| Objective1?The effect of DNA-dependent protein kinase catalytic subunit inhibitor NU7441 on the proliferation,apoptosis and cell cycle of ESCC was observed.2?When DNA-PKcs pathway was inhibited,the other DNA damage repair pathway and cell cycle checkpoint-related protein changed,including homologous recombination repair pathway(HR)protein FANCD2,base excision repair pathway(BER)protein PARP1,nucleotide excision repair(NER)protein RRM1,cell cycle checkpoint related protein WEE1.3?To explore the effect of miR-126,miR-101 and miR-21 on the radiation damage of ESCC and their relationship with DNA-PKcs.Methods1?Comet assay was used to detect the DNA break induced by irinotecan on esophageal squamous cell carcinoma cell line EC109 DNA damage.2?MTT assay was used to assess the proliferation of esophageal squamous cell carcinoma cell line EC109 with irinotecan combined with NU7441.3?The effect of NU7441 on p-DNA-PKcs,t-DNA-PKcs,PARP1,WEE1,FANCD2 and RRM1 in esophageal squamous cell carcinoma cell line EC109 after irradiation was investigated by Western blot.4?The effect of NU7441 on the cell cycle distribution and apoptosis of esophageal squamous cell carcinoma cell line EC109 after irradiation was detected by flow cytometry.5?After different doses of X-Ray radiation(0,2,4,6,8Gy),quantitative real-time PCR was used to detect the expression levels of miR-126,miR-101,miR-21 and DNA-PKcs mRNA.6?24 cases of peripheral blood samples from ESCC patients of Henan t?Mor hospital during September 2015 and March 2016 were collected.5 cases of peripheral blood were collected from healthy subjects.Quantitative real-time PCR was used to analyse the expression levels of miR-126,miR-101,miR-21 and DNA-PKcs mRNA in peripheral blood samples from ESCC patients before radiotherapy.7?Statistical analysisAll experiments were performed at least 3 times,independently.All data were carried out using SPSS17.0.Comparison between groups were analysed by ANOVA analysis,results were shown as mean ± SD.Comparison of independent treatments were analysed by unpaired Student t-test.The correlation analysis between mi RNA and DNA-PKcs mRNA was performed by Spearman.Significant P values are given,P-value less than 0.05 were considered statistically different.Results1?The DNA damage images were analyzed by comet assay software.The results showed that with increasing of irinotecan concentration,the tail length and olive moment of esophageal squamous cell carcinoma cell line EC109 increased.2?Irinotecan and/or NU7441 inhibit the proliferation of esophageal squamous cell carcinoma cell line EC109.3?Compared with the control group,the expression of p-DNA-PKcs in esophageal squamous cell carcinoma cell line EC109 of NU7441 group was decreased(P<0.01),and the expression of p-DNA-PKcs in radiotherapy group was increased(P<0.01).Compared with the control group,the expression of t-DNA-PKcs in esophageal squamous cell carcinoma cell line EC109 did not change significantly.4?Compared with the control group,the expression of WEE1 and PARP1 in esophageal squamous cell carcinoma cell line EC109 of irradiation alone and combined group were significantly lower(P<0.01).The levels of WEE1 and PARP1 in irradiation alone were higher than combined group,the difference was statistically significant(P<0.01).Compared with the control group,the expression of FANCD2 in esophageal squamous cell carcinoma cell line EC109 of irradiation alone and combined groups were significantly higher(P<0.05).The level of FANCD2 in irradiation alone was lower than combined group,the difference was statistically significant(P<0.05).The expression of RRM1 in esophageal squamous cell carcinoma cell line EC109 was increased,but the difference was not statistically significant.5?No significant difference was observed between NU7441 alone and control group;irradiation alone and combined group G2-phase arrest were induced significantly(P<0.05).Compared with irradiation alone,the esophageal squamous cell carcinoma cell line EC109 G2-phase was dramatically blocked(P<0.05).6?Compared with the control group,the apoptosis rate of esophageal squamous cell carcinoma cell line EC109 in NU7441 group was not significantly changed,but the apoptosis rates of esophageal squamous cell carcinoma cell line EC109 in irradiation alone and combined group were significantly increased(P<0.05).Compared with NU7441 alone and irradiation alone,the apoptosis rate of esophageal squamous cell carcinoma cell line EC109 in combined group was significantly higher(P<0.05).7?In EC109 cells treated with X-Ray radiation the expression levels of miR-126 and miR-101 was up-regulated(P<0.01),miR-21 no obvious alternation,compared with that in non-radiation cells.Expression of DNA-PKcs mRNA in cell line EC109 increased after irradiation(P<0.01).miR-21 expression was negatively correlated to DNA-PKcs mRNA expression.In TE7 cells treated with X-Ray radiation the expression levels of miR-126 was up-regulated(P<0.01),mi R-101 down-regulated(P<0.01)and miR-21 no obvious alternation,compared with that in non-radiation cells.Expression of DNA-PKcs mRNA in cell line TE7 increased after irradiation(P<0.01).In EC9706 cells treated with X-Ray radiation the expression levels of miR-126 was up-regulated(P<0.01),miR-101 and miR-21 down-regulated(P<0.01),and DNA-PKcs mRNA up-regulated(P<0.01),compared with that in non-radiation cells.The expression levels of miR-126,miR-101 and miR-21 were not correlated with DNA-PKcs mRNA in esophageal squamous cell carcinoma cell lines EC109,TE7 and EC9706.8?The level of mi R-101 in the peripheral blood of radiosensitive group was higher than radioresistance group(t=2.265,P<0.05).There was no significant difference in miR-126,miR-101,miR-21 and DNA-PKcs mRNA between radiosensitive group and radioresistance group.Conclusions1?With increasing of irinotecan concentration,DNA damage of esophageal squamous cell carcinoma cell line EC109 is more serious.2?NU7441 can enhance the inhibitory effect of irinotecan on EC109 cell proliferation in a concentration-dependent manner by blocking DNA-PKcs pathway.Irradiation can upregulate the expression of p-DNA-PKcs,NU7441 can downregulate the expression of p-DNA-PKcs.Irradiation combined with NU7441 can make esophageal squamous cell carcinoma cell line EC109 G2-phase block,the apoptosis rate increased,thereby improving its radiosensitivity.3 ? NU7441 can activate homologous recombination repair pathway after irradiation by blocking non-homologous end-joining.Moreover irradiation combined with NU7441 can reduce the expression of WEE1 protein,promote EC109 cell mitosis.NU7441 can downregulate the expression of base excision repair pathway protein PARP1.4?The expression levels of miR-126 and DNA-PKcs mRNA was up-regulated,miR-21 was down-regulated in EC109,TE7,9706 cells after irradiation.In TE7 and EC9706 cells treated with X-Ray radiation the expression levels of miR-101 was down-regulated,The expression level of miR-101 was up-regulated in EC109.miR-21 expression was negatively correlated to DNA-PKcs mRNA expression in EC109.5?The level of mi R-101 in the peripheral blood of radiosensitive group was higher than radioresistance group. |
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