Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells

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.

Keywords: Antisense oligonucleotides (ASO); Colon carcinoma; MicroRNA-21; Phosphatase and tensin homolog (PTEN).

Fig. 1

MiRNA-21 ASO reduced the proliferation of human colon carcinoma cells. a The schematic of a eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO). b Human colon carcinoma cell line HCT116 cells were cultured in 96 well plate and transiently transfected with p-miR-21-ASO or p-Cont (5 μg). After 48 h, the expression of GFP protein was observed by fluorescent microscopy. c The expression level of miR-21 in HCT116 cells was also determined by Real-time PCR assay and calculated. d At indicated time point, the proliferation of HCT116 cells in p-miR-21-ASO transfected group and p-Cont group also were detected by CCK-8 assay. One representative of three experiments was shown. *p < 0.05.

Fig. 2

MiRNA-21 ASO reduced the colony formation capacity of human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). a At indicated time point, the colony diameter was analyzed. b After 13 days, then colony numbers were analyzed by crystal staining and calculated (c). Data represent as mean ± SD of three independent experiments. *p < 0.05.

Fig. 3

MiRNA-21 ASO impaired the invasion and migration ability of human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). Then, the ability of invasion of cells was analyzed by Transwell assay (a) and calculated (b). c The ability of migration of cells also was determined by Wound-healing assay and calculated (d). e HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of VEGF was analyzed by western blotting and calculated (f). One representative of three experiments was shown. *p < 0.05.

Fig. 4

MiRNA-21 ASO reversed PTEN expression in human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the protein expression of PTEN was analyzed by Western blotting (a) and calculated (b). c The expression of PTEN also analyzed by FACS and then the mean fluorescence intensity (MFI) was calculated (d). Gray line p-Cont transfected group, black line p-miR-21-ASO transfected group. One representative of three experiments was shown. *p < 0.05.

Fig. 5

MiRNA-21 ASO altered the transduction of AKT and ERK pathways in human colon carcinoma cells. Human colon carcinoma cell line HCT116 cells were transiently transfected with p-miR-21-ASO or p-Cont (5 μg). 48 h later, the level of total and phosphor-AKT and phosphor-ERK1/2 were analyzed by Western blotting (a) and calculated (b). One representative of three experiments was shown.*p < 0.05.