CCN3通过上调微小RNA-29a表达抑制肾小球系膜细胞胞外基质的合成

CCN3 inhibits deposition of extracellular matrix of glomerular mesangial cells via promoting the expression of microRNA- 29a

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.