关键词: 肾肿瘤, 微RNAs, 细胞周期, 细胞增殖, 锌指蛋白8

Abstract: Objective To investigate the effects of microRNA-1291 (miR-1291) on the expression of Zinc finger protein 8 (PHF8) gene in renal cell carcinoma and its effect on cell cycle and proliferation of renal cell carcinoma. MethodsRealtime fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR1291 in renal cell carcinoma cell lines OS-RC-2, ACHN, A498, 786-O and human proximal tubular epithelial cells HK-2. miR-1291 (miR-1291 group) and miR-NC (miR-NC group) were transfected into the renal cell lines with the lowest expression of miR1291. qRTPCR was used to detect the expression of miR-1291 and PHF8 mRNA in the transfected cells. The expression levels of PHF8, Cyclindependent kinase 6 (CDK6) and Cyclin D1 were detected by Western blotting. The effect of miR-1291 on the transcriptional activity of PHF8 was detected by double luciferase reporter gene system. Flow cytometry was used to detect cell cycle distribution. Methyl thiazolyl tetrazolium (MTT) assay and colony formation assay were used to detect cell viability and proliferation. ResultsThe expressions of miR1291 in renal carcinoma cell lines OS-RC-2, ACHN, A498, 786-O and human proximal renal tubular epithelial cell HK2 were 0.64±0.17, 0.60±0.15, 0.29±0.08, 0.63±0.08 and 1.01±0.17 respectively, with a significant difference (F=13.790, P<0.001). Compared with renal carcinoma cell lines OS-RC-2, ACHN and 786-O, the expression level of miR-1291 in A498 cell line was the lowest (P=0.002, P=0.006, P=0.003). The expression levels of miR1291 in A498 cell lines of miRNC group and miR1291 group were 1.00±0.03 and 775.25±329.91 respectively, with a significant difference (t=4.694, P=0.003); and the expression levels of PHF8 mRNA were 1.00±0.11 and 0.57±0.18 respectively, with a significant difference (t=4.122, P=0.006). The results of Western blotting were consistent with the results of qRTPCR, and the expressions of CDK6 and Cyclin D1 were significantly decreased. The double luciferase reporter gene showed that miR1291 could directly inhibit the activity of luciferase in the 3′ untranslated region of target gene PHF8. Compared with miR-NC group, the proportion of renal carcinoma cells in S phase (23.40±4.29 vs. 32.19±2.64; t=3.491, P=0.013) and G2-M phase (14.38±4.05 vs. 25.59±6.01; t=3.095, P=0.021) decreased; and the proportion of cells in G0-G1 phase increased (62.22±7.56 vs. 42.22±5.23, t=4.351, P=0.005). MTT assay showed that the cell viability of miR1291 was significantly decreased. Colony formation experiments showed that the numbers of colonies formed by A498 cells in miR-NC group and miR1291 group were 246.64±39.94 and 87.34±21.93 respectively, with a significant difference (t=6.993, P<0.001). ConclusionThe expression of miR1291 is significantly decreased in renal cancer cell lines. miR1291 can significantly inhibit the proliferation of renal cell carcinoma cells by targeting interfering PHF8 gene expression, which may contribute to the development of new renal cancer target.

Key words: Kidney neoplasms, MicroRNAs, Cell cycle, Cell proliferation, Zinc finger protein 8