抑制miR-17-5p通过靶向调控Mfn2表达对划痕损伤引起大鼠脊髓星形胶质细胞增殖的抑制作用

doi:10.13481/j.1671-587X.20260109

摘要/Abstract

摘要：

目的探讨抑制微小RNA（miR）-17-5p对划痕损伤诱导大鼠星形胶质细胞增殖的影响，并阐明其作用机制。方法原代分离培养大鼠脊髓组织中的星形胶质细胞，将miR-17-5p抑制剂（miR-17-5p inhibitor）及其阴性对照（inhibitor-NC）和线粒体融合蛋白2（Mfn2）干扰质粒（si-Mfn2）及其阴性对照（si-NC）转染至大鼠脊髓星形胶质细胞中，采用划痕法建立体外机械损伤诱导的星形胶质细胞增殖模型。将星形胶质细胞分为对照组（不进行处理）、Scratch组（划痕处理）、Scratch+inhibitor-NC组（转染inhibitor-NC后划痕处理）和Scratch+miR-17-5p inhibitor组（转染miR-17-5p inhibitor后划痕处理）以及空白组（不进行处理）、inhibitor-NC组（转染inhibitor-NC）、miR-17-5p inhibitor组（转染miR-17-5p inhibitor）、miR-17-5p inhibitor+si-NC组（共转染miR-17-5p inhibitor和si-NC后划痕处理）和miR-17-5p inhibitor+si-Mfn2组（共转染miR-17-5p inhibitor和si-Mfn2后划痕处理）。采用细胞划痕愈合实验检测不同时间点划痕愈合率，实时荧光定量PCR（RT-qPCR）法检测各组细胞中miR-17-5p和Mfn2 mRNA表达水平，Western blotting法检测各组细胞中Mfn2、胶质纤维酸性蛋白（GFAP）、增殖细胞核抗原（PCNA）和Ki-67蛋白表达水平，免疫荧光法检测各组细胞中GFAP和Ki-67表达情况，5-溴-2'-脱氧尿苷（BrdU）实验检测各组细胞增殖情况，双荧光素酶报告基因实验验证Mfn2和miR-17-5p之间的靶向关系。结果细胞划痕愈合实验，随着划痕损伤时间（0、12、24和48 h）的延长，大鼠脊髓星形胶质细胞划痕愈合率升高（P<0.05），且呈时间依赖性。RT-qPCR法和Western blotting法，与Scratch 0 h组比较，Scratch 12、24和48 h组大鼠星形胶质细胞中miR-17-5p表达水平以及GFAP、PCNA和Ki-67蛋白表达水平升高（P<0.05），Mfn2蛋白表达水平降低（P<0.05），且呈时间依赖性；与对照组比较，Scratch组星形胶质细胞中GFAP和Ki-67蛋白表达水平升高（P<0.05），Mfn2蛋白表达水平降低（P<0.05）；与Scratch组比较，Scratch+miR-17-5p inhibitor组星形胶质细胞中GFAP和Ki-67蛋白表达水平降低（P<0.05），Mfn2蛋白表达水平升高（P<0.05）；与空白组比较，miR-17-5p inhibitor组星形胶质细胞中miR-17-5p表达水平降低（P<0.05），Mfn2 mRNA和蛋白表达水平升高（P<0.05）；与Scratch+miR-17-5p inhibitor组比较，miR-17-5p inhibitor+si-Mfn2组星形胶质细胞中Mfn2蛋白表达水平降低（P<0.05）。免疫荧光法，与对照组比较，Scratch组GFAP和Ki-67共定位星形胶质细胞数量增加（P<0.05）；与Scratch组比较，Scratch+miR-17-5p inhibitor组GFAP和Ki-67共定位星形胶质细胞数量减少（P<0.05）；与Scratch+miR-17-5p inhibitor组比较，miR-17-5p inhibitor+si-Mfn2组GFAP和Ki-67共定位星形胶质细胞数量增加（P<0.05）。BrdU实验，与对照组比较，Scratch组星形胶质细胞BrdU阳性表达率升高（P<0.05）；与Scratch组比较，Scratch+miR-17-5p inhibitor组星形胶质细胞BrdU阳性表达率降低（P<0.05）；与Scratch+miR-17-5p inhibitor组比较，miR-17-5p inhibitor+si-Mfn2组星形胶质细胞BrdU阳性表达率升高（P<0.05）。双荧光素酶报告基因实验，miR-17-5p和Mfn2 mRNA的3'UTR区存在结合位点，Mfn2是miR-17-5p下游靶基因。结论大鼠脊髓星形胶质细胞划痕损伤后细胞中miR-17-5p表达水平明显升高，细胞增殖活性增加，抑制miR-17-5p通过靶向上调Mfn2表达抑制划痕损伤所致星形胶质细胞的增殖。

关键词: 星形胶质细胞, 划痕损伤, 细胞增殖, 微小RNA-17-5p, 线粒体融合蛋白2

Abstract:

Objective To discuss the effect of inhibiting micro RNA（miR）-17-5p on scratch injury-induced proliferation of rat astrocytes and to clarify its mechanism. Methods The astrocytes were isolated and cultured primarily from rat spinal cord tissue. MiR-17-5p inhibitor and its negative control （inhibitor-NC）， and mitofusin 2 （Mfn2） interfering plasmid （si-Mfn2） and its negative control （si-NC） were transfected into rat spinal cord astrocytes. An in vitro mechanical injury-induced astrocyte proliferation model was established using the scratch method. Astrocytes were divided into control group （no treatment）， Scratch group （scratch treatment）， Scratch+inhibitor-NC group （transfected with inhibitor-NC then scratch treatment）， and Scratch+miR-17-5p inhibitor group （transfected with miR-17-5p inhibitor then scratch treatment）； and also into blank group （no treatment）， inhibitor-NC group （transfected with inhibitor-NC）， miR-17-5p inhibitor group （transfected with miR-17-5p inhibitor）， miR-17-5p inhibitor+si-NC group （co-transfected with miR-17-5p inhibitor and si-NC then scratch treatment）， and miR-17-5p inhibitor+si-Mfn2 group （co-transfected with miR-17-5p inhibitor and si-Mfn2 then scratch treatment）. Cell scratch healing assay was used to detect the scratch healing rates at different time points； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the miR-17-5p and Mfn2 mRNA expression levels in the cells in various groups； Western blotting method was used to detect the protein expression levels of Mfn2， glial fibrillary acidic protein （GFAP）， proliferating cell nuclear antigen （PCNA）， and proliferation marker protein Ki-67 in the cells in various groups； immunofluorescence method was used to detect the expression of GFAP and Ki-67 in the cells in various groups； 5-bromo-2'-deoxyuridine （BrdU） assay was used to detect the proliferation of cells in various groups； the dual-luciferase reporter gene assay were used to verify the targeted regulatory relationship between Mfn2 and miR-17-5p. Results The cell scratch healing assay results showed that as the scratch injury time （0， 12， 24， and 48 h） increased， the scratch healing rate of spinal cord astrocytes of the rats was was increased （P<0.05） in a time-dependent manner. The RT-qPCR and Western blotting results showed that compared with Scratch 0 h group， the miR-17-5p expression level and the protein expression levels of GFAP， PCNA， and Ki-67 in astrocytes in Scratch 12， 24， and 48 h groups were increased （P<0.05）， while the Mfn2 protein expression level was decreased （P<0.05） in a time-dependent manner； compared with Control group， the GFAP and Ki-67 protein expression levels in astrocytes in Scratch group were increased （P<0.05）， while the Mfn2 protein expression level was decreased （P<0.05）； compared with Scratch group， the GFAP and Ki-67 protein expression levels in astrocytes in Scratch+miR-17-5p inhibitor group were decreased （P<0.05）， while the Mfn2 protein expression level was increased （P<0.05）； compared with blank group， the miR-17-5p expression level in astrocytes in miR-17-5p inhibitor group was decreased （P<0.05）， while the Mfn2 mRNA and protein expression levels were increased （P<0.05）； compared with Scratch+miR-17-5p inhibitor group， the Mfn2 protein expression level in astrocytes in miR-17-5p inhibitor+si-Mfn2 group was decreased （P<0.05）. The immunofluorescence assay results showed that compared with control group， the numbers of GFAP and Ki-67 co-localized astrocytes in Scratch group was increased （P<0.05）； compared with Scratch group， the number of GFAP and Ki-67 co-localized astrocytes in scratch+miR-17-5p inhibitor group was decreased （P<0.05）； compared with Scratch+miR-17-5p inhibitor group， the number of GFAP and Ki-67 co-localized astrocytes in miR-17-5p inhibitor+si-Mfn2 group was increased （P<0.05）. The BrdU assay results showed that compared with control group， the BrdU positive expression rate in the astrocytes in Scratch group was increased （P<0.05）； compared with scratch group， the BrdU positive expression rate in astrocytes in Scratch+miR-17-5p inhibitor group was decreased （P<0.05）； compared with scratch+miR-17-5p inhibitor group， the BrdU positive expression rate in astrocytes in miR-17-5p inhibitor+si-Mfn2 group was increased （P<0.05）. The dual-luciferase reporter gene assay results showed that there was a binding site between miR-17-5p and the 3'UTR region of Mfn2 mRNA， and Mfn2 is a downstream target gene of miR-17-5p. Conclusion The miR-17-5p expression level in rat spinal cord astrocytes is significantly increased after scratch injury， and cell proliferation activity is increased. Inhibition of miR-17-5p suppresses the proliferation of astrocytes induced by scratch injury by targeting and upregulating Mfn2 expression.

Key words: Astrocyte, Scratch injury, Cell proliferation, MicroRNA-17-5p, Mitofusin 2

中图分类号:

R681.5

赵焱,吴华伟. 抑制miR-17-5p通过靶向调控Mfn2表达对划痕损伤引起大鼠脊髓星形胶质细胞增殖的抑制作用[J]. 吉林大学学报(医学版), 2026, 52(1): 81-92.

Yan ZHAO,Huawei WU. Inhibitory effect of inhibition of miR-17-5p on proliferation of spinal cord astrocytes of rats induced by scratch injury through targeting Mfn2 expression[J]. Journal of Jilin University(Medicine Edition), 2026, 52(1): 81-92.

图/表 12

图1

图2

图3

图4

图5

图6

图7

图8

图9

图10

图11

图12

参考文献 21

[1]	IZZY S. Traumatic spinal cord injury［J］. Continuum， 2024， 30（1）：53-72.
[2]	ANJUM A， YAZID M D， FAUZI DAUD M， et al. Spinal cord injury： pathophysiology， multimolecular interactions， and underlying recovery mechanisms［J］. Int J Mol Sci， 2020， 21（20）：7533.
[3]	MAO H， CHEN W， CHEN L X， et al. Potential role of mitochondria-associated endoplasmic reticulum membrane proteins in diseases［J］. Biochem Pharmacol， 2022， 199：115011.
[4]	CAO Y， LV G， WANG Y S， et al. Mitochondrial fusion and fission after spinal sacord injury in rats［J］. Brain Res， 2013， 1522：59-66.
[5]	SHI Y L， LUO P， YI C L， et al. Effects of Mitofusin2 on astrocytes proliferation in vitro induced by scratch injury［J］. Neurosci Lett， 2020， 729：134969.
[6]	HONG P W， JIANG M， LI H D. Functional requirement of dicer1 and miR-17-5p in reactive astrocyte proliferation after spinal cord injury in the mouse［J］. Glia， 2014， 62（12）：2044-2060.
[7]	ZHANG L， WANG Z J， LI B， et al. The inhibition of miR-17-5p promotes cortical neuron neurite growth via STAT3/GAP-43 pathway［J］. Mol Biol Rep， 2020， 47（3）：1795-1802.
[8]	MISHRA P S， RAJU T R. A simple and efficient method for concomitant isolation and culture of enriched astroglial and microglial cells from the rat spinal cord［J］. Bio Protoc， 2020， 10（2）：e3501.
[9]	CAI J J， KONG J D， MA S， et al. Upregulation of TRPC6 inhibits astrocyte activation and proliferation after spinal cord injury in rats by suppressing AQP4 expression［J］. Brain Res Bull， 2022， 190：12-21.
[10]	ORR M B， GENSEL J C. Spinal cord injury scarring and inflammation： therapies targeting glial and inflammatory responses［J］. Neurotherapeutics， 2018， 15（3）：541-553.
[11]	MARANGON D， CASTRO E SILVA J H， CERRATO V， et al. Oligodendrocyte progenitors in glial scar： a bet on remyelination［J］. Cells， 2024， 13（12）： 1024.
[12]	刘建春，张红珍，王青，等. 黄芪甲苷对EAE小鼠轴突修复和再生的促进作用机制［J］. 解放军医学杂志，2024， 49（8）： 914-921.
[13]	GONG L L， GU Y， HAN X X， et al. Spatiotemporal dynamics of the molecular expression pattern and intercellular interactions in the glial scar response to spinal cord injury［J］. Neurosci Bull， 2023， 39（2）： 213-244.
[14]	YANG R C， YANG B， LIU W， et al. Emerging role of non-coding RNAs in neuroinflammation mediated by microglia and astrocytes［J］. J Neuroinflammation， 2023， 20（1）：173.
[15]	GAO X， LI S Y， YANG Y J， et al. A novel magnetic responsive miR-26a@SPIONs- OECs for spinal cord injury： triggering neural regeneration program and orienting axon guidance in inhibitory astrocytic environment［J］. Adv Sci （Weinh）， 2023， 10（32）：e2304487.
[16]	WEI F L， WANG T F， WANG C L， et al. Cytoplasmic escape of mitochondrial DNA mediated by Mfn2 downregulation promotes microglial activation via cGas-sting axis in spinal cord injury［J］. Adv Sci （Weinh）， 2024， 11（4）：e2305442.
[17]	HSU C C， WU K L H， PENG J M， et al. Low-energy extracorporeal shockwave therapy improves locomotor functions， tissue regeneration， and modulating the inflammation induced FGF1 and FGF2 signaling to protect damaged tissue in spinal cord injury of rat model： an experimental animal study［J］. Int J Surg， 2024， 110（12）：7563-7572.
[18]	SHI Y L， YI C L， LI X， et al. Overexpression of Mitofusin2 decreased the reactive astrocytes proliferation in vitro induced by oxygen-glucose deprivation/ reoxygenation［J］. Neurosci Lett， 2017， 639：68-73.
[19]	LIU T， XUE C C， SHI Y L， et al. Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro ［J］. Neurosci Lett， 2014， 579：24-29.
[20]	LI Q， BU Y L， SHAO H F， et al. Protective effect of bone marrow mesenchymal stem cell-derived exosomes on cardiomyoblast hypoxia-reperfusion injury through the HAND2-AS1/miR-17-5p/Mfn2 axis［J］. BMC Cardiovasc Disord， 2023， 23（1）：114.
[21]	李聪，刘小慧，熊海容，等. 竹节参皂苷Ⅳa调节miR-17-5p/MFN2信号通路改善非酒精性脂肪性肝炎［J］. 中国中药杂志， 2020， 45（19）：4725-4731.