ABSTRACT
BACKGROUND: Colon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated. METHODS: In present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot. RESULTS: We found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered. CONCLUSIONS: Therefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.
ABSTRACT
This paper was aimed to investigate the effects of ATP-sensitive potassium channels on the proliferation and differentiation of rat preadipocytes. We examined the expression of sulphonylurea receptor 2 (SUR2) mRNA in preadipocytes and adipocytes obtained by inducing for 5 d and the effects of the inhibitor (glibenclamide) and opener (diazoxide) of ATP-sensitive potassium channels on the expression of SUR2 mRNA in preadipocytes by real-time PCR. Preadipocyte proliferation and cell cycle were measured by MTT spectrophotometry and flow cytometer. The content of intracellular lipid was measured by oil red O staining, cell diameter was determined by Image-Pro Plus 5.0 software and the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) mRNA was estimated by RT-PCR. SUR2 mRNA was expressed in both preadipocytes and adipocytes obtained by inducing for 5 d, and the expression in adipocytes was obviously higher than that in preadipocytes. Glibenclamide inhibited the expression of SUR2 mRNA in preadipocyte, promoted preadipocyte proliferation in a dose-dependent manner, increased the cell percentages in G(2)/M + S phase, increased lipid content, augmented adipocyte diameter, and promoted the expression of PPAR-gamma mRNA. But the actions of diazoxide were contrary to those of glibenclamide. These results suggest that ATP-sensitive potassium channels regulate the proliferation and differentiation of preadipocytes, and PPAR-gamma is probably involved in the effect of ATP-sensitive potassium channels.
