| Background and Objectives Esophagus cancer is one of the common gastrointestinal tumors.About 350,000 people die from esophageal cancer every year in the world.The morbidity and mortality rates vary greatly from country to country.Henan Province of China is one of the areas with a high incidence of esophageal cancer in the world.There are more men than women,and the age of onset is more than 40 years old.The typical symptom of esophageal cancer is progressive dysphagia.First,it is difficult to swallow dry food,then semi-liquid food,and finally water and saliva cannot be swallowed.Esophageal cancer is a malignant tumor that occurs in the epithelial tissue of the esophagus.Esophageal cancer is likely to be cured in the early and mid-term,but it is difficult to be cured in the late stage.The main treatment methods include chemotherapy,targeted therapy,surgery,as well as radiotherapy.However,due to traditional treatment methods such as surgery,chemotherapy and radiotherapy still have the problems of high damage and multiple toxic side effects,which brings great pain to patients and the curative effect is not satisfactory.In recent years,with the enrichment of biological treatment methods such as targeted therapy and the improvement of diagnosis and treatment technology,the treatment effect and survival rate of esophageal cancer have improved to a certain extent.TNF-related apoptosis-inducing ligand(TNF-related apoptosis)-inducing ligant,TRAIL)is a member of the TNF family of apoptotic molecules.It plays a role by specifically binding to cell membrane surface receptors.The combination with TRAIL-R1(DR4)and TRAIL-R2(DR5)can transmit death signals and activate Caspase to induce apoptosis,and decopy receptor 1(Dc R1)and decoy receptor 2(Dc R2)can competitively bind with TRAIL to inhibit TRAIL apoptosis induction.Because Dc R1 and Dc R2 are both expressed in normal cells,and only expressed in a few tumor cells.So TRAIL can specifically induce tumor cell apoptosis,but does not affect normal cells.Studies have shown that TRAIL not only promotes colon cancer,lung cancer,liver cancer,ovarian cancer and other solid tumors,also induce apoptosis of blood system tumor cells including multiple myeloma,leukemia and esophageal cancer.At the same time,TRAIL can also enhance the chemotherapy efficacy of esophageal cancer and improve drug sensitivity.However,due to the diverse sources of esophageal cancer cells and a high degree of heterogeneity,different esophageal cancer cells have different sensitivity to TRAIL.Therefore,it is necessary to further carry out relevant research on TRAIL in the treatment of esophageal cancer.In this topic,firstly,q RT PCR,immunohistochemistry and Western blot were carried out to detect the level of TRAIL in esophageal cancer tissues and cells,and the relationship between the expression level of TRAIL in the esophageal tissue and the pathological parameters of patients with esophageal cancer was analyzed,and the esophageal cell lines with stable overexpressing TRAIL were established,and cell function experiments studied that the effect of TRAIL overexpression on esophageal cancer cell proliferation,apoptosis,invasion and EMT.Finally the nude mouse transplantation tumor models were constructed by EC9706 cells,thereby discussing the effect of TRAIL overexpression on growth of esophageal cancer transplantation tumor.The experimental methods and results are as follows: (1)The expression level of TRAIL mRNA in 21 cases of esophageal cancer and paracancerous tissues was detected by q RT-PCR.(2)SPSS software was performed to analyze the relationship between TRAIL expression in esophageal cancer tissues and pathological parameters of patients.(3)The expression of TRAIL protein in esophageal cancer tissues was determined by immunohistochemistry assay.(4)Western blot was implemented to detect TRAIL protein expression in esophageal cancer cell lines TE-7,Eca109,EC9706 and HYSE450 and normal esophageal epithelial cells HET-1A.(5)The TRAIL fragments were amplified by PCR and digested by Eco RI and Not I.The digested TRAIL fragment were ligated into the p CDH-CMV-MCSEF1-cop GFP vector.The ligated product was transfected into DH5α cells,the transformed monoclonal colonies for identification was picked up,and finally Lenti-TRAIL recombinant lentiviral expression vector was extracted from the cells.(6)Packaging plasmids(p CMV-VSVG and p CMV-8.2)and lentiviral vectors(control vector Lenti-con and TRIAL overexpression vector Lenti-TRIAL)were co-transfected into HEK293 to package lentivirus.Lentivirus concentration and titer determination were performed.(7)Lentivirus was applied to infect esophageal cancer cell lines EC-9706 and Eca109,and Western blot were used to measure the efficiency of lentivirus infection.(8)After esophageal cancer cell lines were infected with Lenti-TRAIL viruses,MTT assay was used to detect cell proliferation.(9)After the esophageal cancer cell lines were infected with Lenti-TRAIL,the apoptosis rate was detected by flow cytometry assay.(10)After the esophageal cancer cell lines was infected with Lenti-TRAIL,the change of cell invasion ability was detected by Transwell assay.(11)Western blot was performed to detect the effects of TRAIL overexpression on the expression of PCNA,Caspase-3,Bcl-2,MMP-2,MMP-9 and EMT-related proteins N-cadherin,E-cadherin,and Vimentin in esophageal cancer cell lines.Methods: (12)EC9706 cells were applied to construct the xenograft model in nude mice to analyze the influence of TRAIL overexpression on the growth of esophageal cancer transplantation tumor.(13)The expression level of TRAIL mRNA in the transplanted tumor of esophageal cancer was detected by q RT-PCR.(14)The influence of TRAIL overexpression on the expression of PCNA protein in esophageal cancer transplantation tumor was detected by Western blot.(15)SPSS17.0 software was used to perform statistical analysis.The data were expressed as mean ± standard deviation(Mean ± SD),and analysis of variance or t test was performed.The difference was significant when P < 0.05.Results:(1)Compared with paracancerous tissues,TRAIL mRNA expression level was significantly reduced in esophageal cancer tissues.(2)TRAIL expression had no significant relationship with age,gender and tumor grade of patients,but was related to lymph node metastasis and distant metastasis.(3)TRAIL protein was expressed in the cell membrane,and the positive expression rate in esophageal cancer tissues was significantly lower than that in adjacent tissues.(4)Compared with normal esophageal epithelial cells HET-1A,TRAIL protein expression in esophageal cancer cell lines TE-7,Eca109,EC9706 and HYSE450 was significantly decreased,especially in Eca109 and EC9706 cells.Therefore,these two types of cells were selected for functional study.(5)The recombinant lentivirus expression vector of Lenti-TRAIL was obtained.The Lenti-con titer was 2×107 IFU/m L,and Lenti-TRAIL titer was 2.8×107 IFU/m L.(6)In the esophageal cancer cell lines EC9706 and Eca109,and the proportion of cells infected with the TRAIL overexpression virus Lenti-TRAIL was 83.25 ±3.31%(EC9706)and 86.75 ± 8.31%(Eca109).Western blot detection of TRAIL protein expression level showed that Lenti-TRAIL could significantly increase the TRAIL expression level in esophageal cancer cell lines Eca109 and EC9706 by 2-4 times.(7)MTT assay showed that TRAIL overexpression could significantly affect the activity of EC9706 and Eca109 cells and inhibit cell proliferation.(8)Overexpression of TRAIL gene could significantly promote apoptosis of EC9706 and Eca109 cell compared with the control group.(9)Overexpression of TRAIL gene could obviously inhibit invasion of EC9706 and Eca109 cells compared to the control group.(10)Compared with the control group,PCNA,Bcl-2,MMP-2,MMP-9,N-cadherin and Vimentin protein levels were significantly decreased in EC9706 and Eca109 cells,while Caspase-3 and E-cadherin protein levels were significantly increased.(11)In transplanted tumors of esophageal cancer,TRAIL mRNA level was significantly increased in the overexpression group of TRAIL.Overexpression of TRAIL gene could significantly inhibit the expression of proliferation-related protein PCNA,thus inhibiting the growth of transplanted tumors.Conclusion:The expression level of TRAIL was significantly decreased in esophageal cancer tissues and cells,and the overexpression of TRAIL could inhibit the proliferation and invasion,EMT in esophageal cancer cells and the growth of transplanted tumor,and promote cell apoptosis. |
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