Effect Of DNASE1L3 On Proliferation, Apoptosis And Cisplatin Resistance In Lung Adenocarcinoma

BackgroundLung cancer is one of the most common malignant tumors in the world today.Non-small cell lung cancer（NSCLC）accounts for about 80%of lung cancer cases and is classified into three categories:squamous cell carcinoma（SCC）,adenocarcinoma（LUAD）and large cell carcinoma.Recently,driving genes such as EGFR、c-MET and ALK-EMK4 fusion genes have been reported to be involved in abnormal proliferation and apoptosis of lung adenocarcinoma（LUAD）cells,and multiple drugs targeting these driving genes have also been developed and used in LUAD molecular targeted therapy.Cisplatin（DDP）,as a first-line chemotherapeutic agent for lung cancer,often leads to difficulty in subsequent treatment,drug resistance hinders the efficacy of DDP in the treatment of lung cancer.Hence,it is important to study the way can reverse molecular targets associated DDP drug resistance for chemotherapy patients.At the same time,to explore the biological function and mechanism of LUAD resistance-related molecules,and lay the foundation for the study of the occurrence,development and resistance mechanism of lung cancer.DNASE1L3（deoxyribonuclease 1 analogue 3）is a member of the deoxyribonuclease 1 family,whose encoded protein hydrolyzes DNA,is not inhibited by actin and mediates DNA decomposition during apoptosis.A mutation in this gene is the cause of systemic lupus erythematosus16,so it is also known as SLE16.The existing literature reports show that DNASE1L3 degrades during apoptosis of multiple cell lines DNA,and promotes the division between nucleosomes during apoptosis.and DNASE1L3 biological function in LUAD has not been reported.Research purposes1.Defining the expression of DNASE1L3 in NSCLC lung cancer cells and tissues2.Defining the biological role of DNASE1L3 in LUAD cells3.Definingthe regulation of DNASE1L3 on PI3K/AKT pathway,cellcycle and apoptosis-related factors4.Screening for differential expression mRNA after overexpression DNASE1L35.Defining the biological effects of differential expression mRNA on upstream genesResearch methods1.Defining the expression of DNASE1L3 in non-small cell lung cancer cells and tissues1.1The expression of DNASE1L3mRNA in LUAD tissue and paracancerous lung tissue was analyzed statistically using Graphpad prism 6.1.2The endogenous expression levels of DNASE1L3 protein in immortalized bronchial epithelium 16 HBE and NSCLC lines H1299,A549,SPCA1,H460 and H1975 were examined by Western blot experiments.1.3 The expression of DNASE1L3 protein in LUAD tissue and paracancerous lung tissue was detected by immunohistochemistry,and the correlation between expression level、prognosis and clinicopathological parameters was analyzed.Graphpad prism 6was used for statistical analysis.2.Effects of DNASE1L3 on biological function of LUAD Cells2.1 To construct a stable overexpression cell line for lung adenocarcinoma,infect the lung adenocarcinoma cell lines H1299 and A549 with lentiviral suspensions packed with LV-NC and LV-DNASE1L3,use puromycin for drug screening,and use WB test to detect protein levels Overexpression efficiency;transiently transfected the overexpressed DNASE1L3 plasmid in H1299 and A549 cell lines,respectively,and used WB test to detect protein level overexpression efficiency.2.2 After overexpressing DNASE1L3 in H1299 and A549 cell lines,the effects of DNASE1L3 on cell growth and proliferation ability were observed by CCK8experiment,plate clone formation experiment,cell cycle and nude mouse subcutaneous tumor formation experiment;DNASE1L3 was observed by flow apoptosis and IC₅₀ experiment Effects on apoptosis and drug resistance.2.3 Transient si-DNASE1L3 in stable transfectants H1299-LV-DNASE1L3 and A549-LV-DNASE1L3 respectively,and use WB experiment to detect the interference efficiency of protein level;2.4After transiently transducing si-DNASE1L3 in stable transfectants H1299-LV-DNASE1L3 and A549-LV-DNASE1L3,respectively,the effects of DNASE1L3 on cell proliferation,apoptosis and drug resistance were observed by CCK8 experiment,flow apoptosis and IC₅₀ experiment.3.Defining the regulation of DNASE1L3 on PI3K/AKT pathway,cell cycle and apoptosis-related factors3.1After overexpression of DNASE1L3 in H1299 and A549 cell lines,Western blot experiments were used to detect changes in p-PI3K,p-AKT,c-JUN,apoptosis and cell cycle-related protein expression.3.2After transiently transducing si-DNASE1L3 in stable transfectants H1299-LV-DNASE1L3 and A549-LV-DNASE1L3,respectively,Western blot experiments were used to detect changes in the expression of p-PI3K,p-AKT,c-JUN,apoptosis and cell cycle-related proteins.4.Screening for differential expression mRNA after overexpression DNASE1L34.1 Use cell sequencing to screen for mRNAs that are differentially expressed after overexpression of DNASE1L3.4.2 qPCR experiments select mRNA with differential expression.4.3 After H1299 and A549 cell lines overexpressed DNASE1L3,the protein expression of differentially expressed mRNA was detected by Western blot.5.DNASE1L3 Effects of SOX4 on Proliferation,Apoptosis and Drug Resistance of LUAD Through PI3K-AKT-C-JUN Pathway5.1 Use the bioinformatics website to predict the upstream transcription factor of SOX4,and find a c-JUN binding site in the promoter region upstream of it;5.2 Transient si-DNASE1L3 and si-SOX4 in stable transfectants H1299-LV-DNASE1L3 and A549-LV-DNASE1L3 were:H1299-LV-DNASE1L3+si-DNASE1L3+si-SOX4 group（experiment group 1）,H1299-LV-DNASE1L3group（control group 1）,H1299-LV-DNASE1L3+si-DNASE1L3 group（control group 2）;A549-LV-DNASE1L3+si-DNASE1L3+si-SOX4 group（experiment group 2）,A549-LV-DNASE1L3 group（control group 3）,A549-LV-DNASE1L3+si-DNASE1L3 group（control group 4）.The effects of SOX4 on proliferation,apoptosis and drug resistance of DNASE1L3 in lung adenocarcinoma cells were observed using CCK8 experiment,flow apoptosis experiment and IC₅₀ experiment.Experimental results1.DNASE1L3 expression in non-small cell lung cancer cells and tissues1.1 Bioinformatics analysis of DNASE1L3 mRNA expression in lung adenocarcinoma tissue1.1.1 TCGA data analysis,the expression of DNASE1L3 mRNA in lung adenocarcinoma tissue（n=501）is lower than that in adjacent lung tissue（n=59）,the difference is statistically significant（t=15.14,P<0.0001）;1.1.2 Analysis of statistical results shows that the survival rate of patients with high expression of DNASE1L3 mRNA in lung adenocarcinoma tissue is higher than that of patients with low expression of DNASE1L3 mRNA,the difference is statistically significant（P=0.0022）;1.2 Western blot experiment results showed that the endogenous expression level of DNASE1L3 protein in 16HBE of immortalized bronchial epithelial cells was higher than that of non-small cell lung cancer cell lines;1.3 Detection of DNASE1L3 protein expression in lung adenocarcinoma tissue1.3.1 Immunohistochemical results showed that the protein expression level of DNASE1L3 in lung adenocarcinoma tissue（n=105）was lower than that in adjacent lung tissue（n=70）,the difference was statistically significant（X²=8.072,P=0.0045）;1.3.2 Analysis of statistical results showed that the survival rate of DNASE1L3 at high protein level（n=33）was higher than that of low expression（n=72）in lung adenocarcinoma tissue,and the difference was statistically significant（P=0.0313）;1.3.3 The results of statistical analysis show that the protein level of DNASE1L3 in lung adenocarcinoma is related to the patient’s tumor diameter（P=0.0001）.2.DNASE1L3 inhibits lung adenocarcinoma cell proliferation and cisplatin resistance,promotes lung adenocarcinoma cell apoptosis2.1 The results of CCK8 experiment,plate clone formation experiment,cell cycle and subcutaneous tumor formation in nude mice showed that after overexpressing DNASE1L3,the growth rate was slower than that of the control group（P<0.05）;the results of flow cytometry experiment showed The apoptosis rate was significantly higher than that of the control group（P<0.05）;IC₅₀ test results showed that the overexpression of DNASE1L3 significantly reduced the IC₅₀ value of the control group（P<0.05）.2.2 The results of CCK8 experiment showed that the growth rate was faster than that of the control group after interfering with DNASE1L3（P<0.05）;the results of flow apoptosis experiment showed that the apoptosis rate decreased after interfering with DNASE1L3（P<0.05）;the results of IC₅₀ experiment showed After overexpression of DNASE1L3,the IC₅₀ value was significantly higher than that of the control group（P<0.05）.3.DNASE1L3 regulates cycle and apoptosis-related proteins via PI3K/AKT/c-JUN3.1 Western blot results showed that after overexpression of DNASE1L3,the expression levels of p-PI3K,p-AKT,and c-JUN protein were significantly down-regulated compared to the negative control group,but there was no significant difference in the expression of PI3K and AKT protein;cyclin CDK2,CDK4 protein The expression level was down-regulated,and the p21 and p27 protein expression levels were up-regulated.3.2 Western blot results showed that after interfering with DNASE1L3,the protein expression levels of p-PI3K,p-AKT,and c-JUN were significantly increased compared with the negative control group,but there was no significant difference in the expression of PI3K and AKT protein;the expression of cyclin CDK2,CDK4protein The levels of p21 and p27 were down-regulated;the expression levels of apoptotic proteins caspase3,caspase6,caspase7 and c-PARP were significantly down-regulated,while the expression of PARP protein was not significantly different.4.Transcriptome sequencing to screen out differentially expressed mRNA after overexpression of DNASE1L34.1 Transcriptome sequencing screened the differential expression of 469 mRNAs between H12991-LV-NC and H12991-LV-DNASE1L3 between two groups,of which151 mRNAs were up-regulated and 318 mRNAs were down-regulated;4.2 qPCR results showed that after overexpressing DNASE1L3 in H1299 and A549cell lines,respectively,compared with the negative control group,the expression of SOX4 at the mRNA level was significantly reduced（P<0.05）;4.3 Western blot results showed that after overexpressing DNASE1L3 in H1299 and A549 cell lines,respectively,the expression of SOX4 at the protein level was significantly down-regulated relative to the negative control group.5.DNASE1L3 regulates the effect of SOX4 on proliferation,apoptosis and drug resistance of lung adenocarcinoma through PI3K-AKT-c-JUN pathway5.1 The bioinformatics website predicted that c-JUN is a transcription factor for SOX4;5.2 The results of CCK8 experiment showed that the growth rate of experimental group 1 and 2 was significantly slower than that of control group 2 and 4（P<0.05）;the result of flow apoptosis experiment showed that experimental group 1 and 2 were withdrawn compared with control group 2 and 4 The mortality rate was significantly increased（P<0.05）;IC₅₀ test results showed that the IC₅₀ values of experimental groups 1 and 2 were significantly lower than those of control groups 2 and 4（P<0.05）.Conclusions1.DNASE1L3 is lowly expressed in non-small cell lung cancer cell lines and lung adenocarcinoma tissues.The protein expression level of DNASE1L3 was negatively correlated with T staging in patients with lung adenocarcinoma;2.Overexpression of DNASE1L3 inhibits the proliferation and drug resistance of lung adenocarcinoma cells and promotes apoptosis of lung adenocarcinoma cells;interferes with DNASE1L3 to promote the proliferation and drug resistance of lung adenocarcinoma cells and inhibits apoptosis of lung adenocarcinoma cells;3.DNASE1L3 regulates cycle and apoptosis-related proteins through PI3K/AKT/c-JUN;4.After overexpressing DNASE1L3,the expression of SOX4 can be down-regulated;5.SOX4 can reverse the proliferation,apoptosis and drug resistance of DNASE1L3 in lung adenocarcinoma cells.