The Effect Of Chk1Gene Silencing On Increasing The Chemotherapy Sensitivity Of Human Esophageal Carcinoma Cells

The incidence of esophageal carcinoma in our country ranks first in the world, and the Taihang Mountain area in Henan province is high risk area for esophageal carcinoma in China. Its attack is strong, rapid, easy to relapse and mortality high. Currently, the treatment of esophageal carcinoma includes surgery, chemotherapy, radiation therapy, biological therapy and so on, in which chemotherapy is one of the principal means of treatment in patients with advanced esophageal carcinoma. But in the clinical course of treatment, many patients resistant to chemotherapy. Study found that chemotherapy drugs can activate cell cycle checkpoint signaling pathway, causing tumor cells to self-healing of damaged DNA to apoptosis escape and cause chemotherapy resistance.Activation of cell cycle checkpoint is a cellular self-protection mechanism. After the damage of chemotherapy and other factors, cells can activate multiple DNA damage cycle checkpoints (G1/S, S and G2/M) of the destroyed cells stagnation in the detection point of DNA repair, thus avoiding excessive chilliness apoptosis. Cell cycle checkpoint kinase1(Checkpoint kinase1, Chkl) plays an important role in the cell cycle checkpoint signaling pathway. Chkl is the most prominent point of the cell cycle of the serine/threonine kinase, mainly involved in signal transduction during the G2/M cell cycle. Chkl may be the most suitable target molecules to eliminate G2arrest by far. Contemporary scholars at home and abroad mainly used Chkl siRNA and antigens oligonucleotide technology to silence the expression of Chkl in tumor cells to enhance the radiosensitivity of malignant tumor cells. Reports about silencing Chkl to enhance the sensitivity of tumor cell to chemotherapy are little, and the study which through silencing Chkl to enhance the sensitivity of esophageal carcinoma cell has not been reported.Cisplatin is one of the essential drugs for treatment of esophageal malignancy. However, multiple resistance, caused by platinum-based, makes the clinical efficiency of joint programs which with platinum-based decreased significantly, that became a bottleneck in the clinical progress in the treatment of advanced malignant tumor. Nowadays, clinical and in vitro and in vivo experiments has researched to their resistance extensively, recently found that the chemotherapy drugs cause different types of DNA damage in tumor cells, activated the DNA damage checkpoint, leading to tumor cell DNA damage repair, so that the cell avoiding apoptosis. Therefore, the efficacy of chemotherapy is to a large extent depends on the functional status of the DNA damage checkpoint. This study is proposed by Chkl siRNA silencing the expression of Chkl in esophageal carcinoma cells, observation in vitro and in vivo, the effect of cisplatin on the chemotherapy sensitivity of esophageal carcinoma after the Chkl gene silencing and that lay the theoretical foundation for clinical for the enhanced sensitivity to chemotherapy of malignant tumor of esophageal carcinoma. This research is divided into the following4parts.Part I Chkl expression in esophageal squamous cell carcinoma and its significanceMethods:1. Using immunohistochemistry, Western blot, in situ hybridization, RT-PCR, to detect the expression of RhoC gene in62cases of esophageal squamous cell carcinoma,31cases of adjacent dysplasia and62cases of normal esophageal mucosa. 2. Statistical analysis:Statistics analysis was utilized by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard α=0.05.Results:1. Expression of Chk1protein was predominantly localized in esophageal cancer cell nuclei and cytoplasm, brown yellow granules; Chk1mRNA positive expression was localized in the esophagus cancer cell cytoplasm, purplish blue particles; Chk1protein and Chk1mRNA were an expression no or lower in atypical hyperplasia tissue and normal esophageal mucosa negative.2. The results of a joint test of immunohistochemistry, Western blot, in situ hybridization, RT-PCR:Chk1mRNA and protein expression level gradually increased from normal esophagus mucous membrane tissue, adjacent atypical hyperplasia tissue to ESCC tissue, and there was significant difference among three groups (P<0.05).3. Expressions of Chk1mRNA and protein in the deep layer invaded groups were considerably higher than those in infiltrating shallow layer group, and there was significant difference among three groups (P<0.05).4. Expressions of Chk1mRNA and protein in lymphatic metastasis groups were significantly higher than those without lymphatic metastasis group, and there was significant difference among three groups (P<0.05).5. Expressions of Chk1mRNA and protein in poor differentiated ESCC tissues were markedly higher than that in well differentiated tissues, and the differences were statistically significant (P<0.05)6. Expressions of Chk1mRNA and protein were not related to sex and age of the patients with esophagus cancer (P>0.05).Part Ⅱ Construction of Chk1siRNA expression vectorMethods:1. Construction of recombinant vector pSilencer3.1-Chk1siRNA. 2. Three pairs of anti-sense oligonucleotide fragments and a pair of non-sense sequence was designed and annealed, and then was introduced into RNA interfering expression vector pSilencer3.1, and the recombinant expression vector pSilencer3.1-Chkl siRNA was successfully constructed.3. Using RT-PCR to detect the expression of Chkl mRNA in the cells and utilizing of the Western blotting to detect the expression of Chk1protein in the cells, and then pick the best inhibitory effect of Chkl siRNA.4. Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard a=0.05.Results:1. Three siRNA expression vector pSilencer3.1-Chkl siRNAl, pSilencer3.1-Chkl siRNA2和pSilencer3.1-Chkl siRNA3were successfully constructed.2. Three Chkl siRNA vector-transfected EC9706cells, Chkl protein and mRNA expression levels were significantly lower than untreated EC9706cells and control siRNA group (P<0.05).3. Among the group of Chk1siRNA, compared between Chk1siRNA1and Chkl siRNA3, the expression of Chkl mRNA was no difference (P>0.05), but it was substantially higher than that of Chkl siRNA2Chkl mRNA expression levels (P <0.05).Part Ⅲ Influence of biology behavior Chkl siRNA interference expression vector for cisplatin induced in vitro of EC9706esophageal cancer cellMethods:1. Esophageal cancer cell EC9706group training, namely A groups:normal group, without any treatment; B:Chk1siRNA2carrier transfection people esophageal squamous carcinoma EC9706cell24h, then reprocessing EC9706cell12h using cisplatin of concentration for10μ mol/L; C group:reprocessing EC9706cell12h using cisplatin of concentration for10μ mol/L 2. Using immunohistochemical, Western blot and in situ hybridization, RT-PCR to test the cells Chkl protein and mRNA expression3. Using MMT method to observe the cell proliferation condition.4. Using TUNEL and flow cytometry methods to detect cell apoptosis situation.5.Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, t test and variance analysis and Spearmanm, test standard α=0.05.Results:1. The results of the method of MTT:Compared to the B group (Chkl siRNA2+cisplatin) and C group (cisplatin) and A group (normal control group), the cell growth inhibition rate increased dramatically, the differences were statistically significantly(P <0.05).2. Immunocytochemistry and Western blot test results:B Group (Chkl siRNA2+cisplatin) and C group (cisplatin) and A group (normal control group), compared with EC9706esophageal cancer cells Chkl protein expression clear cut, two groups of difference in all have statistical significance (P<0.05).3. The results of In situ hybridization and RT-PCR:the Chkl mRNA expressed in cells in group B(Chkl siRNA2+cisplatin) was significantly lower than the group C(cisplatin) and group A(normal control group), compared two groups of two differences were statistically significant (P<0.05).4. Apoptosis results of TUNEL method:Cells in group B(Chkl siRNA2+cisplatin) apoptosis index (AI) is obviously higher than that of group C (cisplatin) and A group (normal control group), compared two groups of two differences were statistically significant (P<0.05).5. Flow cell detection instrument apoptosis results:B Group (Chkl siRNA2+cisplatin) early apoptosis cells number and late apoptosis cells number than the C group (cisplatin) and A group (normal control group) increased considerably, and the differences were statistically significant (P<0.05). Part Ⅳ The effect of Chk1siRNA interference expression vector on cisplatin induced esophageal carcinoma transplanted tumor cells biological behavior in vivoMethods:1. Cultivate esophageal cancer EC9706cell, constructing the nude mouse esophageal cancer transplantation tumor model.2. Group breeding tumor-burdened nude mouse, namely A groups:normal group, pseudoscience injection Sterile Saline; B group:pseudoscience injection Chk1siRNA2add concentration for (5mg/kg) of cisplatin, C groups:pseudoscience injection cisplatin (5mg/kg). All group every3days one time, a total of5times.3. Observe the transplantation tumor growth.4. Using immunohistochemical, Western blot and in situ hybridization, RT polymerase chain reaction (PCR) to test the transplantation of Chkl protein and mRNA expression in the tumor tissues.5.Using TUNEL method. to test the apoptosis situation of each cell.6.Statistical analysis:Statistics analysis was used by SPSS13.0software, using chi-square test, T test and variance analysis and Spearmanm, test standard α=0.05.Results:1. Transplant pseudoscience product of the B group (Chkl siRNA2+cisplatin) noticeably less than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P<0.001).2.The results of immunohistochemical and Western blot test:the expression of Chk1protein in the esophageal cancer transplantation tumor tissues of B group (Chk1siRNA2+cisplatin) was significantly lower than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P <0.05).3.The results of in situ hybridization and RT-PCR detection:the expression of Chk1mRNA in the esophageal cancer transplantation tumor tissues of B group (Chk1 siRNA2+cisplatin) was significantly lower than the C group (cisplatin) and A group (normal control group), compared two groups of two, all have statistical difference (P <0.05).4.The results of TUNEL method detection:the cells apoptosis index (AI) of group B(Chk1siRNA2+cisplatin) is obviously higher than that of the group C (cisplatin) and group A(normal control group), compared two groups of two, all have statistical difference (P<0.05).Conclusion1. High levels of Chkl protein and mRNA were observed in esophageal squamous cell carcinoma.2. The expression of Chkl protein and mRNA were closely associated with the invasion and metastasis of ESCC.3. Interfering expression vector pSilencer3.1-Chk1siRNA was successfully constructed.4. Chk1siRNA2has the best inhibiting effect.5. The expression of Chkl protein and mRNA can be cut by Chkl siRNA interference expression vector in vitro for people with esophageal cancer cells EC9706.6. Silencing Chkl expression in vitro can enhance the esophageal cancer EC9706cell proliferation and induce its apoptosis sensitivity of which cisplatin on.7. Successfully constructed nude mouse esophageal cancer transplantation tumor model.8. Silencing Chkl expression can strengthen the body of nude mouse esophageal cancer transplantation tumor growth inhibition which cisplatin on and induce the effect of cell apoptosis of transplantation tumor.