摘要
目的探讨circ_WBSCR17通过调节miR-30a-5p/JAK1轴对高糖诱导的人肾小球系膜细胞纤维化和炎症的影响。方法将人肾小球系膜细胞HMCL分为NG组(5.5 mmol/L葡萄糖处理HMCL细胞)、HG组(30 mmol/L葡萄糖处理细胞)、si-NC组(30 mmol/L葡萄糖+转染si-NC)、si-circ_WBSCR17组(30 mmol/L葡萄糖+转染si-circ_WBSCR17)、si-circ_WBSCR17+inhibitor-NC组(30 mmol/L葡萄糖+si-circ_WBSCR17和inhibitor-NC共转染)、si-circ_WBSCR17+miR-30a-5p inhibitor组(30 mmol/L葡萄糖+si-circ_WBSCR17和miR-30a-5p inhibitor共转染);RT-qPCR检测细胞中circ_WBSCR17、miR-30a-5p的表达;CCK-8法检测细胞增殖;流式细胞仪检测细胞凋亡;ELISA法检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-8表达水平;Western blot检测细胞中JAK1、增殖细胞核抗原(PCNA)、Bax、转化生长因子-β1(TGF-β1)、纤维连接蛋白(FN)、Ⅳ型胶原蛋白(collagenⅣ)、α-平滑肌肌动蛋白(α-SMA)的表达;荧光原位杂交(FISH)实验检测circ_WBSCR17的分布情况,双荧光素酶报告基因实验分别验证circ_WBSCR17和miR-30a-5p、JAK1的关系。结果与NG组比较,HG组HMCL细胞增殖能力降低,TNF-α、IL-6和IL-8水平、p-JAK1/JAK1、p-STAT1/STAT1、p-STAT3/STAT3、Bax、TGF-β1、FN、collagenIV、α-SMA蛋白表达升高(P<0.05);与HG组和si-NC组比较,si-circ_WBSCR17组HMCL细胞中miR-30a-5p表达、OD450值、PCNA表达升高,TNF-α、IL-6和IL-8水平、circ_WBSCR17、p-JAK1/JAK1、p-STAT1/STAT1、p-STAT3/STAT3、Bax、TGF-β1、FN、collagenIV、α-SMA表达降低(P<0.05);抑制miR-30a-5p减弱了敲低circ_WBSCR17对HMCL细胞增殖的促进作用,增强了细胞凋亡、细胞纤维化和炎症反应;FISH实验证实circ_WBSCR17主要分布在细胞质中;双荧光素酶报告基因实验证实circ_WBSCR17、JAK1与miR-30a-5p存在靶向调控关系。结论敲低circ_WBSCR17可通过调节miR-30a-5p/JAK1轴,进而减轻高糖诱导的人肾小球系膜细胞纤维化和炎症。
Objective To investigate the influences of circ_WBSCR17 on high glucose-induced fibrosis and inflammation in human mesangial cells by regulating the miR-30a-5p/JAK1 axis.Methods Human mesangial cells HMCL were grouped into:NG group(5.5 mmol/L glucose-treated HMCL cells),HG group(30 mmol/L glucose-treated cells),si-NC group(30 mmol/L glucose+transfected with si-NC),si-circ_WBSCR17 group(30 mmol/L glucose+transfected with si-circ_WBSCR17),si-circ_WBSCR17+inhibitor-NC group(30 mmol/L glucose+co-transfected with si-circ_WBSCR17 and inhibitor-NC),and si-circ_WBSCR17+miR-30a-5p inhibitor group(30 mmol/L glucose+co-transfected with si-circ_WBSCR17 and miR-30a-5p inhibitor);RT-qPCR was performed to detect the expression of circ_WBSCR17 and miR-30a-5p in cells;CCK-8 assay was performed to detect cell proliferation;flow cytometry was performed to detect apoptosis;ELISA was performed to detect the expression levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 and IL-8;Western blot was performed to detect the expression of JAK1,proliferating cell nuclear antigen(PCNA),Bax,transforming growth factor-β1(TGF-β1),fibronectin(FN),collagen IV,andα-smooth muscle actin(α-SMA);distribution of WBSCR17 was detected by fluorescence in situ hybridization(FISH);dual-luciferase reporter gene experiment was performed to verify the relationship between circ_WBSCR17 and miR-30a-5p,miR-30a-5p and JAK1,respectively.Results Compared with the NG group,the HMCL cell proliferation ability of the HG group decreased,the levels of TNF-α,IL-6 and IL-8,the protein expressions of p-JAK1/JAK1,p-STAT1/STAT1,p-STAT3/STAT3,TGF-β1,FN,collagenIV andα-SMA,and the apoptosis ability increased(P<0.05);compared with HG group and si-NC group,the expression of miR-30a-5p,OD 450 value and PCNA expression in HMCL cells of si-circ_WBSCR17 group increased,the levels of TNF-α,IL-6 and IL-8,the expressions of circ_WBSCR17,p-JAK1/JAK1,p-STAT1/STAT1,p-STAT3/STAT3,Bax,TGF-β1,FN,collagenIV andα-SMA decreased(P<0.05);inhibition of miR-30a-5p attenuated the promoting effect of knockdown of circ_WBSCR17 on proliferation of HMCL cells,and enhanced apoptosis,cellular fibrosis and inflammatory responses;FISH experiment confirmed that WBSCR17 was mainly distributed in the cytoplasm;dual-luciferase reporter gene experiment confirmed that circ_WBSCR17,JAK1 and miR-30a-5p had a targeted regulatory relationship.Conclusion Knockdown of circ_WBSCR17 can reduce high glucose-induced fibrosis and inflammation in human mesangial cells by regulating the miR-30a-5p/JAK1 axis.
作者
董海芸
韩芳
齐一舟
梅峰
Dong Haiyun;Han Fang;Qi Yizhou;Mei Feng(Dept of Nephrology,Qinghai University Affiliated Hospital,Xining 810000;Oral Medicine,Haiyuan College of Kunming Medical University,Kunming 650106)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第10期1756-1762,1768,共8页
Acta Universitatis Medicinalis Anhui
基金
青海省科技项目(编号:2016-ZJ-709)。
