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

Abstract

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

CLC Number:

R743.3

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.

Figures/Tables 14

Tab.1

Tab.2

Fig.1

Fig.2

Tab.3

Fig.3

Fig.4

Tab.4

Tab.5

Fig.5

Tab.6

Tab.7

Fig.6

Fig.7

References 18

1	WANG T， CHEN H B， XIA S Y， et al. Ameliorative effect of Parishin C against cerebral ischemia-induced brain tissue injury by reducing oxidative stress and inflammatory responses in rat model ［J］. Neuropsychiatr Dis Treat， 2021， 17： 1811-1823.
2	ZHANG Q， JIA M， WANG Y F， et al. Cell Death Mechanisms in Cerebral Ischemia-Reperfusion Injury ［J］. Neurochem Res， 2022， 47（12）： 3525-3542.
3	CHENG X L， HU J， LIU X F， et al. Therapeutic targets by traditional Chinese medicine for ischemia-reperfusion injury induced apoptosis on cardiovascular and cerebrovascular diseases ［J］. Front Pharmacol， 2022， 13：934256.
4	ZHAO N， GAO Y H， JIA H T， et al. Anti-apoptosis effect of traditional Chinese medicine in the treatment of cerebral ischemia-reperfusion injury ［J］. Apoptosis， 2023， 28（5-6）： 702-729.
5	NEAG M A， MITRE A O， BURLACU C C， et al. miRNA involvement in cerebral ischemia-reperfusion injury ［J］. Front Neurosci， 2022， 16： 901360.
6	ZOU Z Y， LIU J， CHANG C， et al. Biliverdin administration regulates the microRNA-mRNA expressional network associated with neuroprotection in cerebral ischemia reperfusion injury in rats ［J］. Int J Mol Med， 2019， 43（3）：1356-1372.
7	LI J J， PENG L J， BAI W Y， et al. Biliverdin Protects Against Cerebral Ischemia/Reperfusion Injury by Regulating the miR-27a-3p/Rgs1 Axis ［J］. Neuropsychiatr Dis Treat， 2021， 17： 1165-1181.
8	ZHU L， FENG Z， ZHANG J， et al. MicroRNA-27a regulates ferroptosis through SLC7A11 to aggravate cerebral ischemia-reperfusion injury ［J］. Neurochem Res， 2023， 48（5）： 1370-1381.
9	YU Z T， LIU Y， LI Y， et al. miRNA-338-3p inhibits glioma cell proliferation and progression by targeting MYT1L ［J］. Brain Res Bull， 2022， 179： 1-12.
10	郝婧雯，邓炎尧，谢君，等. microRNA-9-5p通过抑制Tau磷酸化参与阿尔茨海默病的机制［J］. 实用医学杂志， 2022， 38（18）： 2296-2302.
11	YANG Y， HE B， ZHANG X， et al. Geraniin protects against cerebral ischemia/reperfusion injury by suppressing oxidative stress and neuronal apoptosis via regulation of the Nrf2/HO-1 pathway ［J］. Oxid Med Cell Longev， 2022， 2022： 2152746.
12	ZHANG Y， ZHAN B M， HU Y， et al. Sevoflurane inhibits the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes via regulating miR-27a-3p-mediated autophagy ［J］. J Pharm Pharmacol， 2021， 73（11）： 1470-1479.
13	LUO J， LI J J， XIONG L， et al. MicroRNA-27a-3p relieves inflammation and neurologic impairment after cerebral ischaemia reperfusion via inhibiting lipopolysaccharide induced TNF factor and the TLR4/NF-κB pathway ［J］. Eur J Neurosci， 2022， 56（3）： 4013-4030.
14	YANG Q， SUN Q， JIN P. Long non-coding RNA PVT1 regulates LPS-induced acute kidney injury in an in vitro model of HK-2 cells by modulating the miR-27a-3p/OXSR1 axis ［J］. Exp Ther Med， 2022， 24（3）： 552.
15	BALLESTER-LURBE B， POCH E， MOCHOLI E， et al. RhoE is spatiotemporally regulated in the postnatal mouse CNS ［J］. Neuroscience， 2009， 163（2）： 586-593.
16	MA X L， LI X， TIAN F J， et al. Upregulation of RND3 affects trophoblast proliferation， apoptosis， and migration at the maternal-fetal interface ［J］. Front Cell Dev Biol， 2020， 8： 153.
17	DONG H M， SUN Q， ZHANG Y， et al. Genetic deletion of Rnd3 suppresses apoptosis through NF-κB signaling in the brain ［J］. Oncol Rep， 2021， 45（2）： 595-605.
18	GUASCH R M， BLANCO A M， PEREZ-ARAGO A， et al. RhoE participates in the stimulation of the inflammatory response induced by ethanol in astrocytes ［J］. Exp Cell Res， 2007， 313（17）： 3779-3788.

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

Effects of miR⁃27a⁃3p on neuronal apoptosis induced by oxygen glucose deprivation/reoxygenation via targeting Rnd3

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 14

References 18

Related Articles 5

Recommended Articles

Metrics

Comments

组别	Rnd3 mRNA	Rnd3 蛋白
Control组	1.01 ± 0.05	0.11 ± 0.02
OGD/R组	1.52 ± 0.06^*	0.71 ± 0.05^*
shRnd3-NC组	1.02 ± 0.06	0.69 ± 0.02
shRnd3组	0.12 ± 0.00^△	0.22 ± 0.03^△
shRnd3+Inhibitor组	1.02 ± 0.02^▲	0.52 ± 0.07^▲
F值	127.40	41.17
P值	< 0.001	< 0.001

组别	细胞的活力（%）	C-caspase-3	Bax	Bcl-2	细胞总凋亡率（%）
Control组	100.00 ± 3.08	0.07 ± 0.00	0.14 ± 0.01	0.68 ± 0.02	3.38 ± 0.16
OGD/R组	62.86 ± 2.05^*	0.64 ± 0.04^*	0.92 ± 0.04^*	0.36 ± 0.01^*	11.59 ± 0.69^*
shRnd3-NC组	64.77 ± 3.53	0.49 ± 0.03	0.74 ± 0.03	0.18 ± 0.01	12.10 ± 0.73
shRnd3组	88.12 ± 2.74^△	0.17 ± 0.01^△	0.27 ± 0.01^△	0.49 ± 0.02^△	4.09 ± 0.35^△
shRnd3+Inhibitor组	68.16 ± 2.13^▲	0.62 ± 0.04^▲	0.98 ± 0.04^▲	0.46 ± 0.02	8.96 ± 0.85^▲
F值	35.34	81.70	150.8	124.5	44.81
P值	< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

[1]	Yanchao LI,Qiaoli. REN. IL⁃17A secreted by Th17 cells recruited by CXCL16 against B⁃ALL cell apoptosis [J]. The Journal of Practical Medicine, 2023, 39(24): 3163-3168.
[2]	Zeqi WANG,Yihua GAO,Yuetong JIN,Shanshan CONG,Xuanshun. JIN. The effect of schisandrae B on hyperthyroidism heart disease was evaluated by velocity vector imaging combined with NF⁃κB [J]. The Journal of Practical Medicine, 2023, 39(22): 2940-2945.
[3]	XIN Siyuan, YANG Zhixin, LIU Jinying, LIU Hui, GUO Ji⁃ an′en.. Yulura polysaccharide inhibits LPS⁃induced inflammatory damage and apoptosis in thyroid follicular epi⁃ thelial cells by regulating Caveolin⁃1 expression [J]. The Journal of Practical Medicine, 2022, 38(14): 1759-1772.
[4]	LI Liheng, WANG Rui, WANG Qin, ZHANG Zhiyi, AN Feng, ZHANG Xuan, WANG Xiaoming, ZHANG Fan.. Effect of miR ⁃ 498 targeting PCK1 on growth of oral squamous cell carcinoma cells [J]. The Journal of Practical Medicine, 2022, 38(11): 1339-1346.
[5]	ZHENG Ruizhe, MAO Mintao, YANG Xitao, LIU Yumei. Protective effects of coenzyme Q10 on traumatic brain injury by inhibiting inflammatory injury and cell apoptosis [J]. The Journal of Practical Medicine, 2021, 37(4): 446-450.