| Expression of RMP in esophageal squamous cell carcinoma and its effect on cell biological functionObjective:By measuring the expression level of RMP(RPB5-Mediating Protein)in human esophageal squamous cell carcinoma tissues and cell lines,the relationship between RMP and the clinicopathological features of tumor patients was studied.At the same time,the effect of RMP on the biological function of esophageal squamous cell carcinoma was explored,which was a gene target of esophageal squamous cell carcinoma.Provide a strong basis for treatment.In view of these experiments,we examined the expression of RMP in esophageal squamous cell carcinoma,and we have knocked down or overexpressed RMP's effects on esophageal squamous cell carcinoma cell proliferation,invasion and other cell behaviors,and sensitivity to radiotherapy and chemotherapy.At the clinical level,we studied the prognosis of esophageal squamous cell carcinoma and the efficacy of postoperative chemotherapy.Through this study,we are expected to have a deeper understanding of the mechanisms of esophageal squamous cell carcinoma development and provide new evidence for the discovery of new molecular markers and prognosis of esophageal squamous cell carcinoma.Method:1.The m RNA expression levels of RMP in esophageal squamous carcinoma cell lines and normal cell lines were detected by q RT-PCR,and the corresponding protein expression levels of RMP were detected by western blot.2.The m RNA expression level of RMP in human esophageal squamous cell carcinoma tissues and corresponding paracancerous tissues was detected by q RT-PCR,and the corresponding protein expression level of RMP was detected by western blot.3.The expression level of RMP in esophageal squamous cell carcinoma tissueswas detected by q RT-PCR.The relationship between RMP and clinicopathological features was analyzed.4.Fluorescence microscopy was used to detect transient transfection of RMP plasmids in esophageal squamous cell carcinoma cells Eca109 and Ec9706.The transfection efficiency was detected by q RT-PCR and western blot.5.The effect of RMP on proliferation of Eca109 and Ec9706 cells was examined by CCK-8 assay.6.Eca109 and Ec9706 cells transfected with different RMP plasmids were treated with different concentrations of gefitinib.The effects of RMP on the chemosensitivity of Eca109 and Ec9706 cells were detected by CCK-8 assay and flow cytometry.7.The effects of RMP on radiosensitivity of Eca109 and Ec9706 cells were detected by CCK-8 assay and flow cytometry.Western blot was used to detect the protein expression level of apoptosis-related genes.8.Flow cytometry was used to detect the effect of RMP on the cell cycle of Eca109 and Ec9706.Western blot was used to detect the protein expression level of the cycle-related genes.9.The migration and invasion of Eca109 and Ec9706 cells were detected by migration and invasion assays.10.The effect of RMP on the growth of transplanted tumors was observed by subcutaneous xenografts in nude mice.The protein expression of RMP,Bax,Bcl-2 and capase-3 was detected by immunohistochemistry.11.Follow-up analysis of clinical patient data to study the prognosis of squamous cell carcinoma of the esophagus and evaluate the efficacy of postoperative chemotherapy.Result:1.The results of q RT-PCR and western blot showed that RMP was highly expressed in esophageal squamous cell carcinoma cell lines regardless of m RNA and protein expression levels.2.The results of q RT-PCR and western blot showed that the expression levels of m RNA and protein in human esophageal squamous cell carcinoma tissues were significantly higher than those in adjacent tissues.At the same time,it was found that the expression of RMP is related to the lymph node and T-stage status of tumor tissues and not related to the patient's age,gender,and tumor type.3.Fluorescence microscopy,q RT-PCR and western blot experiments showed that the transient transfection of the RMP plasmids in Eca109 and Ec9706 cells was smooth,and the expression levels of m RNA and protein increased correspondingly.4.The CCK-8 test showed that RMP significantly promoted the proliferation of Eca109 and Ec9706 cells.5.The CCK-8 test showed that RMP can still slow the death of Eca109 and Ec9706 cells under increasing dose of chemotherapeutic drugs.Flow cytometry showed that RMP can reduce chemotherapy-induced apoptosis.6.The CCK-8 test showed that RMP could still slow down the death of Eca109 and Ec9706 cells under increasing doses of radiation.Flow cytometry showed that RMP could reduce apoptosis induced by radiotherapy,Western blot showed that,After radiotherapy,RMP can inhibit the expression of apoptosis-related gene proteins in Eca109 and Ec9706 cells,and promote the expression of anti-apoptosis-related gene proteins.7.Flow cytometry showed that: RMP can reduce the cell cycle G2 arrest of Eca109 and Ec9706 caused by radiotherapy.Western blot showed that after radiotherapy,RMP can regulate the expression of cell cycle related genes in Eca109 and Ec9706.8.Migration and invasion experiments showed that RMP can promote the migration and invasion of Eca109 and Ec9706 cells.9.Nude mice subcutaneous xenograft experiments showed that: RMP can promote the growth of transplanted tumors,while immunohisochemistry showed that,RMP can inhibit the expression of apoptosis-related gene protein,promote the expression of anti-apoptosis-related gene protein,thereby promoting tumor growth.10.Statistical analysis of clinical data showed that high RMP expression suggests that the survival of esophageal squamous cell carcinoma is short,and may be an indicator of poor prognosis,and may indicate resistance to platinum drugs.Conclusion:1.RMP is highly expressed in human esophageal squamous cell carcinoma,suggesting that RMP may be closely related to the growth and malignancy of esophageal squamous cell carcinoma,and it is a potential promoter of cancer associated with esophageal squamous cell carcinoma.2.Overexpression of RMP can alter the biological functions of Eca109 and Ec9706 such as cell proliferation,apoptosis,cycle,migration and invasion,promote the growth of xenografts,and further demonstrate that RMP plays the role of oncogene in esophageal squamous cell carcinoma.We also provide a strong target for gene-targeted therapy for esophageal squamous cell carcinoma.The prognosis of esophageal squamous carcinoma and the efficacy of postoperative chemotherapy were evaluated by RMPObjective:The purpose of this study was to analyze the expression rate of RMP in esophageal squamous cell carcinoma,and to explore the effect of its expression on the prognosis of esophageal squamous cell carcinoma and its correlation with the efficacy of chemotherapy for advanced esophageal squamous cell carcinoma.Methods:SPSS13.0 statistical software was used for statistical analysis.Kaplan-meier method was used for survival analysis,and log-rank test was used for comparison between groups.COX risk ratio regression model was used for prognosis analysis.RMP and chemotherapeutic efficacy were compared using X and test.p<0.05 was considered as a statistical standard.Immunohistochemical method was used to detect the expression of RMP in the paraffin specimens of 59 patients,and the expression level of RMP was detected in different patients.Results:This group of 114 cases of esophageal squamous carcinoma patients with single factor analysis of overall survival: the number of lymph node metastasis,tumor new installment,expression of RMP,chemotherapy,tumor differentiation degree,smoking and BMI has a significant effect on the OS,P values were 0.003,0.032,0.039,0.018,0.022,0.022,0.069.Multivariate analysis results: only tumor staging was independent prognostic factor,P value was 0.016.The P value of RMP was 0.089.No statistical difference was found.Analysis of single factor analysis of disease-free survival showed that the number of tumor staging,lymph node metastasis,RMP expression,the P value of the vascular tumor and ECOG scores were 0.034,0.001,0.007,0.038 and 0.058,with statistical difference.Multivariate analysis: only stages and ECOG scores were independent prognostic factors,with P value of 0.019 and 0.037.The results of single factor analysis were no progress in survival: chemotherapyefficacy,number of chemotherapy cycles,whether cisplatin sufficient chemotherapy,gender,smoking,P value were 0.001,0.012,0.045,0.022,0.016.Multivariate analysis: only chemotherapy efficacy,whether cisplatin adequate chemotherapy is independent prognostic factor,P value is 0.000,0.043.RMP expression and advanced esophageal squamous carcinoma with cisplatin chemotherapy curative effect not see statistical correlation,but subgroup analysis cisplatin chemotherapy in patients with full amount RMP expression and curative effect of chemotherapy in chi-square test,p value is 0.038,with a statistically significant difference between,may prompt RMP positie cisplatin resistance,survival period is short,but it remains to be further confirmed.Conclusion:RMP high expression suggests that esophageal squamous cell carcinoma has a short survival period,which may be an indicator of poor prognosis and may prompt drug resistance to platinoids. |
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