MicroRNA-148b is frequently down-regulated in gastric cancer and acts as a tumor suppressor by inhibiting cell proliferation

Abstract

Background: MicroRNAs (miRNAs) are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Our previous studies have revealed that miR-148a and miR-152 are significantly down-regulated in gastrointestinal cancers. Interestingly, miR-148b has the same "seed sequences" as miR-148a and miR-152. Although aberrant expression of miR-148b has been observed in several types of cancer, its pathophysiologic role and relevance to tumorigenesis are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in gastric cancer.

Results: We showed significant down-regulation of miR-148b in 106 gastric cancer tissues and four gastric cancer cell lines, compared with their non-tumor counterparts by real-time RT-PCR. In situ hybridization of ten cases confirmed an overt decrease in the level of miR-148b in gastric cancer tissues. Moreover, the expression of miR-148b was demonstrated to be associated with tumor size (P = 0.027) by a Mann-Whitney U test. We also found that miR-148b could inhibit cell proliferation in vitro by MTT assay, growth curves and an anchorage-independent growth assay in MGC-803, SGC-7901, BGC-823 and AGS cells. An experiment in nude mice revealed that miR-148b could suppress tumorigenicity in vivo. Using a luciferase activity assay and western blot, CCKBR was identified as a target of miR-148b in cells. Moreover, an obvious inverse correlation was observed between the expression of CCKBR protein and miR-148b in 49 pairs of tissues (P = 0.002, Spearman's correlation).

Conclusions: These findings provide important evidence that miR-148b targets CCKBR and is significant in suppressing gastric cancer cell growth. Maybe miR-148b would become a potential biomarker and therapeutic target against gastric cancer.

Figure 1

The expression of miR-148b in tissues and cell lines. (A), MiR-148b was detected in 106 gastric cancer patients by qRT-PCR. Data were presented as log2 of fold change of gastric cancer relative to non-tumor adjacent tissues. The cases below the line (log2 = -1) revealed >50% reduction in the miR-148b level. (B), The relative level of miR-148b in gastric cancer cell lines (MGC-803, SGC-7901, BGC-823, AGS) relative to normal gastric epithelial cell line (GES-1). (C), H & E staining and detection of miR-148b by in situ hybridization in serial sections from gastric cancer tissue and its matched non-tumor adjacent tissue. Images are overlay images with brown color representing miR-148b expression. Data are presented as mean ± SD from at least three separate experiments. *, P < 0.05; **, P < 0.01.

Figure 2

MiR-148b inhibits cell proliferation in vitro. (A), MTT proliferation assay in MGC-803, SGC-7901 and BGC-823. (B), Growth curves by counting cell number in MGC-803, SGC-7901 and BGC-823. (C), MTT proliferation assay in AGS. (D), Anchorage-independent growth assay in SGC-7901 cells. The colonies were counted (top) and captured (bottom). (E), The results of cell cycle analysis in MGC-803 and SGC-7901 cells. All results were reproducible in three independent experiments. *, P < 0.05; **, P < 0.01.

Figure 3

MiR-148b suppresses tumorigenicity in vivo. Three groups of mice (n = 11) were tested. (A) , the number of alive mice and the size of tumors in three groups. (B), the tumor growth curves of three groups during four weeks. (C), Each tumor lump was removed from the body, and three mice with miR-148b mimics didn't form any tumors. (D), the mean tumor weight of three groups at the end of the experiment. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01.

Figure 4

CCKBR is a potential target of miR-148b in SGC-7901, MGC-803, BGC-823 cells. (A), analysis of luciferase activity of five possible target genes with miR-148b mimics or NC in MGC-803 cells. (B), analysis of luciferase activity of pGL3-CCKBR-3'UTR, pGL3-CCKBR-3'UTR-poorly conserved, pGL3-CCKBR-3'UTR-conserved and positive control (PC) with miR-148b mimics or NC in MGC-803, SGC-7901 and BGC-823 cells. (C&D), Western blot and qRT-PCR were used to monitor the expression level of CCKBR in MGC-803 cells 48 h after transfection with miR-148b mimics or NC. (E), Effects of knockdown of the CCKBR gene on cell proliferation in MGC-803 cells.