| BackgroundAccording to“Cancer statistics,2018”,in both male and female,lung cancer deaths is ranking the first place among all the cancer deaths,meanwhile,the five year survival of lung cancer is only 18%.With the development of technology,genomic screening and genetic testing have greatly increased the therapy effect,including target therapy and immunotherapy,which raise the importance of understanding the molecular mechanism in lung cancer development.Emerging evidence have supported the notion that lncRNAs?long noncoding RNA?exhibit essential regulatory function,such as the well characterized H19,Xist,MALAT1,HOTAIR and so on,by complex mechanism lncRNAs exert their function.LncRNAs have been proved to be pivotal regulators in epigenetic modification course,including the human bronchi epithelial cell?16HBE?malignant transformation course triggered by cigarette smoke extract?CSE?.We are curiosity about the mechanism by which lncRNAs attend in lung cancer development induced by cigarette smoke.Lnc-BMP1-1 locates in the intronic are of SFTPC,a gene encodes SPC protein,being relative the normal lung function of new born infant.SPC protein is uniquely synthesized and secreted by type?aveolar cells,which has been reported to be induced to become adenocarcinoma cells.Moreover,SFTPC is revealed to be associated with cigarette smoke and lung cancer,and its reported to be associated with the expression of ABCA3?ATP-binding cassette sub-family A member 3?and Caveolin-1?Cav-1?.Cav-1might be a vital factor in coping with oxidative stress induced by cigarette smoke and epigenetical regulation.It's verified that hyper-methylation and hypo-histoacetylation modification of Cav-1 could depress its transcriptional expression,meanwhile,Cav-1 is a candidate tumor suppressor gene for cancer prevention,being a membrane protein related with chemotherapy response,in which it's possible for lncRNAs to have functions,however,rare clues have been found.Lung adenocarcinoma occupy about 50%of lung cancer,A549,GLC82 and PC9 are representative cell lines of lung adenocarcinoma.Epigenetical regulation is important supplement to traditional genetic regulaiton,they are both common in regulatory network.HypothesisLnc-BMP1-1 is transcribed from SFTPC,a gene associated with normal lung function and lung cancer,we hypothesize that the abnormal expression of lnc-BMP1-1 is associatied with lung cancer development,meanwhile,cigarette smoke might cause the abnormal expression of lnc-BMP1-1,and thus the epigenetically dysregulation of downstream gene of lnc-BMP1-1 to increase the lung cancer risk.AimsAnalyse the expression of lnc-BMP1-1 in lung cancer population,as well its regulatory mechanism towards target gene?s?.Materials&Methods?.The expression of lnc-BMP1-1 in lung cancer population1.With qRT-PCR,the relative expression level of lnc-BMP1-1 were detected in 276 lung cancer tissues and adjacent non-cancerous tissues.2.T test is carried out to certain the different expression of lnc-BMP1-1 between lung cancer tissues and adjacent non-cancerous tissues.With Chi-square test,the expression of lnc-BMP1-1 and clinical characteristics of lung cancer patients were analyzed.?.The function of lnc-BMP1-1 in lung adenocarcinoma cellsTo understand the tumor suppressing function of lnc-BMP1-1,we use the lentivirus vectors of lnc-BMP1-1 over-expression to transfect the human lung cancer cell lines A549,GLC82 and PC9.Then cell viability test,wound scratch,trans well migration and nude mice tumor transplantation tumor experiment were further employed to make clear the influence of lnc-BMP1-1 on different malignant phenotype.?.Screen target genes for lnc-BMP1-1Combining bio-information and literature analysis,potential target genes regulated by lnc-BMP1-1 were detected in lung caner and its noncancerous tissues with qRT-PCR.To make clear the influence of cigarette smoke solution on the expression of lnc-BMP1-1 and target genes in 16HBEs as well as their co-expression relationship.The expression of lnc-BMP1-1 and target genes are detected with 16HBEs treated with cigarette smoke solution for 5th and 10th generations.VI.The influence of lnc-BMP1-1 on the transplanted tumor growth of nude miceTransplanted tumor growth of nude mice is an experiment which could further reinforce the lung tumor associated function of lnc-BMP1-1 in vivo.V.The mechanism of lnc-BMP1-1 in lung adenocarcinoma cells1.A549 cells were treated with 5-AZA and TSA,respectively,then compare the expression of lnc-BMP1-1,target genes,and DNMTs with untreated cells.2.The DNA methylation level in the promoter of target genes and histo-acetylation modification level influenced by overexpressed lnc-BMP1-1 were analyzed by MSP and western blotting assay.VI.The anti-cancer drug sensitivity influenced by lnc-BMP1-1 in A549 cells To understand whether lnc-BMP1-1 could enhance the sensitivity of A549 cells to anti-cancer drug,including DDP and Dox.Cell viability were detected by CCK8 kit after drug treatment.Results?.The expression of lnc-BMP1-1 in lung cancer population1.The expression of lnc-BMP1-1 is relatively decreased in about 83.3%lung cancer patients?P<0.001;P25=0.019,P50=0.044,P75=0.493 of lnc-BMP1-1 expression for lung cancer tissues,P25=0.225,P50=0.977,P75=2.222 of lnc-BMP1-1 expression for adjacent normal tissues?.2.The expression of lnc-BMP1-1 is associated with those patients possessing smoke history?P=0.048?,clinical progression?P=0.025?and distant metastasis?P=0.002?.?.The function of lnc-BMP1-1 in lung adenocarcinoma cellsOver expression of lnc-BMP1-1 had inhibited the cell proliferation,wound scratch and?or?trans well migration,and decreased of Bcl-2 protein of A549 and GLC82 cells;Over expression of lnc-BMP1-1 had inhibited nude mice tumor growth of A549 cells.?.Cav-1 is the target gene for lnc-BMP1-11.Combining bio-information and literature analysis,Cav-1 and NPR1 were potential target genes regulated by lnc-BMP1-1 and their expression were detected by qRT-PCR with tissue samples.The expression of Cav-1 is commonly downregulated in lung cancer patients?P=0.001;P25=0.001,P50=0.003,P75=0.040 for tumor tissues;P25=0.004,P50=0.012,P75=0.321 for normal noncancerous tissues?,being positively correlative with that of lnc-BMP1-1,the correlative efficiency is 0.211 (P=2.873×10-14).However,there's no difference of NPR1 expression distribution between lung cancer tissues and adjacent normal tissues?P=0.145?.2.Compare to NC cells,the Cav-1 protein expression of A549-BMP and GLC82-BMP cells are increased,non-expressed,respectively.The mRNA expression of Cav-1 has been increased and no change by over expressed lnc-BMP1-1.3.The expression of lnc-BMP1-1 and Cav-1 had been gradually decreased in 16HBE cells treated with cigarette smoke solution for 5th and 10th generations,which reinforce the co-expression relationship between lnc-BMP1-1 and Cav-1.IV.The influence of lnc-BMP1-1 on the transplanted tumor growth of nude miceOnly A549-BMP cells and corresponding control cells were selected to carry out the transplanted tumor growth of nude mice experiment.Compare to A549-NC cells,the tumor growth of nude mice has been inhibited by over-expressed lnc-BMP1-1 in A549-BMP cells.V.The mechanism of lnc-BMP1-1 regulating Cav-1 in A549 cells.1.The expression of lnc-BMP1-1,Cav-1 and DNMTs in A549 cells treated with 5-AZA and TSA,respectively,had been detected.Compared with untreated cells,histone-acetylation modification is predominant regulatory manner than DNA methylation modification.DNMT3a is influenced obviously by TSA treatment.2.A549 cells were selected into further understanding of epigenetic regulation of Cav-1 at transcriptional level by lnc-BMP1-1.Though the protein expression of DNMT3a had been decreased in A549-BMP cells,the DNA methylation level of Cav-1 promoter in A549 cells shows no significant difference.The expression of HDAC2 are decreased by lnc-BMP1-1,indicating a specific higher histo-acetylation modification level of Cav-1,which might promote the transcription of Cav-1.VI.The sensitivity of DOX has been increased by lnc-BMP1-1 in A549 cells Lnc-BMP1-1 is helpful to enhance the drug sensitivity of A549 cells to DOX,the cell viability has been decreased from 47%to 34%.Conclusion1.Lnc-BMP1-1 is proved to be relatively decreased in 83.3%patients,especially in those patients possessing smoke history?P=0.048?,being positively correlative with the survival time and the expression of Cav-1?P=0.001,coefficiency=0.211?.2.In A549 cells,Cav-1 is the target gene of lnc-BMP1-1.The co-expression relationship between lnc-BMP1-1 and Cav-1 are supportted by 16HBE cells treated with cigarette smoke solution.3.Up-regulating the expression of lnc-BMP1-1 in A549 cells?A549-lncBMP?could influence the histo-acetylation modification by decreasing the HDAC2 protein to promote the transcription of Cav-1 and increase the sensitivity to DOX,inversely, the cell growth,migration capability,protein expression of DNMT3a and BLC-2,as well as the tumor growth of nude mice are inhibited.Advantage&DisadvantageIt's the first time to report that lnc-BMP1-1 expression is associated with lung cancer development and exerts tumor suppressing function in A549 cells,moreover,cigarette smoke is found to be associated with lnc-BMP1-1 expression;meanwhile,the pro-histo-acetylation mechanism of lnc-BMP1-1 has been explored.However,there's limits in our study that the study between cigarette smoke and lnc-BMP1-1 might be further carried out in lung cancer population.SummaryGenerally speaking,cigarette smoke is probably contributed to the decrease of lnc-BMP1-1 and thus increase lung cancer risk.Lnc-BMP1-1 positively regulates the expression of Cav-1 via lowering the expression of HDAC2 protein and enhances drug sensitivity of DOX for lung cancer A549 cells. |
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