摘要目的 研究微小RNA-34a(miR-34a)对葡萄膜黑色素瘤细胞生物学行为的影响及其机制.方法 采用葡萄膜黑色素瘤M23细胞作为研究对象,在细胞中分别转染miR-34a mimics和mimics阴性对照,分别记作miR-34a转染组和阴性对照组,设置不转染的细胞为正常对照组.采用real-time PCR法检测转染后miR-34a过表达效果,MTT法检测细胞增生情况,Transwell小室试验检测细胞侵袭和迁移.靶基因预测库预测miR-34a的靶基因,荧光素酶报告载体鉴定靶基因,real-time PCR和Western blot法检测靶基因的mRNA和蛋白表达.在M23细胞中共转染miR-34a mimics和小眼畸形相关转录因子(MITF)过表达载体,采用MTT法和Transwell小室试验分别检测细胞增生、侵袭和迁移能力变化,real-time PCR和Western blot法检测MITFmRNA和蛋白表达. 结果 转染miR-34a mimics后的M23细胞中miR-34a表达水平升高.正常对照组、阴性对照组和miR-34a转染组间细胞增生值、侵袭细胞数目和迁移细胞数目总体比较,差异均有统计学意义(F=18.000,P=0.003;F=20.345,P=0.002;F=15.717,P=0.004),其中miR-34a转染组较正常对照组、阴性对照组细胞增生值减小,侵袭细胞数目和迁移细胞数目减少,差异均有统计学意义(均P＜0.05).靶基因预测库及荧光素酶活性报告基因载体显示,MITF为miR-34a的靶基因.miR-34a转染组MITF mRNA和MITF蛋白的相对表达量分别为0.45±0.06和0.36±0.04,阴性对照组分别为0.99±0.11和0.62±0.05,正常对照组分别为1.00±0.07和0.63±0.08,miR-34a转染组MITF mRNA和蛋白表达水平明显低于阴性对照组和正常对照组,差异均有统计学意义(均P＜0.05).miR-34a+MITF组细胞增生值(A570)、侵袭细胞数目和迁移细胞数目分别为0.35±0.02、(29.48±3.20)个和(41.87±5.82)个,明显高于miR-34a+Vector组的0.26±0.03、(18.53±1.47)个和(27.64±2.45)个,差异均有统计学意义(均P＜0.05).结论 miR-34a具有抑制葡萄膜黑色素瘤细胞恶性表型的作用,其作用机制与抑制靶基因MITF的表达有关.

abstractsObjective 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.