Kruppel-like Factor 8 Promote Renal Carcinoma Cell Line 769-P Proliferation Through Up-regulate FLT1 In Vitro

Objective:Knockdown of KLF8 can inhibit the proliferation of 786-O cell line, but the effect of its overexpression in the human renal carcinoma cell remains unclear, especially the important VEGF/VEGFR signal pathway. So we plan to overexpress KLF8 in 769-P cell line through construct recombinant lentiviral vector pLV-EGFP (2A) Puro-KLF8. And research its regulating function to VEGF/VEGFR signal pathway and its proliferation effect on renal cell carcinoma cell line 769-p.Methods:The recombinant lentiviral vector pLV-EGFP (2A) Puro-KLF8 and pLV-EGFP (2A) Puro were individually co-transfected into 293v cells with packaging plasmids pHl and pH2 to assemble viruses.48 hours later,viruses were collected and infected 769-P cells. Then real-time PCR and western blot were used to detect the expression of KLF8. The 769-P cells were divided into two groups according to if they were infected by the recombined KLF8 viruses or empty vector viruses.The effects of KLF8 on proliferation of 769-P cells were analyzed by Colony-forming assay, MTS and flow cytometric analyses. The changes of potential target gene were detected by western blot. Liposome-mediated method was used to transfect siRNA sequence targeting FLT1 and control into 769-P cell line to observe its efficacy in knocking down FLT1, then we use colony-forming assay and MTS assay to measure its proliferation effect on 769-P-KLF8 and 769-P-empty cell.Results:After successful construction of the recombined pLV-EGFP (2A) Puro-KLF8 lentiviral vector, and transfected into 769-P cell line, the expression of KLF8 was significantly upregulated compared with the empty vector group. Colony-forming assay showed that after 10 days'culture, the experimental group proliferated significantly than the control group. There is significant increase in the absorbance of experimental group than that of control group at 48h,72h,96h in the MTS assay.(P<0.05). Flow cytometric analyses showed that the experimental group reduced in the G0/G1 phase accompanied with a increase in the S phase. The western blot shows no difference of VEGF, VEGFC, VEGFR2 and VEGFR3 between the experimental group and control group, except FLT1 which is highly expressed in the experimental group. siFLT1-3 is the most effective small interfering RNA sequence,The proliferation of 769-P-KLF8 and 769-P-empty reduced after treated with siFLT1.Conclusion The overexpression of KLF8 can increase proliferation of 769-P cell line,and its proliferation effect require up-regulate FLT1.