| 【Background】As the major risk factor of lung cancer,smoking plays a non-negligible role in the performance of high mortality of lung cancer in China.4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)is one of the strong carcinogens in tobacco specific nitrosamines(TSNA),which has been proved having the ability to induce multiple cancers,especially lung cancer,in different animals by animal experiments.Also,lots of epidemiological researchs and experimental researchs found that there is a tight connection between the lung cancer’s progress and the exposure of NNK caused by smoking.DNA methylation is a process using DNA methyltransferase to actuate a chemical modification on specific cites of DNA sequence.DNA 5m C methylation widely exists in the occurance and development of lung cancer so that it is the earliest one to be discovered and the deepest one to be studied in the epigenetic regulatory mechanisms.Numerous in vivo and in vitro experiments have proved that promoters’ methylation would change the expression of m RNA then influence the phenotype of cells through some pathways and induces or promotes cancer development eventually.Although there are several possible carcinogenic mechanisms of NNK have been found by researchers currently,evidences we have can only tell us the way NNK play its’ carcinogenic role is complex because we sure that NNK and its’ metabolites take part in the changes of many cancer signaling pathways.How and in which way does NNK regulates signaling pathyways still remains a puzzle,but plenty of study results support that NNK would affect the DNMTs and lead to genomic aberrant methylation and this may be the key mechanism for NNK to impel cancers.In the past decades,with the deepening of scientific researchs,the correlation between non-coding RNA(ncRNA),especially long non-coding RNA(lncRNA),and lung cancer is well kown by people.Abnormal changes in lncRNA are related to the occurrence and development of lung cancer.However,it has not been reported that whether NNK induces the abnormal methylation of lncRNA-encoding genes’ promoter and the role or mechanism in the process of lung cancer induced by NNK.【Objective】In this study,NNK-induced malignant transformation of human lung bronchial epithelial Beas-2B cells was used as the test cells to explore the role and mechanism of NNK-induced abnormal changes of DNA methylation in non-coding RNA genes’ promoter.Specifically investigated the mechanism of NNK-induced malignant transformation of normal lung bronchial epithelial cells from two epigenetic regulations:DNA megtylation and non-coding RNA.In this way,we aim to provide experimental evidences and new research ideas for finding epigenetic biomarkers in the occurrence,development and prognosis of lung cancer.【Methods】1.Using Western blot to verify the expression of DNA methyltransferase DNMT1 in cells.2.Detect the presence of abnormally methylated lncRNA molecules in the promoter region by Arraystar Human 4×180 K ncRNA Promoter Arrays gene chip technology and use Promoter＿Classification,Peak D M’Value,Peak Score as parameters to screen out the lncRNAs which have hypermethylated promoter.3.Examine the expression level of selected lncRNAs by q RT-PCR,screen for highly methylated and low expression lncRNA AC007255.8 and verify in lung cancer tissues.4.Validate the abnormal methylation of lncRNA promoter region by methylation specific PCR(MSP)and determine the approximate methylation site,and verify in lung cancer tissues.5.Analyze the changed m RNA molecules in 2B-NNK cells by RNA-Seq and screen for genes related to apoptosis such as Bcl-2 according to KEGG pathway enrichment analysis and differential expression analysis results.6.Overexpression of lncRNA AC007255.8 by lentivirus transfection in the condition of MOI=200,transfection time=24h.7.q PCR verified the change of m RNA expression levels of Bcl-2 and other apoptosis-related genes in 2B-NNK cells before and after overexpression of AC007255.8;Western blot examined the protein expression of Bcl-2 before and after overexpression of lncRNA AC007255.8 in 2B-NNK cells;Ed U kit detects changes in the proliferation ability of 2B-NNK cells before and after overexpression of AC007255.8;Flow cytometry detects changes in 2B-NNK cell apoptosis before and after overexpression of AC007255.8【Results】1.Western blot results showed that compared with 2B-C group,the expression of DNA methyltransferase DNMT1 in 2B-NNK cells was significantly increased.2.Arraystar Human 4×180 K ncRNA Promoter Arrays genechip outcomes showed that there were 1010 differentially methylated fragments in the lncRNA promoter region of the 2B-NNK group,including 323 low-methylated fragments and 687 high-methylated fragments.3.q RT-PCR results demonstrated that LOC442497,LOC100132354,LINC00638,LOC401557,AC007255.8,BZRAP1-AS1,CEBPA-AS1 showed a low expression trend in the 2B-NNK group compared with the 2B-C group(P < 0.05).4.MSP results supported that the methylation degree of target region 3 in the promoter region of AC007255.8 in 2B-NNK cells is higher than that in 2B-C.5.The expression level of AC007255.8 in lung cancer tissues is lower than that in adjacent tissues(P < 0.05).6.RNA-Seq outcomes revealed that compared with 2B-C cells,there were 1387 differentially expressed m RNAs in 2B-NNK cells,of which 500 were highly expressed and 837 were low expressed.7.Ed U results proved that the proliferative capacity of 2B-NNK cells increased significantly compared with group 2B-C cells,and it decreased after the overexpression of AC007255.8(P < 0.05).8.The results of q RT-PCR and Western blot certified that compared with 2B-C cells,m RNA and protein expression of apoptosis-related protein Bcl-2 in 2B-NNK cell increased significantly,and obyiously decreased after the overexpression of AC007255.8(P < 0.05).【Conclusion】1.NNK induces an increase in the protein level of methyltransferase DNMT1 in Beas-2B cells and promotes abnormal methylation of the lncRNA promoter.2.NNK-induced malignant transformation of the lncRNA AC007255.8 promoter region in Beas-2B cells and lung cancer tissues showed hypermethylation and was associated with a decrease in its expression level.3.lncRNA AC007255.8 can inhibit the proliferation of 2B-NNK cells and play the role of tumor suppressor gene in lung cancer by regulating the expression of anti-apoptotic protein Bcl-2. |
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