吉林大学学报（医学版）2026，Vol.52Issue(1)：81-92,12.DOI:10.13481/j.1671-587X.20260109
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
Inhibitory effect of inhibition of miR-17-5p on proliferation of spinal cord astrocytes of rats induced by scratch injury through targeting Mfn2 expression
摘要
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.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2Key words
Astrocyte/Scratch injury/Cell proliferation/MicroRNA-17-5p/Mitofusin 2分类
医药卫生引用本文复制引用
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):81-92,12.基金项目
湖北省科技厅自然科学基金项目(2023AFB994) （2023AFB994）
