The Expression And Cisplantin-resistant Influence Of High-risk HPV16/18E6 Protein In Lung Adenocarcinoma

Research Background:Lung cancer is one of the top morbidity and mortality among malignant tumors.Besides,the high cost of treatment,low quality of life for patients increased socioeconomic burden.With progressing in medical science and improving of technology facilitates in relevant diagnostic methods,introduction of new chemotherapy drugs,targeted drugs and biological agents,as well as gradually standardized and orderly diagnostic process.It consequently promotes a widespread application of comprehensive treatment in clinical practice.However,due to the environmental changes,individual differences,gene variation,drug resistance,etc.,there is still inadequate early diagnosis,resulting in the loss of the best opportunity for surgery.Therefore,chemotherapy is still the major therapeutic approach for lung cancer patients,and drug resistance poses a major threat to the success of chemotherapy.With respect to the above,it is urgent to clarify the responsible factors for drug resistance of chemotherapeutic agents from many aspects.Human papillomavirus(HPV)was discovered for the first time to be intimately associated with cervical cancer,which has been confirmed as a carcinogenic factor for it.With the development of research,HPV has been found to exist in respiratory system,digestive system,urinary system and reproductive system as well.Simultaneously,previous experiments also support that it may be partially responsible for the occurrence,development and treatment of tumors.Nevertheless,there are few studies focusing on the effect of High-risk HPV16/18E6 protein on the resistance of chemotherapeutic drug in lung adenocarcinoma.Objective:1.To analyze the positive expression and significance of High-risk HPV16/18E6 protein in clinical lung adenocarcinoma tissues and lung paracancer tissues.2.The lung adenocarcinoma A549 cells were transfected with overexpression recombinant plasmid HPV16E6-pEGFP-N1 in the experiment group(E6 group),in addition,the control group was constructed by transfecting corresponding empty plasmid(Ctl group),and without anything was conducted in the blank group(A549group).The present study was carried out to explore the cisplatin-resistant influence of High-risk HPV16/18 protein in lung adenocarcinoma cells.3.The different expression of E6,p53 and HIF-1α proteins were analyzed in E6 group,Ctl group and A549 group which were in order to explore the mechanism of cisplatin-resistant in human lung adenocarcinoma with overexpressing the High-risk HPV16E6 protein.Then,we hope to provide new ideas for enhancing the sensitivity of chemotherapeutic drug and improving the response to chemotherapy.Methods:1.A total of 60 cases with pathologically confirmed lung adenocarcinoma and adjacent specimens were collected in The first affiliated hospital of Chengdu medical college.The expression of HPV16E6 protein and HPV18E6 protein in lung adenocarcinoma and adjacent tissues were analyzed by immunohistochemistry.The correlation between HPV16/18E6 protein and clinical features was investigated by Pearson’s Chi-square(c2)test.2.These study have three different groups.Firstly,human lung adenocarcinoma A549 cells were transfected with overexpression plasmid HPV16E6-pEGFP-N1 in the experiment group(E6 group).In the second place,the control group was constructed by transfecting corresponding empty plasmid(Ctl group),and no treatment was conducted in the blank group(A549 group),and the expression leve was observed under the microscope.Western blot were used to detect the expression level and verify the stable overexpression of target plasmidb.We can choose the stable HPV16E6-overexpression lung adenocarcinoma A549 cell line for the next study.3.The absorbance of the three groups(E6 group,Ctl group,A549 group)of cells at wavelength of 490 nm was measured by MTT after 48 hours of culture by using cisplantin(DDP)at the same concentration gradient and under the same conditions.The inhibition rate and IC50 value of DDP to A549 cells in the three groups were calculated respectively,in order to analyze the relationship between the different outcome and the drug sensitivity of DDP.4.Western blot were used to detect the different expression level of E6,p53,HIF-1α in E6 group,Ctl group and A549 group,in order to explore the influence of cisplantin-resistant mechanism in human lung adenocarcinoma,which was expressed the High-risk HPV16E6 protein.Result :1.The positive expression rate of High-risk HPV16/18E6 protein was48.3%(n=29),however,the positive expression rate of adjacent specimens was18.3%(n=11).Obviously,HPV16/18E6 protein was highly expressed in lung adenocarcinoma tissues,and its expression level was significantly higher than that in adjacent lung tissues.The expression level of HPV16E6 protein was independent with gender,age and smoking,TNM stage and tumor size.2.The inhibition rate of E6 group was lower than Ctl group and A549group;Otherwise,the IC50 value of E6 group was higher than Ctl group and A549 group,there were statistically significant difference(P<0.05).Meanwhile,the difference of inhibition rate and IC50 value between Ctl group and A549 group was no statistically significant(P>0.05).3.There were different expression of E6,p53,HIF-1α in E6 group,Ctl group and A549 group.The expression level of E6 and HIF-1α protein in E6 group were higher than Ctl group and A549 group,but the p53 protein was lower than Ctl group and A549 group,there were statistically significant differencethere(P<0.05);At the same time,the different expression level of E6,p53 and HIF-1α protein between Ctl groupand A549 group was no statistically significant(P>0.05).Conclusion:1.The expression level of HPV16/18 E6 protein in lung adenocarcinoma is significantly higher than that in adjacent lung tissue,and its expression level is unrelated to the clinical characteristics of lung adenocarcinoma,such as: gender,age,smoking,TNM stage and tumor size(P>0.05).This study suggest that High-risk HPV16/18 maybe have some influence in lung adenocarcinoma,laying a foundation for further research on the role of High-risk HPV16/18E6 protien in lung adenocarcinoma.2.Overexpression of HPV16E6 had reduced the inhibition rate of chemotherapy drug and increased the IC50,showing that it can decrease sensitivity to chemotherapeutic drug(DDP)and increase drug resistance(P<0.05).3.E6 and HIF-1α protein were highly expressed in HPV16E6 overexpression group,and p53 proteinwere lowly expressed.It is suggested that the cisplantin-resistant mechanism of High-risk HPV16E6 protein in lung adenocarcinoma maybe:E6 can promote the HIF-1α expression by inhibiting p53.It remains to be further explored concerning the specific mechanism.