miR-27a-3p靶向Rnd3对氧糖剥夺/复氧诱导的神经细胞凋亡的影响

doi:10.3969/j.issn.1006-5725.2023.23.005

摘要/Abstract

摘要：

目的探讨miR-27a-3p调控Rho家族GTP酶3（Rnd3）在氧糖剥夺/复氧（OGD/R）神经细胞凋亡中的作用及机制。方法体外培养大鼠PC12神经细胞，氧糖剥夺2 h后分别复氧3、6、9和12 h，检测各时刻细胞活力、miR-27a-3p和Rnd3 mRNA的表达筛选最适复氧时间点。采用慢病毒分别转染miR-27a-3p Mimic、miR-27a-3p Inhibitor及二者的阴性对照、转染shRnd3及其阴性对照、或共转染shRnd3和miR-27a-3p Inhibitor入细胞后，CCK-8法检测细胞的活力；流式细胞术检测细胞的凋亡率；RT-qPCR检测miR-27a-3p和Rnd3 mRNA的表达；Western blot检测凋亡相关蛋白和Rnd3蛋白的表达；双荧光素酶报告基因实验验证miR-27a-3p和Rnd3的靶向关系。结果上调miR-27a-3p提高了细胞活力，降低了细胞的总凋亡率、抑制了促凋亡蛋白Cleaved Caspase-3（C-caspase-3）、Bax和促进抗凋亡蛋白Bcl-2的表达，差异有统计学意义（P < 0.05）；下调miR-27a-3p则出现相反的结果。双荧光素酶报告基因实验显示Rnd3是miR-27a-3p的靶基因。下调Rnd3提高了细胞活力，降低了细胞的总凋亡率、抑制了促凋亡蛋白C-caspase-3、Bax和促进抗凋亡蛋白Bcl-2的表达，差异有统计学意义（P < 0.05）。然而，miR-27a-3p Inhibitor逆转了shRnd3的保护作用。结论 miR-27a-3p通过靶向抑制Rnd3抑制细胞凋亡减轻PC12神经细胞的OGD/R损伤。

关键词: miR-27a-3p, 氧糖剥夺/复氧, Rnd3, 细胞凋亡

Abstract:

Objective To investigate the effect and mechanism of miR?27a?3p on nerve cell apoptosis induced by oxygen glucose deprivation/reoxygenation （OGD/R） through regulation of Rho family GTPase 3（Rnd3） expression. Methods PC12 neurons were cultured in vitro and reoxygenated for 3， 6， 9 h and 12 h after 2 h oxygen glucose deprivation. Cell viability， miR?27a?3p expression and Rnd3 mRNA expression were assessed at each time point and the optimal reoxygenation time point was screened. After transfection of miR?27a?3p Mimic， miR?27a?3p Inhibitor and their negative control， transfection of shRnd3 and its negative control， or co?transfection of shRnd3 and miR?27a?3p Inhibitor through lentivirus， CCK?8 assay was used to detect cell activity. The apoptosis rate of the cells was detected using flow cytometry. Expression of miR?27a?3p and Rnd3 mRNA was detected by RT?qPCR. Expression of apoptosis?related protein and Rnd3 protein was detected by Western blot. The dual luciferase reporter assay confirmed the targeting relationship between miR?27a?3p and Rnd3. Results Upregulation of miR?27a?3p increased cell viability， decreased total cell apoptosis rate， suppressed pro?apoptotic proteins Cleaved Caspase?3 （C?caspase?3） and Bax， and promoted expression of anti?apoptotic protein Bcl?2 （P < 0.05）； The opposite result was found when down?regulating miR?27a?3p. The double luciferase reporter gene assay showed that Rnd3 was the target gene of miR?27a?3p. Down?regulation of Rnd3 increased cell viability， decreased the total rate of apoptosis， suppressed the pro?apoptotic protein C?caspase?3， Bax， and promoted expression of the anti?apoptotic protein Bcl?2 （P < 0.05）. However， miR?27a?3p Inhibitor reversed the protective effect of shRnd3. Conclusion miR?27a?3p alleviates OGD/R?induced damage to PC12 neurons by targeting Rnd3 to inhibit cell apoptosis.

Key words: miR?27a?3p, OGD/R, Rnd3, cell apoptosis

中图分类号:

R743.3

李俊杰,白文娅,陈文栋,杨伟,邵建林. miR-27a-3p靶向Rnd3对氧糖剥夺/复氧诱导的神经细胞凋亡的影响[J]. 实用医学杂志, 2023, 39(23): 3051-3057.

Junjie LI,Wenya BAI,Wendong CHEN,Wei YANG,Jianlin. SHAO. Effects of miR⁃27a⁃3p on neuronal apoptosis induced by oxygen glucose deprivation/reoxygenation via targeting Rnd3[J]. The Journal of Practical Medicine, 2023, 39(23): 3051-3057.

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组别	细胞的活力（%）	miR-27a-3p	Rnd3 mRNA
Control组	100.00 ± 2.27	1.01 ± 0.08	1.01 ± 0.10
复氧3 h组	90.46 ± 3.83	0.45 ± 0.01^*	2.56 ± 0.14^*
复氧6 h组	66.30 ± 2.61^*	0.31 ± 0.02^*	2.59 ± 0.08^*
复氧9 h组	76.80 ± 3.18^*	0.49 ± 0.02^*	1.36 ± 0.04
复氧12 h组	62.66 ± 2.82^*	0.08 ± 0.01^*	5.85 ± 0.14^*
F值	28.08	79.10	323.10
P值	< 0.001	< 0.001	< 0.001

组别	miR-27a-3p	细胞的活力（%）	C-caspase-3	Bax	Bcl-2	细胞总凋亡率（%）
Control组	1.02 ± 0.14	100.00 ± 1.21	0.42 ± 0.02	0.28 ± 0.04	0.96 ± 0.01	3.55 ± 0.14
OGD/R组	0.27 ± 0.00^*	59.01 ± 1.71^*	1.00 ± 0.04^*	0.78 ± 0.07^*	0.41 ± 0.03^*	11.25 ± 0.09^*
Mimic-NC组	0.23 ± 0.01	60.95 ± 3.08	0.79 ± 0.07	0.89 ± 0.03	0.42 ± 0.02	12.44 ± 0.76
Mimic组	0.70 ± 0.06^△	74.84 ± 2.25^△	0.49 ± 0.06^△	0.40 ± 0.06^△	0.69 ± 0.07^△	5.28 ± 0.43^△
Inhibitor-NC组	0.23 ± 0.01	59.95 ± 2.29	0.82 ± 0.05	0.77 ± 0.05	0.37 ± 0.02	11.93 ± 0.55
Inhibitor组	0.15 ± 0.01^▲	53.58 ± 3.04	1.17 ± 0.08^▲	1.14 ± 0.07^▲	0.30 ± 0.08	15.67 ± 0.50^▲
F值	30.47	52.92	24.28	33.10	29.78	95.94
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

组别	Rnd3 mRNA	Rnd3 蛋白
Control组	1.01 ± 0.08	0.14 ± 0.01
OGD/R组	3.12 ± 0.11^*	0.64 ± 0.05^*
Mimic-NC组	3.72 ± 0.44	0.64 ± 0.05
Mimic组	1.71 ± 0.14^△	0.45 ± 0.04^△
Inhibitor-NC组	3.32 ± 0.12	0.56 ± 0.05
Inhibitor组	5.81 ± 0.14^▲	1.05 ± 0.08^▲
F值	63.35	34.14
P值	< 0.001	< 0.001

组别	荧光素酶相对活性
Rnd3/Wt+Mimic-NC组	1.00 ± 0.02
Rnd3/Wt+Mimic组	0.73 ± 0.06^*
Rnd3/Mut+Mimic-NC组	1.01 ± 0.04
Rnd3/Mut+Mimic组	1.00 ± 0.05
F值	9.303
P值	< 0.001

组别	Rnd3 mRNA	Rnd3 蛋白
Control组	1.01 ± 0.11	0.87 ± 0.12
shRnd3-NC组	1.03 ± 0.04	0.97 ± 0.12
shRnd3-001组	1.28 ± 0.04	1.08 ± 0.19
shRnd3-002组	0.07 ± 0.02^*△▲	0.38 ± 0.03^*△▲
shRnd3-003组	0.63 ± 0.03^*	0.66 ± 0.03^*
shRnd3-004组	0.47 ± 0.09^*	0.63 ± 0.08^*
F值	47.65	5.63
P值	< 0.001	< 0.001