The effects of up-regulation of microRNA-148a on the expression of anti-oncogene preproenkephalin, p16 and ras association domain family 1A in pancreatic carcinoma AsPC-1 cells

Abstract

Objective To investigate the effect of up-regulation of microRNA (miRNA, miR)-148a on the methylation and protein expression of anti-oncogene preproenkephalin (ppENK), p16, ras association domain family 1A (RASSF1A) and the proliferation of pancreatic cancer AsPC-1 cells. Methods MiR-148a mimics were transfected into AsPC-1 cells mediated by Lipofectamine 2000. AsPC-1 cells were devided into three groups, blank group (transfected Lipofectamine), mimincs-NC group (transfected negative mimics) and mimincs-148a group (transfected miR-148a mimics). At 72 h after transfection, the expression of miR-148a was analyzed by real-time quantitative polymerase chain reaction (Real-time PCR). The CpG island methylation status of anti-oncogene ppENK, p16, RASSF1A were measured by methylation specific polymerease chain reaction (MSP). The expression of DNA methy transferas 1 (DNMT1), ppENK, p16 and RASSF1A protein were detected with Western blotting. The cell proliferation activity was examined by methyl thiazol tetrazolium (MTT) assay. Results The expression of miR-148a in mimincs-148a group was significantly higher than those in blank group and mimincs-NC group (4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P=0.002), while the expression of DNMT1 protein in mimincs-148a group was significantly lower than those in blank group and mimincs-NC group (1.00±0.00 vs. 1.04±0.19 vs. 0.27±0.15, P=0.001). ppENK, p16 and RASSF1A genes were positive methylation in blank group and mimincs-NC group, while ppENK and p16 genes were negative methylation, and RASSF1A gene was partial methylation in mimincs-148a group. The expression of ppENK, p16 and RASSF1A protein in mimics-148a group were significantly higher than those in blank group and mimics-NC group (ppENK: 2.06±0.32 vs. 1.00±0.00 vs. 1.01±0.21, P=0.002; p16: 2.02±0.12 vs. 1.00±0.00 vs. 0.96±0.12, P=0.000; RASSF1A: 1.95±0.37 vs. 1.00±0.00 vs. 1.09±0.12, P=0.002). The cell proliferation activities in mimincs-148a group were significantly slower than those in in blank group and mimincs-NC group (P=0.026). Conclusion Up-regulation of miR-148a could lead to demethylation of ppENK, p16 and RASSF1A gene promoter CpG island and up-regulate its protein expression, and inhibit cell proliferation in pancreatic cancer AsPC-1 cells. The mechanism may be related to the inhibition of DNMT1 expression induced by miR-148a. Key words: Pancreatic cancer; MicroRNA-148a; DNA methy transferas 1; Anti-oncogene; Methylation; Cell proliferation