siRNA靶向干扰TRAF6的表达对肺癌细胞增殖与凋亡的影响

doi:10.19586/j.2095-2341.2024.0056

生物技术进展 ›› 2024, Vol. 14 ›› Issue (5): 875-881.DOI: 10.19586/j.2095-2341.2024.0056

• 研究论文 • 上一篇

siRNA靶向干扰TRAF6的表达对肺癌细胞增殖与凋亡的影响

赵轩¹(), 任丽梅¹^,²^,³, 王晓茹¹^,²^,³, 韩广欣¹, 顾丹丹¹, 姚亚森¹, 祁永浩¹^,³^,⁴()

^1.石家庄学院化工学院，石家庄 050050
^2.河北省高校微生物制药应用技术研发中心，石家庄 050050
^3.河北省生物制药国际联合研究中心，石家庄 050050
^4.河北医科大学河北省创新药物研究与评价重点实验室，石家庄 050017

收稿日期:2024-03-19 接受日期:2024-07-02 出版日期:2024-09-25 发布日期:2024-10-22
通讯作者: 祁永浩
作者简介:赵轩 E-mail: xuanzhao@tju.edu.cn；
基金资助:
河北省自然科学基金青年项目(H2022106006);石家庄市科学技术研究与发展计划项目(211200973)

Effects of siRNA Targeting to Interfere with the Expression of TRAF6 on the Proliferation and Apoptosis of Lung Cancer Cells

Xuan ZHAO¹(), Limei REN¹^,²^,³, Xiaoru WANG¹^,²^,³, Guangxin HAN¹, Dandan GU¹, Yasen YAO¹, Yonghao QI¹^,³^,⁴()

^1.School of Chemical Engineering，Shijiazhuang University，Shijiazhuang 050050，China
^2.Research and Development Center of Microbial Pharmaceutical Application Technology in Colleges and Universities of Hebei Province，Shijiazhuang 050050，China
^3.Hebei Biopharmaceutical International Joint Research Center，Shijiazhuang 050050，China
^4.Key Laboratory of Innovative Drug Research and Evaluation of Hebei Province，Shijiazhuang 050017，China

Received:2024-03-19 Accepted:2024-07-02 Online:2024-09-25 Published:2024-10-22
Contact: Yonghao QI

摘要/Abstract

摘要：

研究旨在探究体外干扰肿瘤坏死因子受体相关因子6（tumor necrosis factor receptor-associated factor 6，TRAF6）的表达对肺癌A549细胞增殖与凋亡的影响以及可能的作用机理。siRNA转染A549细胞，运用荧光定量PCR（qPCR）和Western blotting检测siRNA转染后TRAF6的mRNA和蛋白表达水平。CCK-8法检测增殖情况，Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率，Transwell小室探究转染后细胞的侵袭能力，Western blotting检测TRAF6/TAK1通路相关蛋白（TAK1、ub-TAK1、p-TAK1、Bax、Bcl-2）的表达水平。A549细胞经siRNA干扰后，TRAF6 mRNA和蛋白的表达水平与空白对照组相比均显著下降，细胞增殖减少，凋亡增多，且侵袭能力下降明显。同时，干扰TRAF6的表达抑制了TAK1的活化，明显诱导Bax表达，显著抑制了Bcl-2蛋白的表达。结果表明干扰TRAF6的表达可以抑制肺癌细胞的增殖和侵袭，促进凋亡，其作用机制可能与抑制TRAF6/TAK1信号通路活化有关。

关键词: 肺癌, TRAF6, TAK1, 凋亡

Abstract:

The study aimed to investigate the effect of interfering tumor necrosis factor receptor-associated factor 6 （TRAF6） expression on proliferation and apoptosis of lung cancer A549 cells in vitro and its possible mechanism. A549 cells were transfected with siRNA， and the mRNA and protein expression levels of TRAF6 were detected by fluorescence quantitative PCR （qPCR） and Western blottingrespectivly. Proliferation was detected by CCK-8 assay， apoptosis rate was detected by Annexin V-FITC/PI double staining flow cytometry， and invasion ability of transfected cells was investigated by Transwell chamber. Western blotting was used to detect TRAF6/TAK1 pathway related proteins （TAK1，ub-TAK1， p-TAK1， Bax/Bcl-2， P-P65）. After siRNA interference， the expression levels of TRAF6 mRNA and protein in A549 cells were significantly decreased compared with the control group， cell proliferation was reduced， apoptosis was increased， and invasion ability was decreased significantly. Meanwhile， interference with TRAF6 expression inhibited TAK1 activation， significantly induced Bax expression， and significantly inhibited Bcl-2 and P-P65 protein expression. Thus， down-regulation of TRAF6 expression can inhibit the proliferation and invasion of lung cancer cells and promote apoptosis， and its mechanism may be related to the inhibition of TRAF6/TAK1 signaling pathway activation.

Key words: lung cancer, TRAF6, TAK1, apoptosis

中图分类号:

R734

赵轩, 任丽梅, 王晓茹, 韩广欣, 顾丹丹, 姚亚森, 祁永浩. siRNA靶向干扰TRAF6的表达对肺癌细胞增殖与凋亡的影响[J]. 生物技术进展, 2024, 14(5): 875-881.

Xuan ZHAO, Limei REN, Xiaoru WANG, Guangxin HAN, Dandan GU, Yasen YAO, Yonghao QI. Effects of siRNA Targeting to Interfere with the Expression of TRAF6 on the Proliferation and Apoptosis of Lung Cancer Cells[J]. Current Biotechnology, 2024, 14(5): 875-881.

图/表 7

表1 引物序列

Table 1 PCR primer sequence

引物名称（人源）	引物序列（5'→3'）
TRAF6-F	5'-TGGATGCCAAACCAGAGC-3'
TRAF6-R	5'-TCAAAGCGGGTGGAGACC-3'
β-actin-F	5'-CACGAAACTACCTTCAACT-3'
β-actin-R	5'-CATACTCCTGCTTGCTGATC-3'

图1 转染siRNA-TRAF6后细胞TRAF6 mRNA和蛋白的表达水平注：1—Control；2—siRNA-NC；3—转染24 h；4—转染48 h；5—转染72 h。

Fig. 1 Expression levels of TRAF6 mRNA and protein in cells transfected with siRNA-TRAF6

图2 CCK-8法测定siRNA-TRAF6表达对肺癌A549细胞活力的影响

Fig. 2 The effect of siRNA-TRAF6 expression on the viability of lung cancer A549 cells measured by CCK-8

图3 siRNA-TRAF6对A549细胞凋亡的影响A：流式细胞术结果；B:细胞凋亡率

Fig. 3 The effect of siRNA-TRAF6 on apoptosis of A549 cells

图4 Transwell实验检测干扰TRAF6的表达对细胞侵袭能力的影响A：细胞侵袭能力检测结果；B:具有侵袭能力的细胞数量

Fig. 4 Transwell experiment to detect the effect of interference with TRAF6 expression on cell invasion ability

图5 转染siRNA-TRAF6对细胞TAK1磷酸化和泛素化的影响A:TRAF6的表达对磷酸化TAK1的影响；B:TRAF6的表达对泛素化TAK1的影响。IP—免疫共沉淀产物；Input—未做处理的样品上清。*表示与Control组对比具有统计学差异（P<0.05）。

Fig. 5 The effect of siRNA-TRAF6 transfection on cell TAK1 phosphorylation and ubiquitination

图6 干扰TRAF6的表达对凋亡蛋白的影响注：*表示与Control组对比具有统计学差异（P<0.05）。

Fig. 6 The effect of interference by TRAF6 expression on apoptotic proteins

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siRNA靶向干扰TRAF6的表达对肺癌细胞增殖与凋亡的影响

Effects of siRNA Targeting to Interfere with the Expression of TRAF6 on the Proliferation and Apoptosis of Lung Cancer Cells

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