miR-150-5p在糖尿病肾病模型小鼠肾组织中的表达和对小鼠足细胞MPC5损伤的影响及其机制

doi:10.13481/j.1671-587X.20220106

摘要/Abstract

摘要： 目的

探讨微小RNA-150-5p（miR-150-5p）在糖尿病肾病（DN）模型小鼠肾组织中的表达和对高糖诱导的小鼠足细胞MPC5损伤的作用，并阐明其作用机制。

方法

20只昆明小鼠随机分为对照组（n=8，正常饲养）和DN组［n=12，高脂饮食联合腹腔注射链脲佐菌素（STZ）构建DN模型］，采用实时荧光定量PCR（RT-qPCR）法检测2组小鼠肾组织中miR-150-5p表达水平，采用免疫组织化学法和免疫荧光法观察2组小鼠肾组织中脑酸溶性蛋白1（BASP1）表达情况。采用不含干扰素-γ（IFN-γ）的培养基培养MPC5细胞2周，使其分化成熟，分为对照组和高糖处理（HG）组，HG组采用30 mmol·L^－1D-葡萄糖分别处理MPC5细胞12、24、48和72 h，采用RT-qPCR法和Western boltting法分别检测MPC5细胞中miR-150-5p和BASP1 mRNA及蛋白表达水平。采用生物信息学方法和双荧光素酶报告基因实验评估miR-150-5p与BASP1是否具有靶向关系。将miR-阴性对照（NC）、miR-150-5p模拟物（mimic）、miR-150-5p mimic+阴性对照质粒（pc-DNA3.1-SC）和miR-150-5p mimic+BASP1过表达质粒（pc-DNA3.1-BASP1）分别转入已分化成熟的MPC5细胞，分为正常条件+miR-NC（Con+NC）组、HG条件+miR-NC（HG+NC）组、HG条件+miR-150-5p mimic（HG+mimic）组、HG条件+miR-150-5p mimic+pc-DNA3.1-SC（HG+mimic+SC）组和HG条件+miR-150-5p mimic+pc-DNA3.1-BASP1（HG+mimic+BASP1）组；采用CCK-8法检测各组MPC5细胞活性，采用酶联免疫吸附测定（ELISA）法检测各组MPC5细胞中活性氧（ROS）水平和乳酸脱氢酶（LDH）活性，采用流式细胞术检测各组MPC5细胞凋亡率。

结果

与对照组比较，DN组小鼠肾组织中miR-150-5p表达水平明显降低（P<0.01），BASP1蛋白表达量明显增加，且BASP1与足细胞存在共定位。与对照组比较，HG组MPC5细胞中BASP1蛋白表达水平明显升高（P<0.01），miR-150-5p表达水平明显降低（P<0.05或P<0.01）。BASP1是miR-150-5p的直接靶点。与Con+NC组比较，HG+NC组MPC5细胞活性明显降低（P<0.01），ROS水平、LDH活性和细胞凋亡率均明显升高（P<0.01）；与HG+NC组比较，HG+mimic组和HG+mimic+SC组MPC5细胞活性明显升高（P<0.01），ROS水平、LDH活性和细胞凋亡率均明显降低（P<0.01）；与HG+mimic+SC组比较，HG+mimic+BASP1组MPC5细胞活性明显降低（P<0.01），ROS水平、LDH活性和细胞凋亡率均明显升高（P<0.01）。

结论

miR-150-5p表达降低可能是DN模型中足细胞损伤的机制之一。过表达miR-150-5p可通过靶向BASP1抑制HG诱导的足细胞损伤。

关键词: 糖尿病肾病, 足细胞损伤, miR-150-5p, 脑酸溶性蛋白1, 细胞凋亡

Abstract: Objective

To investigate the expression of miR-150-5p in kidney tissue of the diabetic nephropathy （DN） model mice and the effect of miR-150-5p on the MPC5 mouse podocyte injury induced by high glucose， and to elucidate its mechanism.

Methods

Twenty Kunming mice were randomly divided into control group （n=8， normal feeding） and DN group （n=12， high fat diet feeding combined with intraperitoneal injection of streptozotocin to establish the DN models）.The expression levels of miR-150-5p in kidney tissue of the mice in two groups were detected by real-time fluorescence quantitative PCR （RT-qPCR）， and the expressions of brain acid soluble protein 1 （BASP1） in kidney tissue of the mice in two groups were observed by immunohistochemical and immunofluorescence methods.The MPC5 cells were cultured in medium without interferon-γ（IFN-γ） for 2 weeks to differentiate and mature， then the cells were divided into control group and high glucose treatment （HG） group. The cells in HG group were treated with 30 mmol·L^－1 D-glucose for 12， 24， 48 and 72 h， respectively. The expression levels of miR-150-5p and BASP1 mRNA and protein in the MPC5 cells were detected by RT-qPCR and Western blotting methods， respectively. Bioinformatics and luciferase reporter assays were used to evaluate whether miR-150-5p had a targeting relationship with BASP1.The miR-NC， miR-150-5p mimic， miR-150-5p mimic+pc-DNA3.1-SC and miR-150-5p mimic+pc-DNA3.1-BASP1 were transformed into the mature MPC5 cells， respectively， and they were divided into normal condition+miR-NC （Con+NC） group， HG condition+miR-NC（HG+NC） group， HG condition+miR-150-5p mimic （HG+mimic）group，and HG condition +miR-150-5p mimic+pc-DNA3.1-SC（HG+ mimic+SC） group and HG condition +miR-150-5p mimic+pc-DNA3.1-BASP1 （HG+ mimic+BASP1） group. The viabilities of MPC5 cells in various groups were detected by CCK-8 assay； the reactive oxygen species （ROS） levels and lactate dehydrogenase （LDH） activities of MPC5 cells in various groups were detected by ELISA method， and the apoptotic rates of MPC5 cells in various groups were detected by flow cytometry.

Results

Compared with control group， the expression level of miR-150-5p in kidney tissue of the mice in DN group was decreased （P<0.01）， while the expression amount of BASP1 protein was increased， and BASP1 was co-localized with the podocytes. Compared with control group， the expression levels of BASP1 protein in the MPC5 cells in HG group were increased （P<0.01），and the expression levels of miR-150-5p were decreased （P<0.05 or P<0.01） in a time dependent manner. BASP1 was a direct target of miR-150-5p. Compared with Con+NC group， the viability of MPC5 cells in HG+NC group was significantly decreased （P<0.01）， while the ROS level， LDH activity and apoptotic rate were significantly increased （P<0.01）. Compared with HG+NC group， the viabilities of MPC5 cells in HG+mimic and HG+mimic+SC groups were significantly increased （P<0.01）， while the ROS levels， LDH activities and apoptotic rates were significantly decreased （P<0.01）.Compared with HG+mimic+SC group， the viability of MPC5 cells in HG+mimic+BASP1 group was significantly decreased （P<0.01）， while the ROS level， LDH activity and apoptotic rate were significantly increased （P<0.01）.

Conclusion

The decreased expression of miR-150-5p may be one of the mechanisms of podocyte injury in the DN models. Over-expression of miR-150-5p may inhibit the HG-induced podocyte injury by targeting BASP1.

Key words: Diabetic nephropathy, Podocyte injury, MiR-150-5p, Brain acid soluble protein 1, Apoptosis

中图分类号:

R587.1

朱妤,王晶晶,吴芳. miR-150-5p在糖尿病肾病模型小鼠肾组织中的表达和对小鼠足细胞MPC5损伤的影响及其机制[J]. 吉林大学学报(医学版), 2022, 48(1): 44-51.

Yu ZHU,Jingjing WANG,Fang WU. Expression of miR-150-5p in kidney tissue of diabetic nephropathy model mice and its effect on MPC5 mouse podocyte injury and mechanism[J]. Journal of Jilin University(Medicine Edition), 2022, 48(1): 44-51.

图/表 7

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Group	miR-150-5p expression level
Control	100.76±8.25
HG (t/h)
12	83.65±3.78^*
24	71.93±6.84^**
48	49.29±9.88^**
72	25.63±3.02^**

Group	Cell viability	ROS level	LDH activity	Apoptotic rate
Con+NC	101.55±3.16	101.23±18.33	99.43±9.35	2.78±1.73
HG+NC	51.35±7.93^*	814.85±26.81^*	512.89±11.42^*	37.98±3.34^*
HG+mimic	81.14±2.82^△	295.23±21.74^△	208.82±16.59^△	14.73±1.59^△
HG+mimic+SC	83.53±4.19^△	309.00±25.69^△	212.96±18.83^△	15.25±2.03^△
HG+mimic+BASP1	49.13±8.38^#	679.83±22.63^#	469.35±20.18^#	32.59±2.84^#

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