Long Intergenic Non-coding RNA 00467 Promotes Lung Adenocarcinoma Proliferation,Migration And Invasion By Binding With EZH2 And Repressing HTRA3 Expression

BackgroundThe incidence of lung cancer is increasing year by year.Lung cancer is divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Lung adenocarcinoma is the main type of NSCLC.Even though we have made great progress in the therapies including in surgery and chemoradiotherapy in recent years,most patients with lung adenocarcinoma(LAD)have limited survival.With the development of whole transcriptome sequencing,a large number of long non-coding RNAs(lnc RNAs)were discovered and functional annotations were initiated.At present,lnc RNA was closely related to human cancer,and its abnormal expression in cancer had important pathogenic consequences.ObjectiveIn the present study,we analyze the relationship between the expression level of linc00467 and lung adenocarcinoma in clinical tissue samples,and further observe the effect of abnormal expression of linc00467 on the proliferation,migration and invasion of lung adenocarcinoma in vitro.We aim to explore the possible molecular mechanisms to provide a theoretical basis for the pathogenesis of lung adenocarcinoma.MethodsBioinformatics analysis revealed abnormal expression of linc00467 in lung cancer tissues.q RT-PCR was used to detect the expression of linc00467 in paired NSCLC tissues and analyze its expression level and clinicopathological characteristics.We then selected three lung adenocarcinoma cell lines(A549,H1299,PC9)and a normal human normal bronchial epithelial cell line(16HBE)for linc00467 expression.Knockdown and overexpression of linc00467 in lung adenocarcinoma cell lines,the proliferation of cells were observed by MTT and colony formation assays,cells apoptosis were confirmed by flow cytometry,the migration and invasion of tumour cells were observed by transwell assay.Linc00467 was knocked down by Lentiviral(sh RNA)in H1299 cell line,and RNA transcriptome sequencing was used to find the downstream potential target gene HTRA3.The expression of HTRA3 in A549 cells and H1299 cells that linc00467 had been knocked down by lentivirus were further verified by Real Time PCR and western blot.The ability of linc00467 binding with EZH2 was verified by bioinformatics method and RNA immunoprecipitation(RIP)assay.Chromatin immunoprecipitation(Ch IP)assay was used to verify whether linc00467 regulated downstream target gene HTRA3 expression by binding with EZH2.Rescue experiments were used to investigate whether HTRA3 was involved in the linc00467 regulated in lung adenocarcinoma cell proliferation,migration,invasion.Finally,q RT-PCR was performed to detect the relative expression of HTRA3 in paired NSCLC tissues and analyze its expression level and clinicopathological characteristics.Results1.Analysis of the GEO and TCGA databases by bioinformatics revealed that the expression of linc00467 in lung cancer tissues was significantly up-regulated.In 35 pairs of matched lung adenocarcinoma tissues,linc00467 was up-regulated in lung adenocarcinoma tissues compared with normal lung tissues.Up-regulated linc00467 expression in lung adenocarcinoma was associated with larger and the later stage tumours.2.The ability of cell proliferation,migration and invasion was decreased and cell apoptosis was increased in linc00467 knocked down A549 and H1299 cells,which were contrast in linc00467 overexpressed PC9 cells.3.Downstream target gene HTRA3 was identified by RNA transcriptome sequencing,bioinformatics method was used to predict that linc00467 can recruit protein EZH2,RIP assay further verified that linc00467 could bind with EZH2.In addition,a Ch IP assay was used to verify that EZH2 bound to the promoter of HTRA3.The results demonstrated that linc00467 regulated HTRA3 expression by binding with EZH2.In the rescue experiment,HTRA3 knockdown partially reversed linc00467-depletion induced inhibition of LAD cell proliferation,migration and invasion.4.In 35 pairs of matched LAD tissues,HTRA3 was down-regulated in lung adenocarcinoma tissues compared with normal lung tissues.Down-regulated HTRA3 expression in LAD was associated with larger tumour sizes,increased lymph node metastasis and advanced TNM stages.ConclusionThis study showed that the expression of linc00467 in LAD was significantly higher than that in normal tissues,which indicated the linc00467 may serve as a biomarker of detection of LAD.We firstly proved that the linc00467 resulted in the increase of the proliferation,invasion and migration and decrease of apoptosis of LAD cells.It is demonstrated that the tumour-promoting role of linc00467 in LAD.The mechanism of linc00467 to promote proliferation,migration and invasion of LAD cells were through binding with EZH2 and to negatively regulate the expression of HTRA3.This provided a new theoretical foundation for elucidating the molecular mechanism of LAD.