Abstract: Objective To investigate the effects and mechanisms of replication activating factor C2(RFC2) on the proliferation and migration of renal cell carcinoma(RCC) cells.Methods Western blotting was used to determine and compare the expression levels of RFC2 protein in human normal renal tubular epithelial cell lines HK-2 and RCC cell lines ACHN,786-O,Caki-1,KETR3,and OS-RC2.The KETR-3 cell line was transfected with Lipofectamine 3 000 using RFC2 small interfering RNA(siRNA) and control inhibitory sequence scaffold,respectively,and divided into RFC2 silenced expression group(si-RFC2) and inhibitory control group(si-NC).Transfect RFC2 overexpression plasmid(RFC2 plasmid) and blank control plasmid,and divide into RFC2 overexpression group(OE-RFC2) and overexpression control group(Vector).Cell proliferation ability was detected using Methylthiazolyldiphenyl-tetrazolium bromide(MTT) cell proliferation detection kit,cell migration ability was measured using cell scratch assay,and protein blotting assay was used to determine the expression levels of RFC2,p-PI3K,PI3K,p-Akt,and Akt proteins.Results Compared with the normal human renal tubular epithelial cell line HK-2,the RCC cell lines ACHN,786-O,Caki-1,KETR3,and OS-RC2 showed high expression of RFC2 protein(all P<0.001).MTT assay showed that the A_490nm values of the si-RFC2 group were lower than those of the si-NC group at 0,24,48,and 72h(all P<0.05),while the A_490nm values of the OE-RFC2 group were higher than those of the Vector group at 0,24,48,and 72h(all P<0.05).The scratch healing rate of the OE-RFC2 group was higher than that of the Vector group[(89.4±9.4)% vs(15.8±6.3)%,P<0.05].The cell scratch healing rate in the si-RFC2 group was lower than that in the si-NC group[(5.2±1.9)% vs(16.5±5.5)%,P<0.05].After upregulating RFC2 expression in KETR-3 cells,the relative expression level of RFC2 protein in the OE-RFC2 group was higher than that in the Vector group(1.04 ± 0.19 vs 0.25±0.03,P<0.05),the p-PI3K/PI3K ratio in the OE-RFC2 group was higher than that in the Vector group(1.15±0.23 vs 0.34±0.024,P<0.05),and the p-Akt/Akt ratio in the OE-RFC2 group was higher than that in the Vector group(1.26±0.25 vs 0.38± 0.022,P<0.05).After silencing the expression of RFC2,the relative expression level of RFC2 protein in the si-RFC2 group was lower than that in the si-NC group(0.16 ± 0.02 vs 1.08 ± 0.06,P<0.05),the p-PI3K/PI3K ratio in the si-RFC2 group was lower than that in the si-NC group(0.18 ± 0.04 vs 0.86±0.14,P<0.05),and the p-Akt/Akt ratio in the si-RFC2 group was lower than that in the si-NC group(0.10±0.01 vs 0.58±0.13,P<0.05).Conclusion RFC2 promotes the proliferation and migration of renal cell carcinoma cells,and the mechanism may be related to the activation of the PI3K/Akt signaling pathway.

Key words: replication activation factor C2, renal cell carcinoma, cell proliferation, cell migration, mechanism