| Background and purpose:Esophageal cancer is one of the most common digestive tract malignancies worldwide.In recent years,esophageal cancer has greatly advanced in clinical treatment,but its five-year survival rate remains low and the prognosis is poor.Although smoking has been widely shown to promote the occurrence of esophageal cancer,the mechanisms of cigarette smoke-induced malignant transformation have been unclear.In this study,we originally explored the molecular mechanism of cigarette smoke-induced esophageal carcinogenesis by constructing a cell model to provide a scientific theoretical basis for the prevention and treatment of esophageal cancer and its precision treatment.Materials and methods:(1)Establishment of cell model of chronic exposure to cigarette smoke:After the preparation of cigarette smoke extract(CSE),CCK-8 test was used to detect the survival of SHEE cells after acute exposure to CSE to determine the concentration of chronic exposure to cigarette smoke,and construct the SHEE cell model of chronic exposure to cigarette smoke in different passages.SHEE cells(P30-CSE and P30-DMSO)treated for 30passages were examined by clone formation assay,subcutaneous tumorigenicity in nude mice and transwell assay to detect cell malignant transformation.the m RNA relative expression involved in epithelial mesenchymal transition(EMT)were detected by RT-q PCR.(2)High-throughput microarray screening candidate lncRNAs:P30-CSE and P30-DMSO cell lines was extracted RNA,and the changes of lncRNA relative expression were detected by Human lncRNA Expression Microarray V4.0.The differentially expressed lncRNAs were screened by bioinformatics analysis,which were validated using RT q PCR experiments in cell lines of different passages and patient tissues.(3)Analysis of the association between lncRNA H19 and clinicopathological characteristics of patients:Non parametric test was used to perform the association between the relative expression of H19 and clinicopathological characteristics of patients,and Spearman correlation analysis was used to explore the correlation between the relative expression of H19 and tumor size of patients.(4)DNA methylation changes of H19promoter were analyzed:The DNA methylation level of H19 CTCF6 was detected by pyrosequencing technology in both treated cell lines and tissue from patients with ESCC,and the relationship between the relative expression of H19 and the methylation level of H19CTCF6 was explored by Spearman correlation analysis.The relationship between DNA methylation level and clinicopathological characteristics of ESCC patients was performed by t-test.Results:(1)With acute exposure to SHEE cell,the chronic exposure to concentration was determined to be 15mg/ml.Compared with the control group(P30-DMSO),the tumorigenesis ability of the exposed group(P30-CSE)was significantly improved,indicating that cell model of chronic exposure to CSE had been successfully constructed in vitro.(2)High-throughput microarray showed that chronic exposure to cigarette smoke led to different expression profiles compared with normal tumorigenesis cells,and screened out a lot of differentially expressed lncRNAs.Further screening and verification revealed that in cell lines exposed to different passages(10,20,30 and 40),the relative expression of H19 gradually increased during exposure to CSE,while the relative expression of LINC02582 and LINC01133 gradually decreased.The relative expression of LINC00342 showed no significant statistic.(3)In tissue specimens from patients with esophageal squamous cell carcinoma,the relative expression of H19 in smokers was significantly higher than that in normal tissues(P=0.003),while there was no significant difference in non-smokers(P=0.395).There was a statistically significant difference in the relative expression between the two groups(P=0.015).Other three lncRNAs(LINC02582,LINC01133,and LINC00342)showed no significant difference.We also found that the relative expression of H19 was higher in the advanced TNM stage(stage III-IV)in smokers(P=0.045).The relative expression level of H19 was positively correlated with tumor size in smokers(r_s=0.373,P=0.039),while there was no corresponding trend in non-smokers(P=0.212).(4)In the cell model exposed to cigarette smoke,decreased DNA methylation levels at the CTCF6 binding site of the H19 promoter region were found,but no changes in tissue methylation levels were observed in smokers and non-smokers with esophageal cancer.DNA methylation levels were not significantly correlated with clinicopathological characteristics of the patients.Conclusion:Chronic exposure to cigarette smoke can significantly enhance the malignant transformation ability in esophageal epithelial cells.Esophageal immortalized cells chronically exposed to cigarette smoke had significantly different gene expression profiles from natural tumorigenic cells.LncRNA H19 may be involved to the tumorigenesis of esophageal squamous cell carcinoma induced by cigarette smoke,but the relative expression of H19 was not regulated by DNA methylation. |
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