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中华细胞与干细胞杂志(电子版) ›› 2021, Vol. 11 ›› Issue (04) : 215 -221. doi: 10.3877/cma.j.issn.2095-1221.2021.04.004

论著

miR-942-5p靶向TRAF6对病毒性心肌炎细胞损伤的影响
杨婷1,()   
  1. 1. 044000 运城,山西省运城市中心医院心内科
  • 收稿日期:2021-03-22 出版日期:2021-08-01
  • 通信作者: 杨婷

The effect of miR-942-5p on viral myocarditis cell injury by targeting TRAF6

Ting Yang1,()   

  1. 1. Internal Medicine-Cardiovascular Department, Shanxi Yuncheng Central Hospital, Yuncheng 044000, China
  • Received:2021-03-22 Published:2021-08-01
  • Corresponding author: Ting Yang
引用本文:

杨婷. miR-942-5p靶向TRAF6对病毒性心肌炎细胞损伤的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2021, 11(04): 215-221.

Ting Yang. The effect of miR-942-5p on viral myocarditis cell injury by targeting TRAF6[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(04): 215-221.

目的

探讨miR-942-5p靶向肿瘤坏死因子受体相关因子6 (TRAF6)对病毒性心肌炎细胞损伤的影响。

方法

体外分离培养BALB/c小鼠心肌细胞,采用柯萨奇病毒B3 (CVB3)感染心肌细胞,建立病毒性心肌炎细胞模型,将细胞分为对照组(未感染CVB3病毒)、CVB3组(感染CVB3病毒)、CVB3+miR-NC组(感染CVB3病毒,转染miR-NC)、CVB3+miR-942-5p组(感染CVB3病毒,转染miR-942-5p)、CVB3+si-NC组(感染CVB3病毒,转染si-NC)、CVB3+si-TRAF6组(感染CVB3病毒,转染si-TRAF6)、CVB3+miR-942-5p+pcDNA组(感染CVB3病毒,转染miR-942-5p和pcDNA)和CVB3+miR-942-5p+TRAF6组(感染CVB3病毒,转染miR-942-5p和TRAF6)。采用实时定量聚合酶链反应(qRT-PCR)检测细胞中miR-942-5p和TRAF6 mRNA的表达;蛋白免疫印迹法(Western blot)检测TRAF6、B细胞淋巴瘤/白血病-2(Bcl-2)和B细胞淋巴瘤/白血病-2相关蛋白X (Bax)蛋白表达水平;噻唑蓝比色法(MTT)检测细胞活力;流式细胞术检测细胞凋亡情况;ELISA检测肿瘤坏死因子(TNF-α)、白介素-1β(IL-1β)的表达;双荧光素酶报告实验验证miR-942-5p和TRAF6的关系。两组间比较采用独立样本t检验。

结果

与对照组相比,CVB3组的miR-942-5p表达量(0.97±0.06比0.36±0.03)、细胞活力(98.10﹪±8.67﹪比44.61﹪±5.30﹪)、Bcl-2蛋白表达(0.93±0.06比0.38±0.03)均降低;TRAF6 mRNA (0.96±0.08比2.14±0.21)和蛋白表达(0.86±0.07比1.69±0.17)、细胞凋亡率(8.92﹪±1.13﹪比26.27﹪±2.24﹪)、Bax蛋白表达(0.94±0.06比1.67±0.156)、TNF-α(56.70±5.56比97.62±8.89)、IL-1β (38.42±4.01比86.91±8.09)均升高,差异有统计学意义(P均< 0.05);与CVB3+miR-NC组比较,CVB3+miR-942-5p组细胞活力(41.24﹪±4.71﹪比65.32﹪±5.81﹪)、Bcl-2蛋白表达(0.41±0.04比0.64±0.05)均升高,细胞凋亡率(25.67﹪±1.19﹪比14.22﹪±1.46﹪)、Bax蛋白表达(1.70±0.16比1.29±0.12)、TNF-α (103.68±9.82比78.44±6.68)、IL-1β (81.27±7.81比57.43±4.78)均降低(P均< 0.05);与CVB3+si-NC组比较,CVB3+si-TRAF6组细胞活力(50.32﹪±4.94﹪比68.32﹪±6.44﹪)、Bcl-2蛋白表达(0.39±0.04比0.63±0.05)均升高,细胞凋亡率(23.86﹪±1.82﹪比16.34﹪±1.27﹪)、Bax蛋白表达(1.73±0.15比1.26±0.15)、TNF-α (86.08±8.04比63.42±5.89)、IL-1β (74.48±6.88比55.64±5.18)均升高(P均<0.05)。miR-942-5p可以靶向TRAF6的表达,过表达TRAF6可以逆转过表达miR-942-5p对CVB3诱导的病毒性心肌炎细胞的活力、凋亡和炎症反应的影响。

结论

miR-942-5p可以通过靶向调控TRAF6的表达减轻CVB3感染心肌细胞的凋亡和炎症反应。

Objective

To investigate the effect of miR-942-5p on viral myocarditis cell injury by targeting tumor necrosis receptor related factor 6 (TRAF6) .

Method

The cardiomyocytes of BALB/c mice were isolated and cultured in vitro, and Coxsackievirus B3 (CVB3) was used to infect cardiomyocytes to establish a viral myocarditis cell model, the cells were divided into Con group (no CVB3 virusinfected) and CVB3 group (CVB3 virusinfected) , CVB3+miR-NC group (CVB3 virusinfected and miR-NC transfected) , CVB3+miR-942-5p group (CVB3 virusinfected and miR-942-5ptransfected) , CVB3+si-NC group (CVB3 virusinfectedandsi-NC transfected) , CVB3+si-TRAF6 group (CVB3 virusinfected and si-TRAF6 transfected) , CVB3+miR-942-5p+ pcDNA group (CVB3 virusinfected, miR-942-5p and pcDNA transfected) and CVB3+miR-942-5p+ TRAF6 group (CVB3 virusinfected, miR-942-5p and TRAF6 transfected) . Then Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-942-5p and TRAF6 mRNA. Western blot detected the expression of TRAF6, B-cell lymphoma/leukemia-2 (Bcl-2) and B-cell lymphoma/leukemia-2 related protein X (Bax) protein. Thiazole blue colorimetry (MTT) detected cell viability. Apoptosis was assessed by flow cytometry. ELISA was used to detect tumor necrosis factor (TNF-α) , interleukin-1β (IL-1β) expression and dual luciferase report experiment verified the relationship between miR-942-5p and TRAF6. The independent sample t test was used for the comparison between the two groups.

Result

Compared with the Con group, the expression of miR-942-5p (0.97±0.06 vs 0.36±0.03) , cell viability[ (98.10±8.67) ﹪ vs (44.61±5.30) ﹪, (97.51±8.89) ﹪ vs (46.69±5.42) ﹪], and Bcl-2 (0.93±0.06 vs 0.38±0.03, 0.92±0.06 vs 0.41±0.03) protein expression in CVB3 group were significantly reduced. TRAF6 mRNA (0.96±0.08 to 2.14±0.21) and protein expression (0.86±0.07 vs 1.69±0.17) , apoptosis rate [ (8.92±1.13) ﹪ vs (26.27±2.24) ﹪, (7.20±0.94) ﹪ vs (24.58±2.12) ﹪], Bax (0.94±0.06 vs 1.67±0.15, 0.98±0.07 vs 1.77±0.16) protein expression, TNF-α (56.70±5.56 vs 97.62±8.89, 48.52±4.68 vs 81.24±7.67) , IL-1β (38.42±4.01 vs 86.91±8.09, 33.21±4.06 vs 77.39±7.21) were significantly increased. Overexpression of miR-942-5p or interference with TRAF6 can significantly increase cell viability [ (41.24±4.71) ﹪ vs (65.32±5.81) ﹪, (50.23±4.94) ﹪ vs (68.32±6.44) ﹪], Bcl-2 (0.41±0.04 vs 0.64±0.05, 0.39±0.04 vs 0.63±0.05) protein expression, and reduce cell apoptosis rate[ (25.67±1.91) ﹪ vs (14.22±1.46) ﹪, (23.86±1.82) ﹪ vs (16.34±1.27) ﹪], Bax (1.70±0.16 vs 1.29±0.12, 1.73±0.15 vs 1.26±0.15) protein expression, TNF-α (103.68±9.82 vs 78.42±6.68, 86.08±8.04 vs 63.42±5.89) , IL-1β (81.27±7.81 vs 57.43±4.78, 74.48±6.88 vs 55.64±5.18) . miR-942-5p can target the expression of TRAF6, and overexpression of TRAF6 can reverse the miR-942-5p overexpression effecton the viability, apoptosis and inflammatory response of viral myocarditis cells induced by CVB3.

Conclusion

miR-942-5p can target apoptosis and inflammation in CVB3 infected cardiomyocytes by targeting TRAF6 expression.

表1 引物序列信息
图1 TRAF6在CVB3感染心肌细胞后的蛋白表达
表2 miR-942-5p和TRAF6在CVB3感染心肌细胞后的表达( ± s
图2 过表达miR-942-5p对CVB3感染心肌细胞凋亡的影响
表3 过表达miR-942-5p对CVB3感染心肌细胞凋亡和炎症反应的影响( ± s
图3 干扰TRAF6对CVB3感染心肌细胞凋亡的影响
表4 干扰TRAF6对CVB3感染心肌细胞凋亡和炎症反应的影响( ± s
图4 miR-942-5p和TRAF6存在互补的核苷酸序列
表5 双荧光素酶报告实验( ± s
图5 过表达TRAF6可以逆转过表达miR-942-5p对CVB3感染心肌细胞凋亡的影响
表6 过表达TRAF6可以逆转过表达miR-942-5p对CVB3感染心肌细胞凋亡和炎症反应的影响( ± s
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