| Background and purpose of the study:Research purpose and background:Non-small cell lung cancer(NSCLC)is the main cause of death from malignant tumors worldwide.China’s population aging has continued to increase in recent years.Coupled with environmental pollution,the increase in the proportion of smoking population,and the impact of second-hand smoke,the situation of NSCLC in our country was severe,with morbidity and mortality increasing year by year.HMGB1 protein has gradually progressed in oncology research,but many of its related mechanisms have not been carefully elucidated,and it is still in the preliminary exploration stage.In order to clarify the relationship between HMGB1 in NSCLC drug resistance,apoptosis pathway,and the role of HMGB1 expression in the invasion,proliferation,staging and prognostic analysis guidance of NSCLC,Silence or overexpression of HMGB1 explored its effect on the biological activity of lung cancer tumor cells.And according to the mechanism study of lung cancer drug resistance and apoptosis pathway,HMGB1 was used as a gene therapy target in clinical medicine.Its specific function and potential mechanism were still unclear,and we need to do further research.Experimental methods:1)This study collected 23 pairs of non-small cell lung cancer clinical tissues and corresponding paracancerous tissues,46 cases of non-small cell lung cancer patients with malignant pleural effusion and 31 cases of non-malignant pleural effusion,and conducted a general investigation of the patients;The mRNA and protein levels of HMGB1 in clinical tissues of non-small cell lung cancer and corresponding adjacent tissues were detected by western blot and real-time quantitative PCR;IL-1β,IL-6,IL-in pleural effusion were detected by ELISA method 8.The expression levels of HMGB1 and HMGB1 and the correlation analysis;Western blot was used to detect the resistance-related proteins MDR1,MRPs,P-gp,LRP and apoptosis proteins Bcl-2,Bax in the adjacent tissues,preoperative tissues and recurrent tissues,The change level of Caspase3.2)In this study,a drug-resistant cell line A549/DDP of non-small cell lung cancer A549 cells was constructed,and the mRNA and protein levels of HMGB1 in A549 and A549/DDP were detected by real-time quantitative PCR and western blot;cell transfection pcDNA3.1/HMGB1 and the application of G418 drugs were screened and HMGB1 stable transgenic cell lines were constructed in A549 and A549/DDP respectively;the MTT method was used to detect the effects of HMGB1 on cisplatin and doxyl in A549,A549-HMGB1 and A549/DDP cell lines.The influence of metoxetin,gemcitabine,pemetrexed and paclitaxel half maximal inhibitory concentration(IC50),and draw the curve of drug inhibition rate and drug resistance multiple of each group of cells,and analyze the correlation between HMGB1 and drug resistance.3)Screening and constructing a stable transfected cell line with knockdown of HMGB1 through siRNA-HMGB1 RNAi interference technology transfected cells,using real-time quantitative PCR and western blot to detect the knockdown efficiency;A549 and A549/DDP acting on cisplatin and paclitaxel Detect the effect of HMGB1 on cell IC50 and drug resistance index by MTT method;detect the effect of HMGB1 on cell proliferation by colony formation experiment;detect the effect of HMGB1 on cell migration and invasion ability by Transwell;detect HMGB1 by flow cytometry The effect on cell apoptosis;The effect of HMGB1 on cell apoptosis and drug resistance-related protein expression was further verified by western blot.4)A mouse lung cancer in situ model was constructed by subcutaneously injecting tumor cells into BALB/c nude mice.After the model was successfully constructed,cisplatin and paclitaxel were injected continuously for 3 weeks to detect the effect of HMGB1 on cisplatin and paclitaxel.The effect of HMGB1 on the pathological changes of lung cancer in situ in mice under the action of cisplatin and paclitaxel was detected by HE staining;the difference in HMGB1 expression levels in different groups of tissues was detected by immunohistochemistry;the difference in HMGB1 expression levels in different groups of tissues was detected by Tunel method Apoptosis of cells in tumor tissues of different groups of animals was compared with that of Nahua;Western blot was used to further detect the expression levels of apoptosis and drug resistance-related proteins in tumor tissues of the same group of animals.Results:1)The study found that among 46 cases of malignant pleural effusion,38 cases were lung adenocarcinoma and 8 cases were squamous cell carcinoma,and the HMGB1 content was not statistically significant among gender,age,and smoking;IL-6 in malignant pleural effusion group the three indicators of IL-8 and HMGB1 are higher than those of the non-malignant pleural effiusion group.It is believed that IL-6,IL-8 and HMGB1 may be markers of malignant pleural effusion of non-small cell lung cancer;non-small cells were found in clinical tissues HMGB1 mRNA in lung cancer tissue increased by 50%,and the corresponding drug resistance protein and anti-apoptotic protein increased with the increase of HMGB1.Compared with preoperative lung cancer tissue HMGB1,drug resistance protein and anti-apoptosis protein,the prognosis of chemotherapy tissue was Apoptosis proteins were significantly increased,indicating that HMGB1 has a certain relationship with drug resistance and apoptosis.2)In lung cancer resistant A549/DDP cells,the expression level of HMGB1 was significantly higher than the normal level of A549;and with the increase of HMGB1 expression,its resistance to different chemotherapeutic drugs was enhanced to varying degrees.The results showed that A549-The resistance indexes of HMGB1 cisplatin,gemcitabine,docetaxel,pemetrexed,and paclitaxel were 15.262,1.615,1.964,2.202,and 13.809;the resistance indexes of A549/DDP were 19.923,1.751,2.621,and 15.058.3)The IC50 values of A549 cisplatin and paclitaxel in the overexpression HMGB1 system were 1.832 and 0.869,the IC50 values of A549-pcDNA3.1 NC cisplatin and paclitaxel were 1.712 and 0.8257,and the IC50 values of A549-pcDNA3.1-HMGB 1 cisplatin and paclitaxel were 22.316,6.251;The IC50 of A549/DDP drug in interference transfection was 31.568,7.049,the IC50 of A549/DDP-siRNA NC drug was 30.206,6.870,and the IC50 of A549/DDP-si-HMGB1 was 14.895,4.788.Overexpression of HMGB1 enhances the invasion,metastasis and proliferation of lung cancer cells,inhibits the expression of pro-apoptotic proteins,and promotes the expression of resistance-related proteins.Silencing the expression of HMGB1 through RNAi technology can alleviate the above phenomenon.4)In animal experiments on mice,it was further verified that high expression of HMGB1 would enhance tumor size and cell invasion and migration ability,A549,A549-pc-DNA3.1 NC,A549-pc-DNA3.1-HMGB1,A549/DDP,The tumor volume of the mice in the A549/DDP-siRNA NC and A549/DDP-siRNA-HMGB1 groups was 0.315,0.271,0.519,1.063,0.924,0.528,when stimulated by cisplatin.The tumor volume formed under the stimulation of paclitaxel drugs was about 0.395,0.365,0.696,0.926,0.894,0.522;WB results showed that the pro-apoptosis-related protein was inhibited,and the drug resistance-related protein increased with the increase of HMGB1.At the same time,it was found that when HMGB1 is highly expressed,HMGB1 is mainly distributed in the cytoplasm,and the expression of resistance-related protein P-gp is also up-regulated.HMGB1 is mainly located in the cytoplasm when the expression of HMGB1 is low,and the expression of resistance protein is also down-regulated.Conclusion:HMGB1 plays an important role in NSCLC.It regulates the invasion,proliferation,drug resistance,and apoptosis of lung cancer cells.It has an indicative role in clinical staging and was promising as a new target for gene therapy point,contribute to the treatment of lung cancer. |
