Downregulation of miR‑143 modulates KRAS expression in colorectal carcinoma cells

MicroRNAs (miRs) are a class of noncoding small RNAs that have been demonstrated to be involved in the pathogenesis of human cancer. There is even evidence that microRNAs can act as oncogenes or tumor suppressors. Although microRNA expression profiles have been characterized in colorectal carcinoma, their precise physiological functions are largely unknown. It has become clear that the activated KRAS ProtoOncogene, GTPase (KRAS) oncogene plays an important role in colorectal carcinogenesis. In the present study, it was found that the level of mature miR143 was lower in colorectal carcinoma tissues compared with that observed in normal adjacent tissues. A lack of miR143 was detected in human colorectal carcinoma cell lines, SW480, LoVo and HT29, compared to the high expression observed in normal colon epithelial cell line NCM460. pcDNA3.1primiR143 and its mutant were successfully constructed and transfected into colorectal carcinoma cells. Increased accumulation of mature miR143 was observed in the pcDNA3.1primiR143transfected cells. In SW480 cells, transfection of pcDNA3.1primiR143 resulted in a 35 and 47% reduction in cell growth after incubation for 4 and 5 days, respectively, compared with transfection of the pcDNA3.1primiR143 mutant; while in LoVo cells, transfection of pcDNA3.1primiR143 resulted in a 33 and 46% reduction in cell growth respectively. In contrast, transfection of pcDNA3.1primiR143 had no significantly effects on HT29 cell growth. We also found that transfection of pcDNA3.1primiR143 had no effect on levels of KRAS mRNA, but resulted in a 58% decrease in the KRAS protein level in the transfected SW480 cells, while an approximate 54 and 43% KRAS protein reduction in LoVo and HT29 cells, respectively, compared with the pcDNA3.1primiR143 mutant. Two fragments containing the putative complementary site were cloned into the pGL3 vector, constructing the luciferase reporter pGL3KRASCS1 and pGL3KRASCS2. Cotransfection of pcDNA3.1primiR143 with pGL3KRASCS1 and pGL3KRASCS2 respectively resulted in 4.6 and 3.3fold inhibition of luciferase activity in the SW480 cells, while a 4.0 and 3.2fold inhibition of luciferase activity in the LoVo cells, 3.7 and 3.1fold inhibition in the HT29 cells. Differences in pGL3KRASCS1 and pGL3KRASCS2 activity were not significant. Our results revealed that increased accumulation of miR143 is likely to modulate levels of KRAS protein expression at the posttranscriptional level by interacting specifically with the complementary site, and consequently inhibiting proliferation of the transfected cells.