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中华细胞与干细胞杂志(电子版) ›› 2022, Vol. 12 ›› Issue (02) : 79 -85. doi: 10.3877/cma.j.issn.2095-1221.2022.02.003

论著

替米沙坦调控miR-495-3p/心肌细胞增强因子2A通路减轻缺氧诱导的神经细胞PC12损伤
刘宁1, 赵有芳1, 段爱琴1, 刘燕军1,†()   
  1. 1. 443000　宜昌，湖北宜昌国药葛洲坝中心医院三峡大学第三临床医学院神经内科
  • 收稿日期:2021-09-15 出版日期:2022-04-01
  • 通信作者: 刘燕军

Protective effect of telmisartan on hypoxia-induced PC12 cell injury by regulating the miR-495-3p/myocyte enhancer factor 2A pathway

Ning Liu1, Youfang Zhao1, Aiqin Duan1, Yanjun Liu1,†()   

  1. 1. Department of Neurology, Third Clinical Medical College, Three Gorges University, Gezhouba Central Hospital, Yichang 443000, China
  • Received:2021-09-15 Published:2022-04-01
  • Corresponding author: Yanjun Liu
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刘宁, 赵有芳, 段爱琴, 刘燕军. 替米沙坦调控miR-495-3p/心肌细胞增强因子2A通路减轻缺氧诱导的神经细胞PC12损伤[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(02): 79-85.

Ning Liu, Youfang Zhao, Aiqin Duan, Yanjun Liu. Protective effect of telmisartan on hypoxia-induced PC12 cell injury by regulating the miR-495-3p/myocyte enhancer factor 2A pathway[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(02): 79-85.

目的

探讨替米沙坦（TM）调控miR-495-3p/心肌细胞增强因子2A （MEF2A）通路对缺氧诱导PC12细胞损伤的影响和机制。

方法

将PC12细胞进行正常培养（对照），缺氧（缺氧24 h），缺氧+ 0.1、1、10 μg/mL TM、缺氧+ miR-NC （转染miR-NC），缺氧+miR-495-3p （转染miR-495-3p模拟物），缺氧+TM+anti-miR-NC （转染anti-miR-NC，10 μg/mL TM）和缺氧+ TM+anti-miR-495-3p （转染anti-miR-495-3p，10 μg/mL TM）处理。酶联免疫吸附实验（ELISA）检测乳酸脱氢酶（LDH）漏出量和超氧化物歧化酶（SOD）的活性，流式细胞术检测细胞凋亡，实时荧光定量PCR （RT-qPCR）和Western blot检测miR-495-3p、心肌细胞增强因子2A （MEF2A）表达水平。两组间比较采用t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与对照比较，缺氧处理的PC12细胞LDH漏出率[（9.46±0.97）﹪比（45.69±4.31）﹪]、细胞凋亡率[（5.36±0.54）﹪比（28.36±2.41）﹪]、MEF2A mRNA（1.00±0.08比2.74±0.26）和MEF2A蛋白表达水平（0.39±0.03比0.87±0.06）均升高；SOD活性[（12.24 ±1.13）比（5.13±0.52 ）U/mL）]和miR-495-3p表达水平（1.00±0.08比0.35±0.03）均降低，差异有统计学意义（P均< 0.05）。与缺氧处理比较，缺氧+0.1、1、10 μg/mL TM干预的PC12细胞LDH漏出率[（45.69±4.31）﹪比（32.14±3.84）﹪、（23.54±3.17）﹪、（16.87±1.46）﹪]、细胞凋亡率[（28.36±2.41）﹪比（22.46±2.31）﹪、（17.14±1.65）﹪、（10.23±1.12）﹪]、MEF2A mRNA（2.74±0.26比2.26±0.23、1.87±0.19、1.34±0.18）和MEF2A蛋白表达水平（0.87±0.06比0.75±0.05、0.63±0.04、0.46±0.03）均降低；SOD活性[（5.13±0.52）比（6.87±0.69 ）、（8.01±0.81）、（10.12±1.02）U/mL]、miR-495-3p表达水平（0.35±0.03比0.49±0.04、0.61±0.06、0.83±0.07）均升高，差异有统计学意义（P均< 0.05）。与缺氧+ miR-NC比较，缺氧+ miR-495-3p的PC12细胞LDH漏出率[（46.87±4.28）﹪比（19.65±1.87）﹪]和细胞凋亡率[（28.38±2.44）﹪比（12.36±1.25）﹪]均降低，SOD活性[（5.15±0.51）比（9.67±0.97）U/mL]升高，差异有统计学意义（P均< 0.05）。与缺氧+TM+anti-miR-NC比较，缺氧+TM+anti-miR-495-3p的PC12细胞LDH漏出率[（17.64±1.79）﹪比（32.69±3.57）﹪]和细胞凋亡率[（10.98±1.75）﹪比（22.64±2.13）﹪]均升高，SOD活性[（12.63±1.27）比（7.32±0.71）U/mL]降低，差异有统计学意义（P均< 0.05）。

结论

TM通过上调miR-495-3p/MEF2A通路对缺氧诱导的PC12细胞损伤具有保护作用。

关键词: 替米沙坦, miR-495-3p, MEF2A, 缺氧, PC12细胞, 细胞损伤
Objective

To investigate the effect of telmisartan (TM) on hypoxia-induced PC12 cell injury by regulating miR-495-3p/myocyte enhancer factor 2A (MEF2A) pathway and its mechanism.

Methods

PC12 cells were divided into control group (cells in normal culture) , hypoxia group (hypoxia 24 h) , TM+hypoxia-low dose (L) group (0.1 μg/mL TM, hypoxia for 24 h) , TM+hypoxia-medium dose (M) group (1 μg/mL TM, hypoxia for 24 h) , TM+hypoxia-high dose (H) group (10 μg/mL TM, hypoxia for 24 h) , hypoxia+miR-NC group (transfected with miR-NC, hypoxia for 24 h) , hypoxia+miR-495-3p group (transfected with miR-495-3p mimics, hypoxia for 24 h) , hypoxia+TM+anti-miR-NC group (transfected with anti-miR-NC, hypoxia for 24 h) , and hypoxia+TM+anti-miR-495-3p group (transfected with anti-miR-495-3p, hypoxia for 24 h) . Enzyme-linked immunosorbent assay (ELISA) was used to detect the leakage of LDH and the activity of SOD in each group. Apoptosis was detected by flow cytometry. The expression levels of miR-495-3p and cardiomyocyte enhancer factor 2A (MEF2A) were detected by real-time quantitative PCR (qRT-PCR) and Western blotting. The comparison between the two groups was performed by t test, and the comparison between multiple groups was performed by one-way ANOVA and LSD-t test.

Results

Compared with the control group, the LDH leakage rate [ (9.46±0.97) ﹪ vs (45.69±4.31) ﹪], apoptosis rate [ (5.36±0.54) ﹪ vs (28.36±2.41) ﹪], MEF2A mRNA (1.00±0.08 vs 2.74±0.26) and protein (0.39±0.03 vs 0.87±0.06) expression of PC12 cells were increased in the hypoxia group, and SOD activity [ (12.24±1.13) vs (5.13±0.52) U/mL], miR-495-3p expression (1.00±0.08 vs 0.35±0.03) were decreased, and the difference was statistically significant (P < 0.05) . Compared with the hypoxia group, the LDH leakage rate[ (45.69±4.31) ﹪vs (32.14±3.84) ﹪, (23.54±3.17) ﹪, (16.87±1.46) ﹪], apoptosis rate [ (28.36±2.41) ﹪ vs (22.46±2.31) ﹪, (17.14±1.65) ﹪, (10.23 ±1.12) ﹪], MEF2A mRNA (2.74±0.26 vs 2.26±0.23, 1.87±0.19, 1.34±0.18) and protein (0.87±0.06 vs 0.75±0.05, 0.63±0.04, 0.46±0.03) expression of PC12 cells in the low, medium and high dose TM intervention group were decreased, SOD activity [ ( 5.13±0.52) vs (6.87±0.69) , (8.01±0.81) , (10.12±1.02) U/mL], miR-495-3p expression (0.35±0.03 vs 0.49±0.04, 0.61±0.06, 0.83±0.07) were increased, the difference was statistically significant (P < 0.05) . Compared with the hypoxia+miR-NC group, the LDH leakage rate [ (46.87±4.28) ﹪ vs (19.65±1.87) ﹪]and the apoptosis rate [ (28.38±2.44) ﹪ vs (12.36±1.25) ﹪]of PC12 cells were reduced in the hypoxia+miR-495-3p group, and SOD activity [ (5.15±0.51) vs (9.67±0.97) U/mL) ] was increased, and the difference was statistically significant (P < 0.05) . Compared with the hypoxia+TM+anti-miR-NC group, the LDH leakage rate [ (17.64±1.79) ﹪vs (32.69±3.57) ﹪] and cell apoptosis rate [ (10.98±1.75) ﹪ vs (22.64± 2.13) ﹪] were decreased, SOD activity [ (12.63±1.27) vs (7.32±0.71) U/mL] was increased, the difference was statistically significant (P < 0.05) .

Conclusion

Telmisartan has a protective effect on hypoxia-induced PC12 cell injury by activating the miR-495-3p/MEF2A pathway.

Key words: Telmisartan, miR-495-3p, MEF2A, Hypoxia, PC12 cells, Cell damage
表1 引物序列信息
基因 引物序列（5'-3'） 预期扩增产物长度（bp）
miR-495-3p 上游ACACTCCAGCTGGGAAACAAACATGGTGCA 76
  下游TGGTGTCGTGGAGTCG  
U6 （内参） 上游CTCGCTTCGGCAGCACA 94
  下游AACGCTTCACGAATTTGCGT  
MEF2A 上游TCAGCTCTCTTGTTGCTGGA 93
  下游AAATCGGTTCGGACTTGATG  
GAPDH （内参） 上游GGTCTCCTCTGACTTCAACA 116
  下游AGCCAAATTCGTTGTCATAC  
表2 TM对缺氧诱导的PC12细胞LDH漏出率和SOD活性的影响（± s）
分组 实验次数 LDH （﹪） SOD （U/mL）
对照 3 9.46±0.97 12.24±1.13
缺氧 3 45.69±4.31a 5.13±0.52a
缺氧+ 0.1 μg/mL TM 3 32.14±3.84b 6.87±0.69b
缺氧+1 μg/mL TM 3 23.54±3.17bc 8.01±0.81bc
缺氧+10 μg/mL TM 3 16.87±1.46bcd 10.12±1.02bcd
F值   190.713 93.337
P值   < 0.001 < 0.001
表3 TM对缺氧诱导的PC12细胞凋亡的影响（± s）
分组 实验次数 凋亡率（﹪） Bcl-2蛋白 Bax蛋白
对照 3 5.36±0.54 0.77±0.07 0.23±0.03
缺氧 3 28.36±2.41a 0.29±0.03a 0.69±0.03a
缺氧+ 0.1μg/mL TM 3 22.46±2.31b 0.41±0.04b 0.57±0.05b
缺氧+1μg/mL TM 3 17.14±1.65bc 0.53±0.05bc 0.45±0.04bc
缺氧+10 μg/mL TM 3 10.23±1.12bcd 0.64±0.05bcd 0.32±0.03bcd
F值   82.671 42.847 75.706
P值   <0.001 <0.001 <0.001
表4 TM对缺氧诱导的PC12细胞中miR-495-3p和MEF2A表达的影响（± s）
分组 实验次数 miR-495-3p MEF2A mRNA MEF2A蛋白
对照 3 1.00±0.08 1.00±0.08 0.39±0.03
缺氧 3 0.35±0.03a 2.74±0.26a 0.87±0.06a
缺氧+ 0.1 μg/mL TM 3 0.49±0.04b 2.26±0.23b 0.75±0.05b
缺氧+1 μg/mL TM 3 0.61±0.06bc 1.87±0.19bc 0.63±0.04bc
缺氧+10 μg/mL TM 3 0.83±0.07bcd 1.34±0.18bcd 0.46±0.03bcd
F值   58.603 37.287 62.368
P值   < 0.001 < 0.001 < 0.001
图1 TM对缺氧诱导的PC12细胞凋亡相关蛋白表达的影响
图2 TM对缺氧诱导的PC12细胞中MEF2A蛋白表达的影响
图3 MEF2A的3'UTR中含有与miR-495-3p互补的核苷酸序列
图4 miR-495-3p调控MEF2A蛋白的表达
表5 双荧光素酶报告实验（± s）
分组 实验次数 WT-MEF2A MUT-MEF2A
miR-NC 3 1.02±0.09 0.39±0.04
miR-495-3p 3 1.00±0.08 1.04±0.09
t值   19.190 0.997
P值   < 0.001 0.334
图5 miR-495-3p过表达对缺氧诱导的PC12细胞MEF2A和凋亡相关蛋白表达的影响
表7 miR-495-3p过表达对缺氧诱导的PC12细胞损伤的影响（± s）
分组 实验次数 miR-495-3p MEF2A蛋白 LDH （﹪） SOD （U/mL） 凋亡率（﹪） Bcl-2蛋白 Bax蛋白
对照 3 1.00±0.07 0.37±0.03 10.12±1.01 13.64±1.25 6.84±0.64 0.74±0.07 0.22±0.03
缺氧 3 0.38±0.03 0.85±0.07 43.26±4.15 5.21±0.54 26.47±2.54 0.28±0.03 0.66±0.06
t值   14.101 10.917 13.439 10.723 12.980 10.462 11.361
P值   < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
缺氧+miR-NC 3 0.37±0.04 0.86±0.06 46.87±4.28 5.15±0.51 28.38±2.44 0.27±0.03 0.68±0.07
缺氧+miR-495-3p 3 0.78±0.06 0.49±0.04 19.65±1.87 9.67±0.97 12.36±1.25 0.61±0.06 0.37±0.03
t值   9.848 8.887 10.094 7.144 10.121 13.880 7.050
P值   0.001 0.001 0.001 < 0.001 < 0.001 < 0.001 < 0.001
图6 抑制miR-495-3p表达逆转了TM对缺氧诱导的PC12细胞MEF2A和凋亡相关蛋白表达的作用
表8 抑制miR-495-3p表达逆转TM对缺氧诱导的PC12细胞损伤的作用（± s）
分组 实验次数 miR-495-3p MEF2A蛋白 LDH（﹪） SOD（U/mL） 凋亡率（﹪） Bcl-2蛋白 Bax蛋白
缺氧 3 1.00±0.09 0.86±0.07 46.28±4.33 5.06±0.58 27.32±2.77 0.27±0.03 0.71±0.06
缺氧+TM 3 2.56±0.24 0.42±0.04 18.42±1.82 11.21±1.02 11.36±1.05 0.65±0.06 0.31±0.03
t值   10.542 9.453 10.274 9.078 9.332 9.812 10.328
P值   < 0.001 0.001 0.001 0.001 0.001 0.001 < 0.001
缺氧+TM+anti-miR-NC 3 2.59±0.23 0.40±0.03 17.64±1.79 12.63±1.27 10.98±1.75 0.67±0.05 0.30±0.03
缺氧+TM+anti-miR-495-3p 3 1.46±0.15 0.73±0.06 32.69±3.57 7.32±0.71 22.64±2.13 0.38±0.03 0.59±0.05
t值   7.128 8.521 6.527 6.321 7.326 8.614 8.614
P值   0.002 0.001 0.003 0.003 0.002 0.001 0.001
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