| BackgroundIn 2018,the new and death cases of lung cancer accounted for 13% and 18% of malignant tumors respectively in the world,ranking the first among all malignant tumors.Lung cancer is also the most common malignant tumor in China,ranking first among male cancers and second among female cancers.Due to the high incidence and mortality of lung cancer,the disease burden indicators such as potential years of life lost(PYLL)and disability-adjusted life years(DALY)are on the rise,which directly affect the life expectancy of the population.Lung adenocarcinoma(LUAD)accounts for more than 40% of lung cancer,with a poor prognosis.Therefore,it is of great significance to study the molecular mechanism of LUAD.The gene of non-selective endothelin receptor B(EDNRB)is about 24 kb in length,located at 13q22,and contains 7 exons and 6 introns.The encoded protein,namely non-selective EDNRB,belongs to G protein-coupled transmembrane receptor protein.It is widely expressed in vascular endothelial cells such as cardiovascular system,gastrointestinal tract,lung,kidney,adrenal gland,uterus,prostate and brain.In the process of tumorigenesis and development,the chromosomal locus of EDNRB gene is a region where the gene deletion often occurs.Some studies have speculated that some tumor suppressor genes exist in this region and play a key role in the tumorigenesis and development.Up to now,there are few reports on the role of EDNRB in LUAD and its molecular mechanism.The main purpose of ‘Study on the function and mechanism of EDNRB gene in LUAD’ was to analyze the epidemic trend and disease burden of lung cancer deaths in Nanjing residents from 2013 to 2017,and further to predict and analyze lung cancer deaths in Nanjing by ARIMA model.Based on TCGA data analysis,the molecular mechanism of EDNRB gene in LUAD and its hypothesis affecting the survival of patients with LUAD were established.To study the effect of EDNRB on the survival of patients with LUAD by analyzing the clinical data and the expression of EDNRB in LUAD.Cell experiments and animal models were used to study the effects of EDNRB on the growth and migration of lung cancer cells,and the molecular mechanism of EDNRB on LUAD and its possible diagnostic and therapeutic value as a molecular marker were preliminarily discussed,providing scientific basis for exploring new targeted therapeutic drugs for LUAD and improving the outcome of patients with LUAD.Methods(1)Get the basic information of lung cancer deaths in Nanjing from January 1,2013 to December 31,2017 from the ‘China Disease Detection System Death Monitoring Network Report Database’.Calculate the crude mortality rate according to the total population,sex and age.Standardize the population data by sex and age in the fifth national census in 2000.The annual percentage change(APC)of malignant tumor mortality was estimated by Joinpoint software,and the death trend of lung cancer was analyzed;YLL and YLL rate were calculated by GBD’s simplified estimation method.Use ARIMA time series prediction model to predict lung cancer death in Nanjing.(2)Search TCGA data on ucsc(http://xena.ucsc.edu/)website,download RNA-Seq data of 31 kinds of cancers according to cancer classification,and study the expression of EDNRB in 31 kinds of common cancers and normal tissues as well as the normal tissue expression data in Genotype Tissue Expression(GTEx)database(https://gtexportal.org/home).Download relevant clinical information data of patients with LUAD from TCGA database,including survival time,survival state,age,sex,tumor stage,etc.Those patients with lack of prognosis information,incomplete clinical information or severe basic diseases were screened out and the data were deleted,and finally 996 samples were obtained for the following analysis.(3)Gene expression and grouping of EDNRB: The expression of EDNRB in 31 kinds of cancer tissues and normal tissues was analyzed using ggpubr R software package.The top 498 samples were the low expression group of EDNRB gene,and the remained 498 samples after sorting were the high expression group of EDNRB gene.Then,the survival of differentially expressed m RNA was analyzed by SPSS 20.0statistical software,and the survival states were estimated by Kaplan-Meier statistical model.Combining the expression data of EDNRB with relevant clinical data,COX risk ratio model was constructed to study the influence of EDNRB expression on the prognosis of patients.(4)Enrichment analysis: Based on the median expression of EDNRB in LUUDA,patients were divided into two groups,namely high expression of EDNRB and low expression of EDNRB,and then GSEA of EDNRB single gene was performed by cluster Profiler R package.(5)Path analysis: GO analysis and KEGG database analysis were carried out on the selected target genes of EDNRB by MSig DB database,and significance analysis of GO annotation was carried out by Cytoscape＿v.3.0 plug-in Enrichment Map,so as to obtain statistically significant high frequency annotation,signal transduction and disease pathway of gene set.(6)Clinical data analysis of patients with LUDA: The clinical data were collected from 168 patients with LUAD who underwent surgical resection in Zhongda Hospital Affiliated to Southeast University from January 2014 to December 2015.Specimens were handled by trained professionals.Pathological diagnosis was made by two pathologists after operation.The basic clinical data,such as gender,age,degree of differentiation,clinical classification and staging,were summarized by consulting the patient’s medical records.Finally,72 cases were finally included except for the cases without complete data and.In this study,the staging of LUAD was based on the eighth edition of TNM staging of non-small cell lung cancer of AJCC.IHC aasay was used for localization,qualitative and quantitative study,and the expression of EDNRB protein in cancer tissues of patients with LUDA was detected.(7)According to the proportion of positive stained cells in IHC,the patients were divided into two groups,namely EDNRB cells with high expression(positive stained cells > 50%)and EDNRB cells with low expression(positive stained cells ≤50%).For comparison of quantitative data,two independent samples were selected for T test.Qualitative data,including positive rate,demographic variables(gender,age)and clinical characteristics(tumor size,differentiation and stage),were analyzed by Chi-square test.Logistic regression was used for multivariate analysis.Kaplan-Meier and other statistical methods were used to analyze the survival curve.P < 0.05 was regarded as statistically significant.(8)The expression of EDNRB protein in lung cancer cell lines,including H1299,A549,h1650,H23,H460,H520 and h466 and BEAS-2B cells,were detected by Western blot.The expression of EDNRB protein in H1299 and H1299/EDNRB clones and normal human lung epithelial BEAS2 B cells were detected by Western blot.CCK-8 test was used to investigate the effect of EDNRB overexpression on the proliferation of H1299 cells.Scratch test was used to detect the effect of EDNRB overexpression on the migration of lung cancer cells.Furthermore,the difference of protein expression between H1299/EDNRB and H1299/vector was detected to explore whether EDNRB inhibited the activation of ERK and Akt signaling pathways,thereby affecting the migration and invasion of LUDA cells.(9)Tumor formation experiment: nude mice were inoculated subcutaneously with H1299/vector and H1299/EDNRB cell lines to establish tumor bearing model and evaluate tumor formation.Results(1)From January 1,2013 to December 31,2017,the number of deaths due to lung cancer in Nanjing was 14,693,with an average of 8.05 deaths every day,including 10,608 males(72.20%)and 4,085 female(27.80%)s,there was a significant difference between them.Most of them were over 60 years old(85.12%),those who died of lung cancer usually had low education level,and most of their occupations were farmers.The crude mortality rate of lung cancer increased from 43.17/100,000 to 47.21/100,000,and APC was 2.2%(P<0.001),showing an increasing trend of mortality rate year by year.The standardized mortality rate of lung cancer at different ages increased first and then decreased.The standardized mortality rate was the highest in the male at the age of 70-74 years old,and that was the highest in the female at the age of 75-79 years old.(2)In the past five years,the YLL of lung cancer in Nanjing residents was302,078.27 person ever year,and the YLL rate was 9.26 ‰.The YLL of male lung cancer was 218,309.05 person every year,the YLL rate of male lung cancer was13.36‰,the YLL rate of female lung cancer was 83,769.21 person every year,and the YLL rate of female lung cancer was 5.14 ‰.The YLL rate of male increased from12.89‰ to 13.80%(APC% = 1.9,P < 0.001),but there was no significant difference in the YLL rate of female(APC%=0.4,P=0.7).(3)Through expert modeling and manual debugging,the final model parameter was ARIMA(3,1,3),and the model was tested by white noise.The residual sequence ACF and PACF falled within 95% CI;The result of Ljung-Box Q test of residual sequence showed no statistical significance(statistic 11.54,P= 0.644).Based on the above two points,it was considered that the residual error of the model was a white noise sequence.The normalized minimum BIC index value of the model was 5.37,and the stationary R2 was 0.79.There was a significant difference in testing each parameter of the model(P<0.05).(4)A total of 996 specimens of LUAD were screened.The expression of EDNRB in LUA was 5.09±0.06,which was lower than that in normal tissues(7.74±0.13)(P<0.001).Kaplan-Meier results showed that the expression level of EDNRB was positively correlated with the survival curve of patients with LUAD(P=0.034),that is,LUAD patients with high expression of EDNRB may live longer.The results of multivariate analysis of the factors including gender,age,tumor size,TNM,stage and the expression of EDNRB in the EDNRB gene data set showed that clinical stage was related to the expression of EDNRB in patients.Gene enrichment analysis showed that the regulation of Erk and PI3K-Akt pathway ranked the first two places in TCGA data,suggesting that the expression of EDNRB may be closely related to the regulation of Erk and Akt pathways.(5)In 72 cases of LUDA,EDNRB was highly expressed in 15 cases(20.8%).The expression of EDNRB gene was correlated with the clinical stage of patients with LUDA(P<0.001),but there were no significant differences in gender,age,diabetes,hypertension,hyperlipidemia and smoking.Kaplan-Meier analysis showed that there was a positive correlation between EDNRB expression and survival time(P=0.034).(6)The expression of EDNRB protein in seven lung cancer cells(H1299,A549,H1650,H23,H460,H520 and H466 lines)was detected.The expression of EDNRB was the lowest in H1299 and H460 cells,and the highest in H1650 and H520 cells.The effect of EDNRB overexpression on H1299 cell proliferation was detected by CCK-8 assay,and the results showed that the cell proliferation of EDNRB overexpression group was lower than that of control group,there was a statistical difference(P<0.05).Scratch test showed that the migration rate of H1299 cells in EDNRB overexpression group was lower than that in control group(P<0.05).Transwell migration experiment showed that the number of migrating transmembrane cells in the overexpression group of EDNRB in H1299 cells was less than that in the control group(P<0.05).(7)The result of mouse tumorigenesis experiment showed that the subcutaneous tumor volume of mice inoculated with H1299/EDNRB cells was smaller than that of H129/Vector mice.The expression levels of p-Erk,p-Akt,MMP-9,MMP-2,SMA-a,Slug and N-cadherin in H1299/EDNRB group were significantly lower than those in H1299/Vector group.The protein levels of ZO1 and E-cadherin in H1299/EDNRB group were significantly lower than those in H1299/Vector group.Conclusion(1)From January 1,2013 to December 31,2017,lung cancer in Nanjing was mainly in male patients,mostly elderly.In recent years,the standardized mortality rate of lung cancer in Nanjing was stable.The standardized mortality rate increased and then decreased in different age groups,and the YLL rate of lung cancer death increased in different age groups.Notably,ARIMA time series prediction model could be used to predict the monthly death rate of lung cancer.(2)TCGA data mining showed that the expression of EDNRB was related to the prognosis of patients with LUDA.EDNRB is a tumor suppressor gene of LUDA.Overexpression of EDNRB in LUDA weakened cancer cell migration and invasion via the inhibition of Erk and Akt expression,which may be a possible molecular mechanism of EDNRB.TCGA-based data analysis could provide new clues and new ideas for the molecular mechanism research of related tumors.(3)The results of clinical data analysis and IHC showed that the expression of EDNRB in cancer tissues and its correlation with survival of patients with LUDA PAT were consistent with the results of TCGA data mining.The prognosis of LUDA patients with high expression of EDNRB was better than those with low expression.(4)The results of cell and mouse experimental models showed that EDNRB was a tumor suppressor gene of LUDA,through which influence the proliferation,migration and invasion of lung adenocarcinoma cells via Erk and Akt signaling pathways.Thus,he expression level of EDNRB has an important reference value as a molecular marker for the prognosis of LUDA... |
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