CCN3 inhibits deposition of extracellular matrix of glomerular mesangial cells via promoting the expression of microRNA-29a / 中华肾脏病杂志

ABSTRACT

Objective To investigate the effects of nephroblastoma over-expressed 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 immunofluorescence.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,c were down-regulated in TGF-β1 group (all P ＜ 0.05).(2) Compared with the TGF-β1 group,the expressions of FN and COL I were decreased when pretreated with the different doses of exogenous of CCN3 or transfected with pcDNA3.1(+)-CCN3 (all P ＜ 0.05).Meanwhile,the expression of miRNA-29a was significantly increased when pretreated with 50 μg/L and 500 μg/L CCN3 or transfected with pcDNA3.1(+)-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.