miR-520c-3p对糖尿病肾脏病患者近端肾小管上皮细胞焦亡的影响及机制研究

Effect of miR-520c-3p on pyroptosis of proximal tubular epithelial cells in diabetic kidney disease and its mechanism

目的探讨miR-520c-3p对糖尿病肾脏病患者近端肾小管上皮细胞焦亡的影响及其分子机制。方法收集2019年6月至2020年12月在郑州大学第一附属医院就诊的糖尿病肾脏病患者(DKD组,4例)和糖耐量正常肾脏肿瘤手术者(正常对照组,4例)的肾脏组织。HE染色、过碘酸-希夫染色(PAS)和透射电镜观察肾脏组织病理学变化。取生长状态良好的人近端肾小管上皮(HK-2)细胞传代至培养板内,并分成如下8组:正常葡萄糖组(NG,5.6 mmol/L葡萄糖)、高糖组(HG,30.0 mmol/L葡萄糖)、抑制物组(正常葡萄糖+转染miR-520c-3p抑制物)、抑制物对照组(正常葡萄糖+转染miR-520c-3p抑制物阴性对照物)、模拟物组(高糖+转染miR-520c-3p模拟物)、模拟物对照组(高糖+转染miR-520c-3p模拟物阴性对照物)、模拟物+TXNIP共转染组(高糖+共转染miR-520c-3p模拟物+TXNIP)、共转染阴性对照组(高糖+共转染miR-520c-3p模拟物+TXNIP阴性对照物)。活细胞动态实时监测细胞活性,碘化丙啶(PI)染色检测细胞焦亡情况;免疫荧光染色、荧光原位杂交、实时定量PCR和Western blotting法检测miR-520c-3p、硫氧还蛋白相互作用蛋白(TXNIP)和细胞焦亡分子NOD样受体蛋白3(NLRP3)、活化半胱氨酸蛋白酶-1(cl-caspase-1)、消皮素D(GSDMD)的mRNA和蛋白表达水平;酶联免疫吸附测定法(ELISA)检测细胞上清液白细胞介素-1β(IL-1β)含量;双荧光素酶报告基因实验明确miR-520c-3p和TXNIP之间的相互作用。两组间比较采用t检验,多组间比较采用单因素方差分析。结果与对照组相比,DKD组肾脏组织肾小管炎性细胞增多,伴随miR-520c-3p表达水平下降,TXNIP表达升高(均P<0.05);肾小管特异性标志物水通道蛋白1(AQP1)与NLRP3和GSDMD焦亡蛋白共定位均增加。在HK-2细胞中,与NG组相比,HG组焦亡细胞增多,伴随miR-520c-3p表达水平下降,TXNIP、NLRP3、cl-caspase-1、GSDMD的蛋白水平和细胞上清液IL-1β浓度升高(均P<0.05);与模拟物对照组相比,模拟物组中miR-520c-3p表达量更高,TXNIP mRNA表达量更低,TXNIP、NLRP3、cl-caspase-1、GSDMD的蛋白水平和IL-1β浓度更低(均P<0.01),TXNIP和NLRP3共表达定位减少(P<0.05);与抑制物对照组相比,抑制物组中miR-520c-3p表达量更低,TXNIP mRNA表达量更高,TXNIP、NLRP3、cl-caspase-1、GSDMD的蛋白水平和IL-1β浓度更高(均P<0.01)。与共转染阴性对照组相比,模拟物+TXNIP共转染组的TXNIP和NLRP3共表达定位增加,TXNIP、NLRP3、cl-caspase-1、GSDMD的蛋白水平更高(P<0.05)。双荧光素酶报告基因实验表明,TXNIP是miR-520c-3p直接作用靶点,miR-520c-3p通过靶向调节TXNIP表达,抑制高糖诱导的细胞焦亡。结论miR-520c-3p通过抑制TXNIP/NLRP3途径减轻高糖诱导的近端肾小管上皮细胞焦亡。

Objective To investigate the protective effect of miR-520c-3p on cell pyroptosis of proximal tubular epithelial cell(PTEC)in diabetic kidney disease(DKD)and its molecular mechanism.Methods Human kidney specimens from patients with DKD(DKD group,n=4)were obtained from the First Affiliated Hospital of Zhengzhou University from June 2019 to December 2020,and the kidney specimens from unaffected portions of the kidney tumor with normal glucose tolerance were served as normal controls(NC group,n=4).Renal histopathological changes were examined by hematoxylin and eosin(HE)staining,periodate-Schiff staining(PAS)and transmission electron microscope(TEM).PTEC(HK-2 cell line)were cultured with normal glucose(NG group,5.6 mmol/L glucose)and high glucose(HG group,30.0 mmol/L glucose),and transfected with miR-520c-3p mimics,miR-520c-3p inhibitor,miR-520c-3p negative control(NC)plasmid,thioredoxin-interacting protein(TXNIP)plasmid and TXNIP negative control(NC)plasmid.Cell pyroptosis was determined by dynamic real-time monitoring of living cells and propidium iodide(PI)staining.The expression of miR-520c-3p,TXNIP,NOD-like receptor thermal protein domain associated protein 3(NLRP3),cleaved caspase-1,Gasdermin D(GSDMD)were detected by immunofluorescence staining,fluorescence in situ hybridization(FISH),real time-PCR and Western blotting.The IL-1βconcentration in cell supernatant was detected by enzyme-linked immunosorbent assay(ELISA)kit.Interaction between miR-520c-3p and TXNIP was analyzed by dual luciferase activity assay.Data were presented as the mean±standard deviation(SD),and the significance was determined using unpaired Student′s t-test for two groups and one-way analysis of variance(ANOVA)for multiple groups.Results Compared with the NC group,inflammatory cells were increased in the DKD group,accompanied with reduction of miR-520c-3p expression level and increased of TXNIP level(P<0.05).The co-localization between renal tubule specific marker aquaporin(AQP1)and NLRP3 and that between AQP1 and GSDMD were both increased.In HK-2 cells,compared with NG group,cell pyroptosis was increased in HG group,accompanied with reduction of miR-520c-3p level and increases of TXNIP,NLRP3,cl-caspase-1 and GSDMD levels,as well as IL-1βconcentration in cell supernatant(P<0.05).Compared with miR-520c-3p mimics NC plasmid,overexpression of miR-520c-3p inhibited cell pyroptosis,decreased the level of TXNIP mRNA,TXNIP,NLRP3,cleaved caspase-1,GSDMD and IL-1βprotein levels induced by high glucose(P<0.01),and reduced the co-localization between TXNIP and NLRP3(P<0.05).However,compared with miR-520c-3p inhibitor NC plasmid,inhibition of miR-520c-3p promoted cell pyroptosis and increased TXNIP mRNA level as well as TXNIP,NLRP3,cleaved caspase-1,GSDMD protein levels and IL-1βconcentration(P<0.01).Additionally when compared with TXNIP NC plasmid,overexpression of TXNIP abolished the inhibitory effects on TXNIP,NLRP3,cleaved caspase-1 and GSDMD by miR-520c-3p mimics and increased the co-localization between TXNIP and NLRP3(P<0.05).Dual luciferase reporter assay indicated that TXNIP was the direct target of miR-520c-3p.Meanwhile,miR-520c-3p inhibited cell pyroptosis induced by high glucose through regulating TXNIP expression.Conclusion miR-520c-3p alleviated PTEC pyroptosis induced by high glucose through inhibition of the TXNIP/NLRP3 pathway.