The Change Of Cell Cycle-related Proteins Of Esophageal Squamous Cell Carcinoma In Vitro And In Vivo After RNA Interference Of Checkpoint Kinase MDC1 And 53BP1

Objective: To investigate the change of p53,CHK1,CHK2 and CHK2-T68 protein of esophageal squamous cell carcinoma in vitro and in vivo after the expression of checkpoint kinase MDC1 and 53BP1 are blocked by RNAi.Methods: Construct control Eca109,negtive Eca109,MDC1-/- Eca109,53BP1-/-Eca109 cell strain firstly. The aim of this study is to: (1) Inoculate these four kinds of human esophageal carcinoma cell lines on Six-well cluster dishes respectively.Prepare cell-anchored slips, cells were harvested at 0h,0.5h,1h,2h,4h and 12h after exposing to 5 Gy ofγ-rays. Measure the expression of CHK2-T68 protein at each time point by S-P immunohistochemical method. (2) Culture those four kinds of human esophageal carcinoma cell lines to best tumorigenesis cell population. Set up esophageal carcinoma nude mouse animal models which were treated with 15 Gy ofγ-rays after forming tumor, divided them for group A(control Eca109),group B(negtive Eca109 with empty vector),group C(Eca109 with 53BP1 RNAi),group D(Eca109 with MDC1 RNAi),group E(control Eca109+ IR),group F(negtive Eca109 with empty vector +IR),group G(Eca109 with 53BP1 RNAi+ IR),group H(Eca109 with MDC1 RNAi+ IR),each group has six nude mouse. Sacrifice the nude mouse at 1h after exposing to IR, stripping tumour, Measure the expression of p53,CHK1,CHK2 protein by S-P immunohistochemical method. Statistical analyses were performed using univariate ANOVA, repeated measure ANOVA, with spss13.0 software. Using±s express test data. P<0.05 was considered statistically significant.Results:Vitro experiment: The number of positive expression of CHK2-T68 in control Eca109 at 0h,0.5h,1h,2h,4h,12h after exposing to IR (/200) were 7±3.08,72±9.54,90±11.31,79±12.08,55±11.27,6±2.64;The number of positive expression of CHK2-T68 in negtive Eca109 with empty vector at each time point after exposing to IR (/200) were 8±3.31,72±10.17,89±14.40,76±11.76,53±8.69,6±2.04.They increased within 1 hour after irradiation, and then fell off, dropped to normal level at 12 hours after irradiation. The tendency of control Eca109 was the same as that of negtive Eca109 with empty vector. The number of positive expression of CHK2-T68 in MDC1-/-Eca109 at each time point after exposing to IR (/200) were 6±2.16,55±8.91,72±10.78,61±9.54,42±6.63,5±1.75. The expression of CHK2-T68 protein was slightly reduced after MDC1 transfection. But the tendency was the same as that of control Eca109.The number of positive expression of CHK2-T68 in 53BP1-/-Eca109 at each time point after exposing to IR (/200) were 6±2.17,40±9.30,49±10.37,41±10.80, 29±11.20,6±2.16; The peak expression of CHK2-T68 protein in Eca109 with 53BP1 transfection was lower than that of Eca109 with MDC1 transfection. But the tendency was the same.Vivo experiment: (1) The positive number of p53 protein in group A,B,C,D (/200) were 54±13.43,57±14.47,25±14.61,48±17.22, there were significant difference between interclass (P=0.01). The expression of P53 protein was depressed with 53BP1 gene knocked out (P=0.01). The positive number of CHK1 protein in group A,B,C,D (/200) were 54±13.25,50±14.67,78±19.39,59±16.11,there were significant difference between interclass (P=0.03). The expression of CHK1 protein was elevated with 53BP1 gene knocked out (P=0.04). The positive number of CHK2 protein in group A,B,C,D (/200) were 62±14.56,57±14.08,82±11.72,55±13.44,there were significant difference between interclass (P=0.01). The expression of CHK2 protein was elevated with 53BP1 gene knocked out (P=0.04). (2)The positive number of p53 protein in group E,F,G,H (/200) were 94±15.25,88±25.05,60±16.18,81±20.57,there were significant difference between interclass (P=0.04). The expression of P53 protein was depressed in group G (P=0.02). The positive number of CHK1 protein in group E,F,G,H (/200) were 35±14.32,30±12.52,56±12.33,38±13.85,There were significant difference between interclass (P=0.02). The expression of CHK1 protein was elevated in group G (P=0.03). The positive number of CHK2 protein in group E,F, G,H (/200) were 41±13.30,34±14.66,63±15.67,36±12.09,there were significant difference between interclass (P=0.01).The expression of CHK2 protein was elevated in group G (P=0.04).Conclusion: (1) Eca109 cell treated with plasmid-53BP1 or plasmid-MDC1 down-regulate the CHK2-T68 protein expression; (2)Mouse treated with plasmid-53BP1 down-regulate the p53 protein expression and up-regulate CHK1,CHK2 protein expression; (3)Mouse treated with plasmid-MDC1 have no effect on p53,CHK1,CHK2 protein expression.