Effect of miR-34a-5p on cell proliferation by targeting GMFB in Hirschsprung disease

Abstract

Objective To investigated the effect of miR-34a-5p on proliferation of SH-SY5Y cells by targeting GMFB in Hirschsprung disease (HSCR). Methods The expression of miR-34a-5p in HSCR and normal colon tissues was detected by Q-PCR. The downstream target gene of miR-34a-5p was detected by dual luciferase reporter gene assay. SH-SY5Y cells were transferred with miR-NC, miR-34a-5p mimics and miR-34a-5p mimics+GMFB. The cell viability was detected by CCK8 assay. The expression of GMFB protein was detected by western blot. CCK8 and Western blot results were compared using t test and one-way ANOVA. Results The results of Q-PCR showed that the relative expression of miR-34a-5p in the HSCR colon was 0.43±0.10, which was significantly lower than that in the normal colon (1.15±0.18, t = 3.50, P < 0.01). CCK8 results showed that the cell A450 values of miR-34a-5p mimics group was 0.53±0.03 and 0.87±0.04 respectively at 24 h and 48 h, significantly lower than those of the miR-NC control group (0.87±0.03, 1.42±0.04). The dual luciferase reporter assay results showed that the fluorescence intensity of miR-34a-5p mimics co-transfected with GMFB 3'UTR WT vector was 0.44±0.03, which was significantly lower than that of the control miR-NC and WT vector co-transfection group 1.02±0.06. CCK8 results showed that after transfection of miR-34a-5p mimics + GMFB, cells at 24 h and 48 h had significantly higher A450 values 0.99±0.02 and 1.50±0.03, respectively, compared to 0.53±0.03 and 0.87±0.04 in the miR-34a-5p mimics group (t = 7.07, P < 0.01; t = 9.14, P < 0.01). Western blot results showed that the expression of GMFB protein in the miR-34a-5p mimics group was 0.25±0.01, which was significantly lower than that in the miR-NC control group (0.90±0.03, t = 35.60, P < 0.01). The expression of GMFB protein in the miR-34a-5p mimics+GMFB group was significantly higher than that in the miR-34a-5p mimics group (1.03±0.03 versus 0.25±0.01, t = 42.74, P < 0.01). Conclusion miR-34a-5p inhibited the proliferation of melanoma SH-SY5Y cells by regulating the expression of target gene GMFB. Key words: Hirschsprung disease; miR-34a-5p; Proliferation; Glia Maturation Factor