Construction And Identification Of Recombined STAT1 And H2AX-shRNA Lentivirus And Transfection Of Esophageal Carcinoma Cells In Vitro

Objective: To construction transfected esophageal carcinoma cell lines which targeting STAT1 and H2AX gene. To observe the expression of STAT1 and H2AX mRNA and protein after irradiated, and measure the location and quantity of STAT1 and H2AX nuclear foci. In order to provide a based for changing the radiosensitivity of TE13 and ECA109 esophageal caicinoma cells by RNAi targeting STAT1 and H2AX gene.Methods: Four specific target sequences were selected according to mRNA sequence of STAT1 and H2AX gene respectively. A non-specific control group was designed. The complementary DNA which contained both sense and antisense oligonucleotides were synthesized. These double strands DNA were cloned to AgeI and EcoRI sites of pGCSIL-H1-GFP after phosphorylation and annealing. The products were pGCSIL-STAT1-shRNA1, pGCSIL-STAT1-shRNA2,pGCSIL-STAT1-shRNA3,pGCSIL-STAT1-shRNA-4 and pGCSIL-H2AX-shRNA1, pGCSIL-H2AX-shRNA2, pGCSIL-H2AX- shRNA3, pGCSIL-H2AX-shRNA4 and pGCSIL-negative was confirmed by electrophoresis and sequencing.Compared the RNAi effect of four specific plasmids harboring different shRNA expressing cassette by western blotting. pGCSIL-STAT1-shRNA and pGCSIL-H2AX-shRNA were cotransfected along with pPGCLH1-Lentivector Packaging system into 293T to package lentivirus particles. The effects of RNAi were identificated by Real-time RT-PCR and western blotting after transfection 72h with specific or control lentiviral vectors. The location and quantity with nuclear foci of STAT1 and H2AX were detected with Zeiss fluorescence microscope.Resμlt:①It was confirmed by digestion and sequencing that STAT1 and H2AX shRNA expression structure was correctly cloned to pGCSIL-H1-GFP. ②Expression of protein STAT1 and H2AX genes were obviously inhibited after transfected with TE-13 and ECA109 cells. The rates of inhibaition with pGCSIL-STAT1-shRNA1,pGCSIL-STAT1-shRNA2,pGCSIL-STAT1-shRNA3, pGCSIL-STAT1-shRNA4 were 75%, 60%, 26%, 22%with TE-13, and 81%, 56%, 21%, 33%with ECA109, respectively.While the rates of inhibaition with pGCSIL-H2AX-shRNA1,pGCSIL-H2AX-shRNA2, pGCSIL -H2AX-shRNA3, pGCSIL-H2AX-shRNA4 were 34%, 22%, 72%, 61%in TE-13, and 29%, 34%, 74%, 62%in ECA109.③S uccessfμlly established the STAT1 and H2AX knocking down cell lines: TE-13/STAT1,ECA109/STAT1,TE-13/H2AX and ECA109/H2AX.④The mRNA level and protein expression of STAT1 and H2AX genes were obviously inhibited in TE-13/STAT1, ECA109/STAT1, TE-13/H2AX and ECA109/H2AX. The inhibition rates of mRNA and protein expression of TE-13/STAT1 were 73%and 69%, while ECA109/STAT1 were 66%and 65%. But TE-13/H2AX were 66%and 83%, ECA109/H2AX were 68%and 63%.⑤The formation of STAT1 nuclear foci were also inhibited after treated with cisplatin. The same of H2AX nuclear foci were formated by 4Gy irradiated.Conclusion:①Transfected with STAT1 and H2AX shRNA,mRNA expression of STAT1 and H2AX genes were obviously inhibited in TE-13 and ECA109 cells.②Successfμlly established the STAT1 and H2AX knocking down cell lines: TE-13/STAT1,ECA109 /STAT1,TE-13/H2AX and ECA109/H2AX.③the gene P-STAT1 and P-H2AX were markedly inhibited after irradiated by 4Gy. The formation of STAT1 nuclear foci were also inhibited after treated with cisplatin. The same of H2AX nuclear foci were formated by 4Gy irradiated.