Inhibitory effect of miR-34a on biological behavior of uveal melanoma cells and its mechanism / 中华实验眼科杂志

ABSTRACT

Objective To study the effect of microRNA-34a(miR-34a) on the biological behavior of uveal melanoma cells and its mechanism.Methods Uveal melanoma M23 cells were used as research objects,miR-34a mimics and mimics negative control were transfected into the cells respectively as miR-34a transfection group and the negative control group,and the non-transfected cells served as the normal control group.The overexpression effect was validated by real-time PCR.MTT assay was used to detect cell proliferation.Cell invasion and migration were detected by Transwell test.Target gene prediction library predicted target genes of miR-34a,and the target gene was identified by luciferase activity report.Real-time PCR and Western blot were used to detect the mRNA and protein expression of target genes.MiR-34a mimics and microphthalmia-associtated transcription factor (MITF) overexpression vectors were cotransfected into M23 cells.Cell proliferation,invasion and migration abilities were detected by MTT assay and Transwell test,respectively.The mRNA and protein expressions of MITF were detected by real-time PCR and Western blot.Results The expression of miR-34a in M23 cells transfected with miR-34a mimics increased.The cell proliferation (A570),number of invasive cells and migrating cells were significantly different among the miR-34a transfection group,negative control group and normal control group (F =18.000,P =0.003;F =20.345,P =0.002;F=15.717,P=0.004).The proliferation,invasion and migration ability of M23 cells in the miR-34a transfection group were significantly decreased compared with the negative control group and normal control group (all at P＜0.05).Target gene prediction library and luciferase activity report showed that MITF was the target gene of miR-34a.The relative expression levels of MITF mRNA and protein were 0.45 ±0.06 and 0.36± 0.04 in the miR-34a transfection group,0.99± 0.11 and 0.62 ± 0.05 in the negative control group,1.00 ± 0.07 and 0.63 ± 0.08 in the normal control group,respectively,and compared with the negative control group and normal control group,the expression of MITF in miR-34a transfection group were significantly decreased (all at P＜0.05).Cell proliferation (A570),the number of invasived cells and the number of migrated cells were 0.35±0.02,29.48±3.20 and 41.87±5.82 in the miR-34a + MITF group,0.26 ± 0.03,18.53 ± 1.47 and 27.64 ± 2.45 in the miR-34a + Vector group,respectively,the proliferation,invasion and migration ability of the cell.s in the miR-34a+MITF group was significantly higher than that in the miR-34a+Vector group (all at P＜0.05).Conclusions miR-34a can inhibit the malignant phenotype of uveal melanoma cells by inhibiting the expression of the target gene MITF.