Effects of miR-200c-3p on proliferation and apoptosis of nephroblastoma SK-NEP-1 cells / 中华实用儿科临床杂志

ABSTRACT

Objective:To investigate the effect of miR-200c-3p on the proliferation and apoptosis of nephroblastoma SK-NEP-1 cell and its mechanism.Methods:From January 2015 to August 2019, nephroblastoma tissue and peritumoral tissue of 30 patients in Shenzhen Children′s hospital were collected.The experimental group of mimic negative control, miR-200c-3p and miR-200c-3p inhibitor was set up.The expression of miR-200c-3p in 30 paired nephroblastoma tissues and adjacent kidney tissues were detected by real-time fluorescence quantitative PCR.The differential expression of miR-200c-3p was also detected in SK-NEP-1 and 293FT cell lines.The effects of miR-200c and miR-200c-3p inhibitor on the proliferation, cell cycle distribution and apoptosis of SK-NEP-1 cells were detected by cell counting kit-8(CCK-8)and flow cytometry assays, respectively.Xenograft tumors were generated by peri-renal adipose tissue injection to assess the effect of miR-200c-3p on tumor growth in vivo.The pathological morphology of xenograft tumors was observed by HE staining.The proliferation index of Ki-67 were detected by immunohistochemistry.Western blot method was used to detect the B-cell lymphoma 2(Bcl-2), Bcl-2 related X protein (Bax) and cleaved cysteinyl aspartate specific proteinase-3(Caspase-3)expression level on xenograft tumors. Results:The expression level of miR-200c-3p in nephroblastoma(0.420±0.587)was significantly lower than that in matched normal kidney(1.500±0.504)( t=8.613, P<0.001). The expression level of miR-200c-3p in SK-NEP-1 cells (0.363±0.006) was significantly lower than that in human embryonic kidney 293FT cells (0.807±0.186) ( t=4.136, P<0.05). The group and time interaction results of CCK-8 proved that miR-200c-3p could inhibit the proliferation of SK-NEP-1 cells( F=16.81, P<0.001). The flow cytometer test of cell cycle and apoptosis showed that miR-200c-3p could block in G0/G1 and S phase( t=-7.770, P<0.01; t=11.501, P<0.001). Moreover, it increased the early apoptosis rate and decreased the late apoptosis rate ( t=-22.270, P<0.001; t=4.612, P<0.01). A orthotopic transplantation assay was employed to evaluate the effect of miR-200c-3p and miR-200c-3p inhibitor on the proliferation of SK-NEP-1 cells.The final volume of tumor miR-200c-3p group [(0.419±0.16) cm 3]was significantly lower than those in the control group [(2.469±0.914) cm 3, t=0.507, P<0.001]. However, the miR-200c-3p inhibitor group had no significant difference [(1.627±0.189) cm 3; t=2.209, P=0.052]. miR-200c-3p overexpression upregulated expression levels of apoptotic proteins cleaved Caspase-3 and Bax ( t=-47.000, -82.730, all P<0.001), but downregulated the expression level of anti-apoptotic protein Bcl-2( t=53.740, P<0.001). Conclusions:The overexpression of miR-200c-3p can inhibit the proliferation, promote the apoptosis of the SK-NEP-1 cells and partially inhibited tumorigenicity of nude mouse acted as a tumor suppressor gene.