Experimental Research On Radiosensitivity Of Esophageal Carcinoma Cells By RNAi On STAT3

Objective:Construct recombinant plasmid expressing siRNA targeting STAT3, transfect it into esophageal carcinoma cell line Eca-109 to inhibit the expression of STAT3,irradiate the cells with different doses of X-rays to study the effect of cell proliferation,cell cycle,apoptosis and sensitivity of radiotherapy,explore the mechanisms of radiosensitization,provide evidence for improving radiosensitivity and radiotherapeutic effect in esophageal carcinoma.Methods:1.Immunocytochemistry analysis and Western blot analyses the expression of STAT3 protein in esophageal carcinoma cell line Eca-109.2.Three short hairpin sequences targeting STAT3 were desined and three recombinant plasmids expressing STAT3 siRNA were constructed: pRNAT-U6.1-siRNA1,pRNAT-U6.1-siRNA2,pRNAT-U6.1-siRNA3,and a negative recombinant plasmid pRNAT-U6.1-negative which didn't interfere with STAT3 was constructed meanwhile.The recombinant plasmids were identified by sequencing.3.Recombinant plasmids were transfected into Eca-109 cells,stabilized transfected cells were selected with G418.Inhibitory effect of STAT3 mRNA and protein was detected by RT-PCR and Western blot respectively.4.Aider transfection of Eca-109 cells with STAT3 siRNA,the proliferation of cells was analysed by MTT assay,the cell cycle distribution and apoptosis were analysed with flow cytometry.5.Irradiated with 0,2Gy,4Gy,6Gy,8Gy X-rays,the survival fraction of Eca-109 cells were ascertained by MTT and clonogenic assays,the cell cycle and apoptosis were detected by flow cytometry.6.Stabilized transfected cells were exposed to 0,2Gy,4Gy,6Gy,8Gy X-rays respectively,the survival fraction of cells were determined by MTT and clonogenic assays,flow cytometry was applied to analyse cell cycle distribution and apoptosis at the dose of 4Gy.Results:1.Immunocytochemistry analysis and Western blot analyses showed that STAT3 protein expressed in esophageal carcinoma cell line Eca-109.2.Three recombinant plasmids expressing STAT3 siRNA were constructed and identified as correct by sequencing.3.The results from RT-PCR analysis and Western bolt analyses demonstrated that the plasmids containing pRNAT-U6.1-siRNA3 could specifically reduce the expression of STAT3 mRNA and protein.However,the other plasmids containing pRNAT-U6.1-siRNA1 and pRNAT-U6.1-siRNA2 had no inhibitory effect on STAT3.4.MTT assay explained that the growth-inhibition rate in siRNA3 group was 35.68%,significantly higher than that of the control(p＜0.01).5.Flow cytometry detection confirmed that the percentage of G₀/G₁ phase cell in siRNA3 group was 79.51%,significantly increased compared with the control (p＜0.05);the apoptosis rate in siRNA3 group was 13.26%,increased obviously than that of the control(p＜0.01).6.Irradiated with 6Gy,8Gy X-rays,the survival fraction of Eca-109 cells was 44.28%and 16.74%respectively,decreased significantly compared with 2Gy and 4Gy(p＜0.01).7.Exposed to 4Gy,6Gy,8Gy X-radiation,the percentage of G₀/G₁ phase cell in Eca-109 wasl 1.7%,14.08%and 24.27%respectively,significantly higher than that of 0 and 2Gy(p＜0.01).There were no significant differences in apoptosis rate induced by irradiation(p＞0.05).8.Irradiated with different doses,the survival fraction in siRNA3 group was significantly lower than that of the control(p＜0.01).9.Irradiated with 4Gy X-rays,the percentage of G₀/G₁ phase cell in siRNA3 group was 83.79%,significantly increased compared with the control(p＜0.05);the apoptosis rate in siRNA3 group was 16.42%,increased significantly than that of the control (p＜0.01).10.The sensitization enhancement ratio(SER)in this study was 1.334,showed that RNAi on STAT3 had effect on radiosensitization.Conclusion:1.STAT3 protein expressed in esophageal carcinoma cell line Eca-109.2.Three recombinant plasmids expressing STAT3-siRNA were constructed and identified as correct by sequencing.3.The plasmids containing pRNAT-U6.1-siRNA3 could obviously reduce the expression of STAT3 mRNA and protein.4.STAT3-siRNA can inhibit the proliferation of Eca-109 cells,result in cell cycle arrest at G₀/G₁ phase and induce apoptosis.5.Single irradiation can inhibit the proliferation of Eca-109 cells,result in cell cycle arrest at G₂/M phase,but induce little apoptosis.6.Radiotherapy combined with STAT3-siRNA can significantly inhibit the proliferation of Eca-109 cells,result in cell cycle arrest at G₀/G₁ phase and induce apoptosis.7.RNAi on STAT3 had effect on radiosensitization,it improved the radiosensitivity in Eca-109 cells.