毒表达载体稳定感染BEAS-2B细胞株的建立

doi: 10.16462/j.cnki.zhjbkz.2016.02.021

1. 安徽医科大学基础医学院病理学与病理生理学, 安徽合肥 230032;
2. 合肥市第一人民医院内分泌科, 安徽合肥 230061

基金项目:

国家自然科学基金（81272258）

详细信息

作者简介:
董伟(1986-),男,安徽合肥人,在读硕士研究生。主要研究方向：肿瘤分子生物学。

中图分类号: R322.34;R394.33
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- 被引次数: 0
出版历程
- 收稿日期: 2015-09-01
- 修回日期: 2015-11-12

Establishment of BEAS-2B stable cell lines with lentiviral-based hsa-miR-100-3p inhibitor

1. Department of Pathology and Pathophysiology, School of Basic Medicine Anhui Medical University, Hefei 230032, China;
2. Department of Endocrinology, the First people's Hospital of Hefei City, Hefei 230061, China

摘要

摘要: 目的建立稳定感染人hsa-miR-100-3p抑制剂慢病毒表达载体的人支气管上皮（human bronchial epithelial，BEAS-2B）细胞株，为研究miR-100的功能奠定基础。方法根据hsa-miR-100-3p的成熟序列，设计其反向互补序列，用聚合酶链式反应（polymerase chain reaction，PCR）的方法扩增目的基因，将目的基因与慢病毒载体GV280经双酶切后，进行定向连接，产生GV280-hsa-miR-100-3p-inhibitor慢病毒表达载体。将制备好的重组慢病毒质粒与两种辅助包装原件载体质粒共转染人胚肾上皮细胞293（human embryo kidney epithelial cell，HEK-293）T细胞，包装产生病毒颗粒并以最适滴度感染BEAS-2B细胞以获得稳定感染的细胞株。倒置荧光显微镜观察感染效率，用逆转录聚合酶链式反应（reverse transcription-polymerase chain reaction，RT-PCR）的方法检测重组慢病毒感染后BEAS-2B细胞中miR-100的相对表达量。结果测序结果表明，目的基因成功的连接到慢病毒载体上，重组慢病毒高效的感染了BEAS-2B细胞。RT-PCR检测发现，BEAS-2B细胞中miR-100的相对表达明显的下调了。结论稳定感染hsa-miR-100-3p抑制剂的BEAS-2B细胞株构建成功，敲低了细胞中内源性miR-100的表达。
- 聚合酶链式反应 /
- 慢病毒属 /
- 微RNAs
Abstract: Objective To establish the human bronchial epithelial (BEAS-2B ) stable cell lines with lentiviral-based hsa-miR-100-3p inhibitor and lay a foundation for the study of miR-100. Methods The reverse complementary sequence was designed based on the mature sequence of hsa-miR-100-3p. The purpose gene was amplified by polymerase chain reaction (PCR) and inserted directly into lentiviral plasmid vector GV280 after the double enzyme digestion, generatting GV280-hsa-miR-100-3p-inhibitor lentiviral expression vector. Then human embryo kidney epithelial cell (HEK-293) T cells was transfected with recombinant lentiviral plasmid along with two packaging vectors to produce virus particles. BEAS-2B cells were infected with the optimal titer of virus particles to obtain stable infected cell lines. At last, the infection efficiency was observed by fluorescence inverted microscope and the relative expression of miR-100 in BEAS-2B cells after transfecting with recombinant lentivirus was determined using reverse transcription-polymerase chain reaction (RT-PCR). Results DNA sequencing demonstrated that the lentiviral vector was made successfully. The target BEAS-2B cells were efficiently infected. More importantly, the expression of miR-100 in BEAS-2B cells was reduced significantly, measured by RT-PCR. Conclusions We successfully established the BEAS-2B stable cell lines with lentiviral-based hsa-miR-100-3p inhibitor to reduce the endogenous expression of miR-100 in BEAS-2B cells.
- Polymerase chain reaction /
- Lentivirus /
- MicroRNAs

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参考文献(19)

崔花芹,汪心怡,陈洁,等. 环境污染物SHP2D61G1+激活对小鼠成纤维细胞mTRNA表达谱的影响 [J]. 中国比较医学杂志, 2013,23(7)：1-6.

Carthew RW, Sontheimer EJ. Origins and mechanisms of miRNAs and siRNAs [J]. Cell, 2009,136(4):642-655.

Bushati N, Cohen SM. microRNA functions [J]. Annu Rev Cell Dev Biol, 2007,23:175-205.

Sun J, Chen Z, Tan X, et al. MicroRNA-99a/100 promotes apoptosis by targeting mTOR in human esophageal squamous cell carcinoma [J]. Medical oncology, 2013,30(1):411-420.

Kolokythas A, Miloro M, Zhou X. Review of microRNA deregulation in oral cancer. Part I [J]. J Oral Maxillofac Res, 2011,2(2):1-35.

Nagaraja AK, Creighton CJ, Yu Z, et al. A link between miR-100 and frap1/mtor in clear cell ovarian cancer [J]. Mol Endocrinol, 2010,24(2):447-463.

Torres A, Torres K, Pesci A, et al. Deregulation of miR-100,miR-99a and miR-199B in tissues and plasma coexists with increased expression of mTOR kinase in endometrioid endometrial carcinoma [J]. BMC Cancer, 2012,12:369-382.

Wang S, Xue S, Dai Y, et al. Reduced expression of microRNA-100 confers unfavorable prognosis in patients with bladder cancer [J]. Diagn Pathol, 2012,7:159-166.

Chen P, Zhao X, Ma L. Down regulation of miR-100 correlates with tumor progression and poor prognosis in hepatocellular carcinoma [J]. Mol Cell Biochem, 2013,383(1-2):49-58.

Liu J, Lu KH, Liu ZL, et al. MicroRNA-100 is a potential molecular marker of non-small cell lung cancer and functions as a tumor suppressor by targeting polo-like kinase1 [J]. BMC cancer, 2012,12:519-530.

Luo D, Wilson JM, Harve N, et al. A systematic evaluation of miRNA-mRNA interactions involved in the migration and invasion of breast cancer cells [J]. J Transl Med, 2013,11:57-71.

Marshall G, Ferreccio C, Yuan Y, et al. Fifty-year study of lung and bladder cancer mortality in Chile related to arsenic in drinking water [J]. J Natl Cancer Inst, 2007,99(12):920-928.

Chang Q, Pan J, Wang X, et al. Reduced reactive oxygen species-generating capacity contributes to the enhanced cell growth of arsenic-transformed epithelial cells [J]. Cancer Res, 2010,70(12):5127-5135.

Carpenter RL, Jiang Y, Jing Y, et al. Arsenite induces cell transformation by reactive oxygen species,AKT,ERK1/2, and p70S6K1 [J]. Biochem Biophys Res Commun, 2011,414(24):533-538.

Garzon R, Calin GA, Croce CM. MicroRNAs in cancer [J]. Annu Rev Med, 2009,60:167-179.

Slack FJ, Weidhaas JB. MicroRNA in cancer prognosis [J]. N Engl J Med, 2008,359(25):2720-2722.

付海龙,徐广峰,史春梅,等. hsa-miR-100的生物信息学分析 [J]. 国际检验医学杂志, 2012,33(18)：2177-2180.

Simmons A, Whitehead RP, Kolokoltsov AA, et al. Use of recombinant lentivirus pseudotyped with vesicular stomatitis virus glycoprotein G for efficient generation of human anti-cancer chimeric T cells by transduction of human peripheral blood lymphocytes in vitro [J]. Virol J, 2006,3(8):1-10.

Bouvard V, Baan R, Straif K, et al. A review of human carcinogens-Part B: biological agents [J]. Lancet Oncol, 2009,10(4):321-322.

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