首页|LncRNA MEG3调控糖尿病肾病足细胞损伤的机制研究

LncRNA MEG3调控糖尿病肾病足细胞损伤的机制研究

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目的 探讨长链非编码RNA母系表达基因3(LncRNA MEG3)调控糖尿病肾病足细胞损伤的机制.方法 采用葡萄糖诱导小鼠肾足细胞(MPC5)构建足细胞损伤模型.将细胞分为高糖组、高糖+短发夹RNA的阴性对照(sh-NC)组、高糖+干扰LncRNA MEG3表达的短发夹RNA(sh-LncRNA MEG3)组、高糖+sh-LncRNA MEG3+3-甲基腺嘌呤(3-MA)组、高糖+sh-LncRNA MEG3+干扰结节性硬化症复合体1表达的短发夹RNA(sh-TSC1)组和对照组.采用四甲基偶氮唑盐法检测MPC5活性,采用流式细胞术检测MPC5凋亡率,采用实时荧光定量PCR检测LncRNA MEG3相对表达水平,采用Western blot检测自噬相关蛋白[重组人自噬效应蛋白(Beclin-1)、结节性硬化症复合体1(TSC1)、选择性自噬接头蛋白(p62)和微管相关蛋白轻链3(LC3)]表达,采用荧光原位杂交技术检测LncRNA MEG3的表达定位.结果 高糖组MPC5活性低于对照组,高糖+sh-NC组MPC5 活性低于高糖+sh-LncRNA MEG3组,高糖+sh-LncRNA MEG3+sh-TSC1 组、高糖+sh-LncRNA MEG3+3-MA组 MPC5活性低于高糖+sh-LncRNA MEG3组(均P<0.01).高糖组MPC5凋亡率高于对照组,高糖+sh-NC组MPC5凋亡率高于高糖+sh-LncRNA MEG3 组,高糖+sh-LncRNA MEG3+sh-TSC1 组、高糖+sh-LncRNA MEG3+3-MA 组 MPC5 凋亡率高于高糖+sh-LncRNA MEG3组(均P<0.01).高糖组MPC5的LncRNA MEG3相对表达水平高于对照组,高糖+sh-NC组MPC5的LncRNA MEG3相对表达水平高于高糖+sh-LncRNA MEG3组,差异均有统计学意义(均P<0.01).高糖组LC3、Beclin-1、TSC1蛋白表达低于对照组,p62蛋白表达高于对照组,高糖+sh-LncRNA MEG3组LC3、Beclin-1、TSC1蛋白表达高于高糖+sh-NC组,p62蛋白表达低于高糖+sh-NC组,高糖+sh-LncRNA MEG3+sh-TSC1组TSC1蛋白表达低于高糖+sh-LncRNA MEG3组,高糖+sh-LncRNA MEG3+3-MA组、高糖+sh-LncRNA MEG3+sh-TSC1 组的 LC3、Beclin-1 蛋白表达低于高糖+sh-LncRNA MEG3组,p62蛋白表达高于高糖+sh-LncRNA MEG3组,差异均有统计学意义(均P<0.01).高糖+sh-LncRNA MEG3组TSC1蛋白表达高于高糖+sh-NC组,差异有统计学意义(P<0.01).LncRNA MEG3在细胞核和细胞质中均可表达.结论 LncRNA MEG3可通过靶向TSC1介导MPC5自噬,进而减轻糠尿病肾病足细胞损伤程度.
Mechanism of LncRNA MEG3 regulating podocyte injury in diabetic nephropathy
Objective To investigate the mechanism of long non-coding RNA maternally expressed gene 3(LncRNA MEG3)on podocyte injury in diabetic kidney disease(DKD).Methods Mouse podocyte clone-5(MPC5)cells were used to construct the glucose-induced podocyte injury model.The MPC5 cells were treated with high glucose,high glucose+short hairpin RNA-negative control(sh-NC)group,high glucose+sh-LncRNA MEG3 group,high glucose+sh-LncRNA MEG3+3-methyladenine(3-MA)group,high glucose+sh-LncRNA MEG3+sh-tuberous sclerosis complex 1(TSC1)group and control group.Cell viability and apoptosis rate were detected by MTT assay and flow cytometry.The relative expression level of LncRNA MEG3 was detected by real-time PCR.Western blot was used to detect autophagy-related proteins recombinant Beclin 1(Beclin-1),TSC1,selective autophagy joint protein(p62)and microtubule-associated protein light chain 3(LC3).Fluorescence in situ hybridization was used to detect the expression and localization of LncRNA MEG3 in cells.Results The MPC5 activity of the high glucose group was lower than that of the control group,the MPC5 activity of the high glucose+sh-NC group was lower than that of the high glucose+sh-LncRNA MEG3 group,the MPC5 activity of high glucose+sh-LncRNA MEG3+sh-TSC1 group and high glucose+sh-LncRNA MEG3+3-MA group was lower than that of high glucose+sh-LncRNA MEG3 group(all P<0.01).The apoptosis rate of MPC5 in the high glucose group was higher than that in the control group,the apoptosis rate of MPC5 in the high glucose+sh-NC group was higher than that in the high glucose+sh-LncRNA MEG3 group,the apoptosis rate of MPC5 in high glucose+sh-LncRNA MEG3+sh-TSC1 group and high glucose+sh-LncRNA MEG3+3-MA group was higher than that in high glucose+sh-LncRNA MEG3 group(all P<0.01).The relative expression level of LncRNA MEG3 in the high glucose group was higher than that in the control group,the relative expression level of LncRNA MEG3 in the high glucose+sh-NC group was higher than that in the high glucose+sh-LncRNA MEG3 group(all P<0.01).The level of LC3,Beclin-1 and TSC1 in the high glucose group were lower than those in the control group,the level of p62 were higher than those in the control group.The level of LC3,Beclin-1 and TSC1 in the high glucose+sh-NC group were higher than those in the high glucose+sh-NC group,the level of p62 were lower than those in the high glucose+sh-NC group.The level of TSC1 in high glucose+sh-LncRNA MEG3+sh-TSC1 group was lower than that in high glucose+sh-LncRNA MEG3 group.The level of LC3 and Beclin-1 in high glucose+sh-LncRNA MEG3+3-MA group and high glucose+sh-LncRNA MEG3+sh-TSC1 group were lower than those in high glucose+sh-LncRNA MEG3 group,the level of p62 was higher than that in high glucose+sh-LncRNA MEG3 group(all P<0.01).The level of TSCI in high glucose+sh-LncRNA MEG3 was higher than that in high glucose+sh-NC group(P<0.01).LncRNA MEG3 was expressed in both nucleus and cytoplasm.Conclusion The study indicates that LncRNA MEG3 alleviating podocyte damage in DKD may be mediated by MPC5 autophagy through targeting TSC1.

Diabetic kidney diseaseMouse podocyte clone-5Long non-coding RNA maternally expressed gene 3Tuberous sclerosis complex 1Autophagy

马翠、龚蜇强、高玉章、温秀梅

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311199 杭州市余杭区第一人民医院内分泌科

311199 杭州市余杭区第一人民医院消化内科

糖尿病肾病 小鼠肾足细胞 长链非编码RNA母系表达基因3 结节性硬化症复合体1 自噬

杭州市医药卫生科技计划项目浙江省医学会临床医学科研专项资金项目浙江省中医药科技计划项目

B202200422022ZYC-A1462023ZL612

2024

浙江医学
浙江省医学会

浙江医学

CSTPCD
影响因子:0.428
ISSN:1006-2785
年,卷(期):2024.46(16)