摘要
目的研究肾母细胞瘤过度表达蛋白(nephroblastoma overexpressed protein,CCN3)对转化生长因子-β1(TGF—β1)诱导的肾小球系膜细胞胞外基质(ECM)合成的影响及微小RNA-29(miRNA-29)在其中的作用机制。方法体外培养人肾小球系膜细胞(HMC),观察外源给予不同浓度(5、50、500μg/L)重组CCN3蛋白或内源转染pcDNA-3.1(+)-CCN3质粒对TGF-β1诱导的ECM和miRNA-29家族(a、b、c)表达的影响。HMC转染miRNA-29a抑制物和类似物,探讨miRNA-29a在CCN3调节TGF-β1诱导的ECM表达中的作用。实时荧光定量PCR检测各组纤连蛋白(FN)、I型胶原蛋白(COLI)的mRNA及miRNA-29家族(a、b、c)的表达,Western印迹检测各组FN、COLI的蛋白表达,细胞免疫荧光检测各组FN、COLI的表达。结果(1)与正常对照组比较,TGF-β1干预后HMC的FN、COLI蛋白及mRNA表达均增加,miRNA.29a、b、c的表达均降低(均P〈0.05)。(2)不同浓度CCN3重组蛋白干扰和转染CCN3质粒后TGF-β1诱导的FN、COLI蛋白及mRNA的表达均低于TGF-β1组(均P〈0.05);而50、500μg,LCCN3重组蛋白干预组和转染CCN3质粒组miRNA-29a的表达较TGF.Bl组增加(均P〈0.05),其余miRNA-29家族表达与TGF-β1组比较差异无统计学意义(均P〉0.05)。(3)与TGF-β1+重组CCN3蛋白组比较,TGF-β1+重组CCN3蛋白+miRNA-29a类似物转染组FN及COLI的表达减少(均P〈0.05),而TGF-β1+重组CCN3蛋白+miRNA-29a抑制物转染组FN及COLI的表达均增加(均P〈0.05)。结论CCN3可通过上调miRNA-29a抑制TGF-β1诱导的HMC合成ECM。
Objective To investigate the effects of nephroblastoma overexpressed protein (CCN3) on the formation of extracellular matrix (ECM) induced by transforming growth factor -β1 (TGF-β1) in human mesangial cells (HMCs) and its underlying signal transduction mechanism related with microRNA- 29(miRNA- 29). Methods HMCs were pretreated with different doses of exogenous CCN3 (5 μg/L, 50 μg/L and 500 μg/L) or transfected with pcDNA3.1(+)-CCN3 before exposed to TGF- β1(2 μg/L), to observe the expression of fibronectin (FN), type I collagen (COL I ) and miRNA-29a, b and c. The mimics or inhibitor of the miRNA- 29a were transfected into HMCs to analyze whether miRNA-29a affect CCN3. The expressions of FN mRNA, COL I mRNA and miRNA-29 family were detected by real time PCR. The protein expressions of FN and COL I were detected by Western blotting and cell immunofluorescenee. Results (1) Compared with the normal control group, the expressions of FN and COL I were up-regulated in TGF-β1 group, while the expressions of miRNA- 29a, b, e were down-regulated in TGF-β1 group (all P 〈 0.05). (2) Compared with the TGF-β1 group, the expressions of FN and COL | were decreased when pretreated with the different doses of exogenous of CCN3 or transfeeted with pcDNA3.1(+)-CCN3 (all P 〈 0.05). Meanwhile, the expression of miRNA- 29a was significantly increased when pretreated with 50 txg/L and 500 μg/L CCN3 or transfected with peDNA3.1(+)-CCN3 (all P 〈 0.05); whereas miRNA-29b and c had no statistical difference (all P 〉 0.05). (3) Compared with TGF-β1+CCN3 group, the expressions of FN and COL I were decreased in CCN3+TGF-β1+miRNA-29a mimics group (all P 〈 0.05), whereas the expressions of FN and COL I in CCN3+TGF-β1+miRNA-29a inhibitors group were increased (all P 〈 0.05). Conclusions CCN3 reduces the TGF-β1-induced production of ECM by the up-regulation of miRNA-29a.
出处
《中华肾脏病杂志》
CSCD
北大核心
2017年第7期531-537,共7页
Chinese Journal of Nephrology
基金
国家自然科学基金青年科学基金(81300596)
江苏省自然科学基金(BK20161437)
江苏省第五期“333高层次人才培养工程”第三层次培养对象项目