RESUMEN
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.
RESUMEN
Objective To investigate the effects of nephroblastoma overexpressed (NOV) on proliferation,adhesion,migration and invasion of remal cell curcinomai (RCC) cells. Methods We constructed a NOV expression plasmid and transfected the plasmid into RCC cell line 786-O and analyzed the effects of NOV expression on proliferation,adhesion,migration and invasion of RCC cells by growth curve assay,WST-1 assay,cell adhesion assay,matrigel invasion assay and transwell migration assay. Results The stable NOV transfected 786-O cells (786-O-NOV) showed decreased growth rate,at 48 h and 72 h,the proliferation activities of 786-O-NOV cells were inhibited by 29.14％ and 32.46％ the proliferation activities of empty vector cells were inhibited by 9.25％ and - 8.16％,respectively,compared to 786-O cells (P ＜0.05); while the 786-O cells transfected with empty vector (786-O-mock) had no difference with 786-Ocells.Adhesion assay indicated significantly increased adhesion of 786-O-NOV cells to fibronectin (0.26 ±0.03) and laminin (0.28 ±0.04),compared to 786-O cells (0.15 ±0.01,0.12±0.10) and 786-O-mock cells (0.14 ±0.02,0.13 ± 0.08).Invasion assay displayed that the numbers of cells penetrated through matrigel membrane were significantly higher in 786-O-NOV cells (240.25 ± 23.12) compared to 786-O cells ( 56.16 ± 6.25 ) and 786-O-mock cells ( 50.28 ± 7.13 ).Migration assay displayed that the numbers of cells passed through polycarbonate filters were significantly higher in 786-O-NOV cells (267.25 ± 20.94) compared to 786-O cells ( 66.10 ± 5.68 ) and 786-O-mock cells ( 56.28 ± 4.11 ).Conclusion NOV exhibits anti-proliferative effects on RCC cells; however,it promotes adhesion,migration and invasion of RCC cells.