慢病毒载体介导RNA结合蛋白-5对肺腺癌细胞系H1299的增殖和凋亡的影响

doi:10.16098/j.issn.0529-1356.2020.01.007

解剖学报 ›› 2020, Vol. 51 ›› Issue (1): 40-46.doi: 10.16098/j.issn.0529-1356.2020.01.007

慢病毒载体介导RNA结合蛋白-5对肺腺癌细胞系H1299的增殖和凋亡的影响

方可欣¹ 王昕晨¹ 洪开听²竺王玉^1*

1. 浙江省舟山医院细胞分子生物学实验室，浙江舟山 316012;2. 浙江省舟山市妇女儿童医院检验科，浙江舟山 316000

收稿日期:2018-11-20 修回日期:2019-02-13 出版日期:2020-02-06 发布日期:2020-04-21
通讯作者: 竺王玉 E-mail:zhuwangyu24@sina.cn
基金资助:
浙江省自然科学基金项目;浙江省医药卫生科技计划;舟山市卫生局攻关项目

Effect of lentiviral vector mediated RNA binding protein quaking-5 on the proliferation and apoptosis of lung adenocarcinoma cell line H1299

FANG Ke-xin¹ WANG Xin-chen¹ HONG Kai-ting² ZHU Wang-yu^1*

1. Cellular and Molecular Laboratory, Zhoushan Hospital, Zhejiang Zhoushan 316021, China;2. Clinical Laboratory, Zhoushan Women’s and Children’s Hospital, Zhejiang Zhoushan 316021, China

Received:2018-11-20 Revised:2019-02-13 Online:2020-02-06 Published:2020-04-21
Contact: ZHU Wang-yu E-mail:zhuwangyu24@sina.cn

摘要/Abstract

摘要：

目的探讨慢病毒载体介导RNA结合蛋白-5（QKI-5）对肺腺癌细胞凋亡的影响。方法应用GV358（过表达）和GV248（抑制表达）载体分别构建针对QKI-5基因的慢病毒过表达载体和抑制表达载体，转染293T细胞产生慢病毒颗粒，收集并测定慢病毒滴度。将经测序鉴定的QKI-5过表达的慢病毒或抑制表达的慢病毒转染到肺腺癌细胞H1299，Real-time PCR检测QKI-5 mRNA表达，集落形成实验检测H1299细胞增殖情况，膜联蛋白V（annexin V）、碘化丙啶（PI）检测H1299细胞凋亡情况，Western blotting检测促凋亡蛋白Caspase-3和Caspase-8表达。结果成功构建QKI-5过表达慢病毒载体和抑制表达慢病毒载体，与对照组比较，QKI-5过表达后，QKI-5 mRNA表达增加，细胞集落形成减少，肺腺癌细胞早期凋亡增加，促凋亡蛋白Caspase-8表达增加；QKI-5抑制表达后，QKI-5 mRNA表达降低，细胞集落形成增加，但肺腺癌细胞凋亡无明显变化，然而促凋亡蛋白Caspase-8表达减少。结论慢病毒介导 QKI-5抑制增殖，可能通过Caspase-8促进肺腺癌细胞系H1299的凋亡。

关键词: 肺腺癌, RNA结合蛋白-5, H1299细胞系, 载体构建, 慢病毒感染, 实时定量聚合酶链反应

Abstract:

To investigate the proliferation and apoptosis of lung adenocarcinoma cells line H1299 by the lentiviral vector mediated RNA binding protein quaking-5 (QKI-5). Methods GV358 (up-regulation) and GV248 (down-regulation) vectors were used to construct the lentiviral QKI-5 up-regulation vector and down-regulation vector, respectively. The vectors were transfected into 293T cells for lentiviral packaging and viral titer were then determined. Gene sequencing was performed to screen the sequence of vectors. Then Real-time PCR was used to evaluate the expression of QKI-5 mRNA and the proliferation of H1299 cells was examined by colony forming assay after transfection. The apoptosis of H1299 cells was determined by the detection of the expression membrane protein Ⅴ (annexin Ⅴ) and propyl iodide ingot (PI) by flow cytometry. Pro-apoptotic protein Caspae-3 and Caspase-8 were evaluated by Western blotting. Results QKI-5 up-regulation and down-regulation lentiviral vectors were constructed successfully. Compared with the controls, the expression of QKI-5 mRNA of H1299 cells was up-regulated, the cell colony formation was decreased, and the early apoptosis of H1299 cells was increased with the over-expression of Caspase-8 after transfected with up-regulated vector, whereas transfecting with QKI-5 down-regulated vector had opposite effect. Conclusion Lenviral vector mediated QKI-5 could inhibit proliferation and promote apoptosis of lung adenocarcinoma cells through Caspase-8.

Key words: Lung adenocarcinoma, RNA binding protein quaking quaking-5, H1299 cell line, Vector construction, Lentiviral transfeted, Real-time PCR

中图分类号:

R730.7

方可欣王昕晨洪开听竺王玉. 慢病毒载体介导RNA结合蛋白-5对肺腺癌细胞系H1299的增殖和凋亡的影响[J]. 解剖学报, 2020, 51(1): 40-46.

FANG Ke-xin WANG Xin-chen HONG Kai-ting ZHU Wang-yu. Effect of lentiviral vector mediated RNA binding protein quaking-5 on the proliferation and apoptosis of lung adenocarcinoma cell line H1299[J]. Acta Anatomica Sinica, 2020, 51(1): 40-46.

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慢病毒载体介导RNA结合蛋白-5对肺腺癌细胞系H1299的增殖和凋亡的影响

Effect of lentiviral vector mediated RNA binding protein quaking-5 on the proliferation and apoptosis of lung adenocarcinoma cell line H1299

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