微小RNA let-7c通过抑制血小板源性生长因子受体表达促进小鼠血管平滑肌细胞衰老  

MicroRNA let-7c promotes senescence of mouse vascular smooth muscle cells by inhibiting expression of platelet derived growth factor

作　　者：崔星钢 余逸杰 姜瑜 李波[1] 赵倩茹 朱灿宏[2] 仲威[1] 卞石惠 Cui Xinggang;Yu Yijie;Jiang Yu;Li Bo;Zhao Qianru;Zhu Canhong;Zhong Wei;Bian Shihui(Department of Cardiology,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,Jiangsu Province,China)

机构地区：[1]江苏大学附属医院心内科,镇江212001 [2]江苏大学附属人民医院老年科

出　　处：《中华老年心脑血管病杂志》2023年第6期629-633,共5页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：国家自然科学基金(82000261);江苏省高等学校自然科学研究项目(20KJB320013);江苏大学医教协同创新基金项目(JDY2022007);镇江市社会发展项目(SH2019071);江苏省老年健康科研项目(LD2021039)。

摘　　要：目的探讨微小RNA(miRNA)let-7c参与小鼠血管平滑肌细胞(VSMC)衰老的机制。方法选择C57BL/6J小鼠40只,将小鼠分为对照组、老年组各20只,通过qPCR技术检测小鼠血清及主动脉血管中let-7c miRNA表达水平。从C57BL/6J小鼠主动脉中提取并原代培养VSMC。通过博来霉素体外诱导细胞衰老模型。使用Lipo2000脂质体转染法过表达或抑制细胞let-7c miRNA表达。将VSMC分为阴性对照(NC)组、博来霉素组、博来霉素+let-7c过表达组、博来霉素+let-7c抑制组,通过衰老相关β半乳糖苷酶(SA-β-gal)染色评估组织或细胞衰老水平。将VSMC分为控制组、血小板衍生生长因子(PDGF)组、PDGF+let-7c过表达组,采用Western blot检测各组细胞蛋白表达。将VSMC分为空白组、let-7c过表达组、突变组、突变+let-7c过表达组,使用双荧光素酶报告基因验证let-7c靶点。结果老年组小鼠主动脉和血清中let-7c miRNA相对表达水平显著高于对照组(6.37±0.81 vs 1.00±0.00,P<0.05;5.23±0.44 vs 1.00±0.00,P<0.05)。博来霉素+let-7c过表达组中SA-β-gal染色阳性比例较博来霉素组增多,而博来霉素+let-7c抑制组中SA-β-gal染色阳性比例较博来霉素组明显减少(P<0.05)。与PDGF+let-7c过表达组比较,PDGF组血清反应因子表达显著升高,α平滑肌肌动蛋白及平滑肌肌球蛋白重链表达显著降低,差异有统计学意义(P<0.05)。双荧光素酶实验显示,let-7c过表达组中荧光素酶活性水平较空白组显著下降(0.43±0.05 vs 1.00±0.00,P<0.05)。结论miRNA let-7c通过抑制PDGF受体表达促进小鼠VSMC衰老。Objective To investigate the mechanism of microRNA(miRNA)let-7c involved in the senescence of mouse vascular smooth muscle cells(VSMC).Methods Forty C57BL/6J mice were equally divided into control group and elderly group.The expression level of miRNA let-7c in the serum and aorta of mice was detected by qPCR.VSMC were isolated from C57BL/6J mouse aorta and then cultured primarily.Cell aging model was induced by bleomycin treatment.Lipo2000 liposome transfection was used to overexpress or inhibit the expression of miRNA let-7c.Then VSMC were divided into negative control(NC)group,bleomycin group,bleomycin+let-7c overexpression group and bleomycin+let-7c inhibition group.β Galactosidase(SA-β-Gal)staining was used to evaluate the aging level of tissues or cells.VSMC was divided into control group,PDGF group and PDGF+let-7c overexpression group.The expression of related proteins in each group was detected by Western blotting.VSMC was divided into blank group,let-7c overexpression group,mutation group,and mutation+let-7c overexpression group,and the let-7c target was verified with dual luciferase reporter system.Results The miRNA let-7c levels in the serum and aorta were significantly higher in the aging mice than the control group(6.37±0.81 vs 1.00±0.00,5.23±0.44 vs 1.00±0.00,P<0.05).The number of SA-β-Gal positive cells was obviously larger in the bleomycin+let-7c overexpression group than the bleomycin group,but smaller in the bleomycin+let-7 inhibition group when compared with the bleomycin group(P<0.05).Compared with the PDGF+let-7c overexpression group,the expression level of SRF was increased notably,and those ofα-SMA and SM-MHC were decreased in the PDGF group(P<0.05).Dual luciferase assay showed that the luciferase activity in the let-7c overexpression group was significantly lower than that in the blank group(0.43±0.05 vs 1.00±0.00,P<0.05).Conclusion MiRNA let-7c promotes VSMC senescence by inhibiting PDGFR expression.

关 键 词：循环微RNA 血小板源性生长因子 肌细胞 平滑肌 

分 类 号：R543[医药卫生—心血管疾病]