Study On The Relationship Between Methylation Status Of KLF2 Gene And Non-small Cell Lung Cancer And Its Function

Lung cancer is still one of the most common malignancies in the world,and non-small cell lung cancer is the main type(about 80%).The number of new cases of lung cancer is 1.8 million per year,accounting for 13% incidence of cancer,therefore lung cancer has become a serious threat to our health.At present,most of lung cancer cases are diagnosed as advanced stage,which means limitations of treatment and poor prognosis.Therefore,searching for new biomarkers of lung cancer may be important for improving lung cancer early diagnosis,treatment options and clinical outcomes.Recent study has reported that most of cancers result from DNA replication errors,but lung cancerogenesis is a multi-process involving inheritance and environmental factors,and environmental factors is considered as the main cause of lung cancer.As known to all,epigenetic changes are closely related to the environmental factors.In addition,epigenetic changes are generally more than genetic changes,and these changes are mostly caused by environmental effect,which means that it can partially represent pathological changes caused by environmental factors.At the same time,tumorigenesis is also strongly associated with epigenetic changes.Tumorigenesis results from a large number of epigenetic changes,these changes regulate gene expression,and then change signal pathways thus leading to cells malignant transformation.DNA methylation is a common epigenetic regulation model,which can lead to gene silencing.DNA methylation can regulate gene expression in an epigenetic way,and this process is dynamically reversible,which suggests that changes in DNA methylation status can be used to monitor gene regulation and to be biomarkers for early diagnosis,prognosis and therapeutic response in cancer.Multiple tumor suppressor genes are hypermethylated in most precancerous lesions,which indicates that changes of methylation status have occurred in the early stages of tumorigenesis.As an early event in tumorigenesis,hypermethylation in gene CpG islands occurs before morphology and function changes of normal cell.KLF2,which is also known as the lung kruppel-like factor(LKLF),plays an important role in the late development of human lungs,which suggests that functional loss of KLF2 may associate with lung lesions.At the same time,the KLF2 gene is usually down regulated in various cancer tissues,and it can be used as a potential tumor suppressor gene to maintain cells normalization.Therefore,the lack of expression of KLF2 may be one of the tumorigenesis drivers.With bioinformatics analysis,we found that there is a Cp G island containing 267 Cp G sites in KLF2 gene sequence,which suggests that methylation modification in this island may regulate this gene expression.Therefore,this study will focus on exploring whether methylation of KLF2 CpG islands involved in the regulation of its expression in non-small cell lung cancer and also provide clues for KLF2 methylation status as an early diagnosis of non-small cell lung cancer.Methods:1.Fluorescence quantitative PCR was used to detect KLF2 expression in 47 non-small cell lung cancer tissues and 6 cell lines.2.Bisulfite sequencing PCR(BSP)was used to explore differential methylation region of KLF2 in non-small cell lung cancer cells and normal bronchial cells,and then methylation status was further confirmed by monoclonal sequencing.3.Demethylating reagent 5-aza-2-deoxycytidine was used to treat non-small cell lung cancer cell lines A549 and HCC827.Then expression of KLF2 was analyzed by fluorescence quantitative PCR.4.Double luciferase reporter assay was used to confirm function of KLF2 gene region 4(+ 567 to +906).5.Methylation-specific PCR(MSP)was used to detectet the methylation status of KLF2 gene in non-small cell lung cancer tissues and corresponding normal tissues.6.CCK-8 cell proliferation kit and cell clone formation assay was used to detect non-small cell lung cancer cell line survival and cloning ability changes after knockdown or re-expression of KLF2.7.Flow cytometry was used to evaluate the effect of KLF2 on cell cycle distribution and apoptosis in non-small cell lung cancer.8.Western blotting was used to detect the expression of p15 and p21 in non-small cell lung cancer cells after knockdown or over-expression of KLF2.Results:1.The expression of KLF2 in 31(31/47,65.96%)cases cancer tissues was lower than that in normal tissues,and it was correlated with lymph node metastasis and TNM staging.Compared with normal bronchial cells,KLF2 was also down-regulated in non-small cell lung cancer cell lines.2.Although the KLF2 gene promoter region did not show differential methylation in non-small cell lung cancer cell lines and normal bronchial cells,the KLF2 gene region 4(+567 to +906)had a differential methylation status.The monoclonal sequencing of BSP technology further confirmed that the methylation level of KLF2 gene region 4 in non-small cell lung cancer cell lines was much higher than that in normal bronchial cells.3.With the treatment of 5-methyl-2'-deoxycytidine,KLF2 was re-expressed both in A549 and HCC827 cell lines.4.Double luciferase reporter gene assay confirmed that KLF2 region 4 can promote luciferase gene expression,and when it was methylated,then inhibited gene expression.5.KLF2 gene region 4 was methylated in 27(27/47,57.44%)cases non-small cell lung cancer tissues.Expression levels of KLF2 in 26 of 27 cases were lower than that in the normal tissues.Therefore hypermethylation of KLF2 region 4 in non-small cell lung cancer was associated with down-regulation of its expression.At the same time,the methylation status of KLF2 region 4 was also associated with lymph node metastasis and TNM staging of non-small cell lung cancer.6.KLF2 over-expression can inhibit proliferation and clonal formation of non-small cell lung cancer cells.Correspondingly,sh RNA interference of KLF2 can promote cells proliferation and clonal formation.7.KLF2 can arrest non-small cell lung cancer cell lines in G0 / G1 phase,and also promote lung cancer cells apoptosis,KLF2 knock down will play the opposite role of cells.8.KLF2 can arrest cell cycle by induction of p15 and p21 expression in non-small cell lung cancer cell lines.Conclusion:In this study,we first assessed KLF2 m RNA level and methylation in NSCLC tissue s and cell lines.We then associated KLF2 methylation with clinicopathological data from NSCLC patients.After that,we focused on the region of KLF2 methylation as a cause of KLF2 silence in NSCLC cells and tissue samples and then manipulated KLF2 expression to determine the role of KLF2 in NSCLC cells in vitro.We found that KLF2 level was significantly decreased in NSCLC cells and tissue samples,while KLF2 was frequently methylated in NSCLC cells and tissue samples.Moreover,reduced KLF2 expression was associated with KLF2 region 4 hypermethylation in majority of NSCLC tissues,while KLF2 methylation at the region 4 was associated with NSCLC lymph node metastasis and advanced TNM stage.In addition,re-expression of KLF2 in NSCLC cell lines significantly suppressed tumor cell viability,arrested tumor cells at G0/G1 cell cycle via p15 and p21 expression,and promoted tumor cells to apoptosis.In contrast,knockdown of KLF2 expression had the opposite effects on SK-MES-1 cell line.Our current data indicate that KLF2 protein could function as a tumor suppressor in NSCLC and that detection of KLF2 methylation should be further evaluated as a tumor or prognostic biomarker for NSCLC.