Post-transcriptional suppression of G protein-coupled receptor 15 (GPR15) by microRNA-1225 inhibits proliferation, migration, and invasion of human colorectal cancer cells

Abstract

The G protein-coupled receptors (GPRs) have been shown to regulate several cancer related processes. The aberrant expression of GPRs has been linked to the development of several cancers. The present study was designed to examine the expression and decipher the role of GPR15 in the development of human colorectal cancer. The results revealed GPR15 to be significantly (P < 0.05) upregulated in colorectal cancer cells. The silencing of GPR15 inhibited the growth of the colorectal cancer cells via induction of apoptosis. Induction of apoptosis in colorectal cancer cells was associated increase in Bax and decrease in Bcl-2 expression. The silencing of GPR-15 also caused a significant (P < 0.05) decline in the migration and invasion of the colorectal cancer cells. Bioinformatic analysis and luciferase assay revealed that the expression of GPR15 to be post-transcriptionally regulated by microRNA-1225 (miR-1225). The expression of miR-1225 was found to significantly (P < 0.05) downregulated in colorectal cancer cells and its overexpression caused suppression of GPR15 and inhibited the proliferation of the colorectal cancer cells. Nonetheless, overexpression of GPR15 could avoid the growth inhibitory effects of miR-1225. The results suggest that the GPR15/miR-1225 axis play an important role in the development of colon rectal cancer and exhibit therapeutic implications for its treatment.

Keywords: Apoptosis; Colorectal cancer; G protein-coupled receptor; GPR15; Micro-RNA; miR-1225.

Fig. 1

GPR15 is upregulated in colorectal cancer. a Gene expression of GPR15 in colorectal cancer and normal tissues, b expression of GPR15 gene in colorectal cancer and corresponding normal adjacent tissues, c immuno-histochemical fluorescence of GPR15 from normal and colorectal cancer tissues, d expression of GPR15 protein in colorectal cancer cell lines (HCT116, SW620, HT-29 and SW480) and normal colorectal epithelial cell line, CCD-18Co. The experiments were performed in triplicate and presented as mean ± SD (*P < 0.05)

Fig. 2

GPR15 repression induced apoptosis and inhibited colorectal cancer cell growth. a Expression levels of GPR15 in si-GPR15 and si-NC transfected HCT116 cells, b MTT assay of HCT116 cancer cells transfected with si-GPR15 or si-NC, c AO/EB staining of HCT116 cancer cells transfected with si-GPR15 or si-NC, d DAPI staining of HCT116 cancer cells transfected with si-GPR15 or si-NC, e flow cytometry of HCT116 cancer cells transfected with si-GPR15 or si-NC, f Bax and Bcl-2 protein levels in HCT116 cancer cells transfected with si-GPR15 or si-NC. The experiments were performed in triplicate and presented as mean ± SD (*P < 0.05)

Fig. 3

GPR15 silencing restricts colorectal cancer cell migration and invasion. Transwell chambers assays for the estimation of migration and invasion of HCT116 cancer cells transfected with si-GPR15 or si-NC. The experiments were performed in triplicate and presented as mean ± SD (*P < 0.05)

Fig. 4

miR-1225 targets GPR15 to regulate colorectal cancer cell growth. a Target scan analysis of GPR15 showing miR-1225 as its post-transcriptional repressor, b dual luciferase assay, c miR-1225 expression in colorectal cancer cell lines (HCT116, SW620, HT-29 and SW480) and normal colorectal epithelial cell line, CCD-18Co, d expression of GPR15 protein in HCT116 cancer cells transfected with miR-1225 mimics or miR-NC, e MTT assay of HCT116 cancer cells transfected with miR-1225 mimics or miR-NC, f MTT assay of HCT116 cancer cells transfected with miR-1225 mimics, miR-1225 mimics + pcDNA-GPR15 or miR-NC. The experiments were performed in triplicate and presented as mean ± SD (*P < 0.05)