The Expression Of DNA Repair Protein Ku80 In Esophageal Squamous Cancer And Its Effects On Sensitivity Of Cisplatin Chemotherapy

Background and ObjectiveIn 2011, the number of new diagnosed esophageal squamous cell carcinomas （ESCC） patients was 482,000 in the world, more than half of ESCC cases were occurred in China. In recent decades, the incidence of ESCC was decreased in the world. But in the northeast Asia, especially China, still has a high incidence. ESCC patients age was increasingly young.For half a century, early diagnosis technology and comprehensive treatment of tumor have been improved. But 5 years overall survival of ESCC patients remains 20% to 30%. Chemotherapy is an important part of comprehensive treatment. Cisplatin-based combination chemotherapy is an standard regimen for ESCC. ESCC has genetic variation and tumor heterogeneity. So, the effect of cisplatin chemotherapy have significant individual differences, and clinical response rate is only 60%. It’s urgent to explore more effective treatment strategy for ESCC. The tumor biological therapy or gene therapy has been the focus of studies, and the one of most promising methods.In recent years, many studies have shown that ESCC is a multi-factor and multi-steps malignant disease involved in multi-gene alterations. The specific and high effective targeted gene have not been found. The progress gene therapy of ESCC is very slow. At present, the biggest obstacle of ESCC gene therapy is the choice of effective targeted gene.Endogenous and exogenous DNA damage play important roles in the process of the carcinogenesis and progress of ESCC. Double stranded break （DSB） is the most dangerous DNA damage. There are two DSB repair methods:DNA homologous recombination repair （HRR） and non-homologous DNA end connection （NHEJ）. The two repair methods is very different, but their function is complementary. The simple eukaryotes （Yeast） rely mainly on HRR. However, NHEJ is the most important method of DSB repair in superior organisms. Ku protein is early response protein in NHEJ and the most important NHEJ repair factor. Abnormal alternation of Ku protein could lead to the DSB repair defect, genome instability, the tendency of carcinogenesis and immune deficiency. Ku protein is dimer formed by Ku70 and Ku80. Ku70 is highly homologous to LOC389901, also called X-ray repair complementing defective repair in Chinese hamster cells 6 pseudogene 2. Silencing or overexpression of Ku70 would have unknown influence in LOC389901. Ku80 not only repairs the DSB rapidly through NHEJ, but also have wide biological functions, including telomere stability, immunoglobulin V （D） J chain rearrangement, cell proliferation, apoptosis and cell cycle, downstream gene transcription and intracellular signal transduction. Moreover, Ku80 is abnormally expressed in a variety of human solid tumors, including lung cancer, colon cancer, liver cancer, breast cancer, which is closely related to the occurrence and development of cancers. Therefore, this study is focused on Ku80. Our previous study showed that overexpression of Ku80 is associated with the progression of ESCC. However, the effect of Ku80 expression on sensitivity of cisplatin chemotherapy is unknown. The biological function of Ku80 in ESCC needs further exploration.This study detected Ku80 mRNA and protein expression in ESCC tissues and explored the clinical relevance of Ku80 with clinicopathologic features and patients’ survival. Through RNA interference （RNAi）, we specifically silenced Ku80 gene and explored the biological function of Ku80 in ESCC. By cell culture and xenograft tumor experiment, we studied the effect of Ku80 on sensitivity of ESCC to cisplatin in vitro. We also established patient-derived esophageal squamous cell carcinoma xenograft （PDECX） model. Based on PDECX model, we further evaluated the effect of Ku80 silencing on ESCC tumor growth and cisplatin resistance in vivo, through local injections of lentivirus mediated short hairpin RNA （shRNA）. This study aims to evaluate potential significance of targeted Ku80 gene therapy, and explore the new method of gene therapy in ESCC.Part I The expression of Ku80 in esophageal squamous cancer tissues and its clinicopathological significanceObjective1. To explore the expression of Ku80 in esophageal squamous cancer tissues.2. To investigate the correction of Ku80 expression and in clinicopathological features and prognosis of ESCC patients.Methods1. We selected 119 cases of ESCC patients from January 2003 to June 2003 in Shandong Provincial Hospital affiliated to Shandong University to establish ESCC clinical database. At the same time,109 cases of simple gastritis patients who underwent gastroscopy was selected as the negative group to establish negative control clinical database. Another cohort of 217 pT2N0M0 ESCC patients who received Ivor-Lewis esophagectomy were selected from February 2000 to February 2004 in Shandong Provincial Hospital affiliated to Shandong University to establish pT2N0M0 ESCC clinical database. ESCC tissues and corresponding healthy mucosa （CHEM） were collected in ESCC patients. Normal esophageal mucosa （NEM） was collected in negative control group. The clinical tissue specimens library was established.2. RT-PCR and immunohistochemistry were used to test Ku80 mRNA and protein expression in 119 ESCC patients and 109 cases in control group. Immunohistochemistry and Western blotting were used to detect Ku80 protein expression in 217 pT2N0M0 ESCC patients.3. The cutoff scores for Ku80 overexpression were screen based on receiver operating characteristics （ROC） curve analysis. Survival curves were calculated by the Kaplan-Meier method. Univariate log-rank test and Cox regression model analysis were performed to identify prognostic factors.Results1. Ku80 mRNA expression in 119 cases ESCC tissues （5.348 ± 1.480） is significantly higher than CHEM （3.327+1.106） and 109 cases NEM （3.149±1.092） （ANOVA, P< 0.001, P<0.001）. ROC curve analysis showed that the threshold of Ku80 mRNA overexpression was 4.35 with a maximum sensitivity （74.8%） and specificity （87.2%）, the area under the curve （AUC） was 0.878 （95% CI,0.829-0.918）. Ku80 immunohistochemical score in 119 cases ESCC tissues （9.656±4.633） is significantly higher than CHEM （5.608+3.759） and 109 cases of NEM （5.532±3.741） （the Mann-Whitney U test; P< 0.001, P< 0.001）. ROC curve analysis showed that:the threshold of Ku80 immunohistochemical score is 9 with the biggest sensitivity （67.0%） and specificity （87.2%）.2. The immunohistochemical score of Ku80 in 217 cases pT2NOMO ESCC was significantly higher than that of CHEM （the Mann-Whitney U test; P< 0.001）. Western blotting results showed Ku80 protein expression （0.907±0.086） in ESCC was significantly increased than CHEM （0.364±0.052） （t test; P=0.001）. ROC curve analysis showed that the threshold was 9 with the biggest sensitivity （67.9%） and specificity （85.1%）, AUC was 0.866 （95% CI,0.8070.912）.3. In 119 cases ESCC patients, Chi-square analysis showed that Ku80 mRNA expression was associated with tumor differentiation （P= 0.016）, local invasion （P= 0.016）, lymph node metastasis （P= 0.002） and TNM stage （P= 0.001）; Ku80 protein expression was related to tumor differentiation （P= 0.001）, local invasion （P= 0.004）, lymph node metastasis （P= 0.007） and tumor stage （P= 0.002）. In 217 cases pT2N0M0 ESCC patients, Ku80 expression was closely related to tumor size （P= 0.018）, differentiation degree （P= 0.010） and TNM stage （P= 0.001）.4. In 119 ESCC patients, Cox multicovariate analysis showed local invasion （P= 0.011）, lymph node metastasis （P= 0.009）, TNM stage （P< 0.001）, Ku80 mRNA and protein expression level （P= 0.024, P= 0.024） were independent prognostic factors of overall survival （OS）. In 217 cases pT2N0M0 ESCC patients, Cox proportional hazards model showed that local invasion, lymph node metastasis, TNM stage and Ku80 expression were independent prognostic factors of OS and disease-free survival （DFS）.Conclusion1. Ku80 is abnormally overexpressed in ESCC tissues.2. The overexpression of Ku80 is closely related to poor clinicopathological features of ESCC patients.3. Ku80 expression level could be used to predict the survival of ESCC patients, and is the independent impact factor for the prognosis of ESCC patients.Pad â…¡ Effects of Ku80 gene silencing on biological behavior of esophageal squamous cancer cells and sensitivity to cisplatin chemotherapy in vitroObjective1. To explore the biological functions of Ku80 in ESCC cells.2. To clarify the effects of silencing Ku80 on cisplatin sensitivity of ESCC cells in vitro.Methods1. We cultured 293T, ECA109 and KYSE150 cells and e. coli strain DH5a in vitro. BALB/cNude rat were bred in specific pathogen free （SPF） conditions.2. Genebank （http://www.ncbi.nlm.nih.gov/） database was used to retrieve Ku80 （NM₀21141） gene sequence design and screen multiple targets siRNA interference and scramble sequences. Oligo DNA was synthesized and connected to liner carrier. Through e. coli strain DH5 alpha transformation,293 T cells transfection, viruses packaging, we built lentivirus GV115 mediated shRNA.3. According to the multiplicity of infection （MOI） value, ECA109 and KYSE150 ESCC cells were transfected with lentivirus GV115 mediated shRNA. We screened successfully transfected ECA109 and KYSE150 cells.4. RT-PCR and Western blotting was used to validate the efficiency of Ku80 gene silencing in ECA109 and KYSE150 cells and select the best shRNA sequences.5. CCK-8 kits was used to test proliferation ability of ECA109 and KYSE150 cells before and after transfection and draw cell growth curve.6. Colony forming experiment was performed to test colony forming ability of ECA109 and KYSE150 cells.7. Cell scratch and transwell experiments were performed to detect ECA109 and KYSE150 cells movement, migration and invasion.8. Flowcytometry was used to detect ECA109 and KYSE150 cell apoptosis and cell cycle.9. BALB/c nude mice xenograft tumor experiment was performed to evaluate the tumor growth of ECA109 and KYSE150 cells.10. Through culture of ECA109 and KYSE150 cells in gradient concentration of cisplatin, we detected the cisplatin sensitivity of ESCC in vitro.11. All statistical analyses were performed using SPSS.17.0 software （SPSS, Chicago, IL, USA）. The one-way analysis of variance was used to examine differences among different groups. The t test was used to examine differences among two groups. A significant difference was defined as a two-tailed P value of less than 0.05.Results1. After screening three siRNA sequences with the best kinetic parameters, we successfully built lentivirus GV115 mediated shRNA:shRNAl, shRNA2 and shRNA3. According to scramble sequence, we built lentivirus GV115 mediated shRNA scramble as negative control. Lentivirus GV115 mediated shRNA transfected ECA109 and KYSE150 cells were cultured in well condition, and the infection rate was more than 90%.2. RT-PCR showed non-transfected and transfected shRNA-scramble, shRNA-1, shRNA 2, shRNA-3 ECA109 cells Ku80 mRNA expression was 1,0.912 ± 0.061,0.584 ± 0.038,0.429±0.027,0.112±0.015, respectively. Non-transfected and transfected shRNA-scramble, shRNA-1, shRNA-2, shRNA-3 KYSE150 cells Ku80 mRNA expression wasl,0.923 ± 0.071,0.596 ±0.047,0.445 ± 0.037,0.173 ± 0.025. Ku80 mRNA expression in shRNA-3 transfected ECA109 and KYSE150 cells decreased compared with shRNA-scramble transfected cells （ANOVA; P< 0.001, P< 0.001）.3. Western blotting showed non-transfected and transfected shRNA-scramble, shRNA-1, shRNA-2, shRNA-3 ECA109 cells Ku80 protein expression was 0.845 ± 0.088, 0.713 ± 0.083,0.427±0.053,0.316±0.041,0.198 ± 0.03; non-transfected and transfected shRNA-scramble, shRNA-1, shRNA-2, shRNA-3 KYSE150 cells Ku80 protein expression was 0.823 ± 0.091,0.745 ± 0.069,0.448 ± 0.053,0.326± 0.047,0.182±0.027. Ku80 protein expression in shRNA-3 transfected ECA109 and KYSE150 cells was worst, compared with shRNA-scramble transfected cells （ANOVA; P< 0.001, P< 0.001）. So, we choose the shRNA-3 as the shRNA Ku80 to function analysis.4. CCK-8 showed the proliferation ability of shRNA Ku80 transfected ECA109 and KYSE150 cells was decreased obviously compared to shRNA scramble transfected cells. The cell proliferation of shRNA scramble transfected ECA109 and KYSE150 has no obvious difference with non-transfected cells.5. The number of clones of non-transfected and transfected shRNA-scramble, shRNA Ku80 ECA109 was 481 ± 43,398 ± 45,187 ± 32. The number of clones of non-transfected and transfected shRNA-scramble, shRNA Ku80 KYSE150 cells was 543±52,406±47,224 ± 38. The difference between non-transfected ECA109 and KYSE150 cells and shRNA Ku80 transfected cells have statistical significance （ANOVA; P< 0.001, P< 0.001）.6. Scratch experiments showed the number of shRNA Ku80 transfected ECA109 and KYSE150 cells which moved to wound decreased significantly compared to non-transfection cells （ANOVA, P< 0.001）, shRNA scramble transfection group did not change significantly. Transwell experiments confirmed that migration and invasion ability of shRNA Ku80 transfected ECA109 and KYSE150 cells were decreased significantly. But, the migration and invasion ability of shRNA scramble transfected cells did not change obviously.7. Flowcytometry confirmed apoptosis rate of shRNA Ku80 transfected ECA109 and KYSE150 cells increased significantly, compared to the non-transfection cells. The proportion of G2/M phase in shRNA Ku80 transfected ECA109 and KYSE150 cells increased, and the proportion of G1/G0 reduced（x square; P< 0.001, P< 0.001）, compared to the non-transfection cells.8. Xenograft tumor experiment showed that the volume of non-transfected and transfected shRNA-scramble, shRNA Ku80 ECA109 xenograft tumor was 3923+418 mm3,3462+357 mm3,923+89 mm3. The volume of non-transfected and transfected shRNA-scramble, shRNA Ku80 KYSE150 xenograft tumor was 3698 ± 384 mm3, 3395±341 mm3,846±74 mm3. Compared to the non-transfection cells, the volume of shRNA Ku80 transfected ECA109 and KYSE150 xenograft tumor was decreased significantly.9. Concentration gradient cisplatin culture experiment showed that the half inhibitory concentration （IC50） to cisplatin of non-transfected and transfected shRNA-scramble, shRNA Ku80 ECA109 cells was 85.97μg/ml,79.49μg/ml,8.22μg/ml. The IC50 of non-transfected and transfected shRNA-scramble, shRNA Ku80 KYSE150 cells was 48.17μg/ml,55.06μg/ml. IC50 of non-transfected ECA109 and KYSE150 cells was 75.45μg/ml,72.40μg/ml,9.59μg/ml. Compared with non-transfected cells, cisplatin sensitivity of shRNA Ku80 transfected ECA109 and KYSE150 cells was improved significantly.Conclusion1. Ku80 silencing significantly inhibits the proliferation, clone formation, movement, migration, invasion and xenograft tumor growth.2. Ku80 silencing significantly induced apoptosis and cell cycle arrest in ESCC cells.3. The Ku80 silencing significantly improved cisplatin chemotherapy sensitivity of ESCC cells in vitro.Part â…¢ Establishment of patient-derived esophageal squamous cell carcinoma xenograft（PDECX） model to evaluate the effects of Ku80 silencing on sensitivity to cisplatin chemotherapy in vivoObjective1. To establish PDECX model and evaluate the efficacy of Ku80 silencing in ESCC by local injections of lentivirus mediated shRNA in vivo.2. Based on PDECX model, to explore the effects of Ku80 silencing on sensitivity of ESCC to cisplatin chemotherapy in vivo.Methods1. We selected ESCC patients who received surgery from October 2014 to November 2014 in Shandong Provincial Hospital affiliated to Shandong University, and collected fresh ESCC tissues. We selected Ku80 mRNA overexpression ESCC patients through RT-PCR. ESCC tissues were cut into pieces and then transplanted into BALB/c nude mice subcutaneously. After three generations of rat-rat successive transplantation, PDECX models were built.2. PDECX models were randomly divided into 3 groups, shRNA Ku80 experimental group:tumors was locally injected with lentivirus GV115 mediated shRNA Ku80; negative control group:tumors was locally injected with lentivirus GV115 mediated shRNA scramble; blank control group:tumors was locally injected with saline solution.3. After PDECX tumor growth of 30 days, BALB/c nude mice received cisplatin injection. The volume of tumor was measured. After PDECX tumor growth of 60 days, tumors were removed and the weight and volume was measured. The cisplatin chemotherapy sensitivity of ESCC was detected in vivo.4. RT-PCR, immunohistochemistry and Western blotting were used to evaluate the efficacy of Ku80 silencing in PDECX model.Results1. We collected 98 cases of fresh ESCC tissues and transplanted in 93 cases of BALB/c nude mice successfully. The number of first, second and third generation of rat-rat transplantation tumors was 53,48 and 45. Consecutively, there were 45 cases of PDECX models. The 45 PDECX models were divided into 3 groups randomly:shRNA Ku80 experimental group （n= 15）, scramble group （n= 15） and blank control group （n = 15）.2. RT-PCR showed Ku80 mRNA expression of shRNA Ku80 experimental group, scramble group and blank control group was 0.254 ± 0.028,0.876+0.057,0.903 ± 0.061. Immunohistochemistry showed Ku80 was overexpression in blank control group and scramble group, which the cellular nucleus were stained yellow. Ku80 expression was reduced in shRNA Ku80 experimental group significantly. Western blotting showed Ku80 protein expression of shRNA Ku80 experimental group, scramble group and blank control group was 0.268 ± 0.019,0.793 ± 0.054,0.912 ± 0.071. Compared with the blank control group, Ku80 protein expression of shRNA Ku80 experimental group decreased significantly（ANOVA, P< 0.001）.3. After PDECX tumors growth of 5 weeks, tumor volume of shRNA Ku80 experimental group, scramble group and blank control group was 3291±156 mm3,6653±317 mm3, 7247+428 mm3, respectively. After PDECX tumors growth of 8 weeks, tumor volume of shRNA Ku80 experimental group, scramble group and blank control group was 483 ±72mm3,3974±189mm3,4526±247mm3, respectively. The tumor weight of shRNA Ku80 experimental group was significantly decreased compared to the blank control group （ANOVA, P< 0.001）.Conclusion1. Local injection of GV115 lentivirus mediated shRNA could silence Ku80 gene expression effectively in PDECX model.2. Ku80 silencing could inhibit the growth of PDECX in vivo.3. The targeted Ku80 gene silencing significantly increased cisplatin chemotherapy sensitivity of ESCC in vivo.