The Long Non-coding RNAs HNF1A-AS1、GAS5-AS1 Regulate The Proliferation And Metastasis In Non-small Cell Lung Cancer

Lung cancer remains the most common cause of global cancer-related mortality with an approximate 5-year survival rate of 15%,and non-small cell lung cancer(NSCLC)is its most common histological type,which accounts for 85%of lung cancer cases.Most of NSCLC are advanced or local advanced at the time of diagnosis because of the abnormal proliferation and metastasis of lung cancer cells.In the past decades,numerous studies have indicated that the mutations or increased copy number in certain protein-coding genes play an important role in the pathogenesis of NSCLC.However,recent advances in genome sequencing technology have showed that non-coding genes may also be emerging as new players in cancer development.Recent evidence indicates that long noncoding RNAs(IncRNAs)have a critical effect on the regulation of cellular processes,such as differentiation,proliferation,apoptosis and metastasis.Some lncRNAs are found to be dysregulated in lung cancer.The clinical significance of HNF1A-AS1 and GAS5-AS1,and their molecular mechanisms controlling NSCLC proliferation,migration and metastasis are unclear.Therefore,This study aims to investigate the role of HNF1A-AS1 and GAS5-AS1 in NSCLC development.In the first part,we investigate the expression pattern of HNF1A-AS1 in lung adenocarcinoma and its clinical significance as well as its biological role in tumor progression.In the second part,we demonstrate that decreased GAS5-AS1 expression may be regulated by histone modifications.We also investigated the expression pattern of GAS5-AS1 in NSCLC as well as its biological role in tumor progression.These data suggest that IncRNA could regulate gene expression and contribute to cancers development.This study will increase our knowledge to better understand the pathogenesis and development of NSCLC,which facilitates the development of lncRNA-directed diagnostics and therapeutics against NSCLC.Part Ⅰ The long non-coding RNA HNF1A-AS1 regulates proliferation and metastasis in lung adenocarcinomaAim:Long noncoding RNAs(lncRNAs)have emerged as key regulators of tumor development and progression.The IncRNA HNF1A-antisense 1(HNF1A-AS1)is a 2455-bp transcript on chromosome 12 with a potential oncogenic role in esophageal adenocarcinoma.Nevertheless,current understanding of the involvement of HNF1A-AS1 in lung adenocarcinoma tumorigenesis remains limited.The aim of this study is to examine the expression pattern of HNF1 A-AS 1 in lung adenocarcinoma and its clinical significance as well as its biological role in tumor progression.Methods and Materials:Real-time quantitative PCR and bioinformatics analysis was performed to detect the relative expression of HNF1A-AS1 in lung adenocarcinoma tissues and analyze its relationship with the prognosis of patients.We use loss of function approaches to investigate the biological functions of HNF1A-AS1.The effect of HNF1A-AS1 on proliferation was evaluated by MTT,colony formation assays,cell-cycle analysis and apoptosis.And transwell assays and vein injection of cells were used to study the invasion and metastasis of lung adenocarcinoma cells.Subcellular fractionation location of HNF1A-AS1 was determined by PARIS Kit.ChIP was used to investigate role of DNMT1 on regulation of E-cadherin in A549 cell.Bioinformatics analysis,qRT-PCR,RIP,western blot assays and immunofluorescence assays were used to investigate the underlying targets of HNF1A-AS1 in lung adenocarcinoma cells.Results are shown as means ± S.D.and differences were tested for significance using two-tailed student’s t-tests.Results:Our results showed that HNF1A-AS1 was significantly up-regulated in lung adenocarcinoma tissues compared with corresponding non-tumor tissues,and its expression level was significantly correlated with TNM stage,tumor size,and lymph node metastasis.The UCSC Cancer Genomics Browser’s Kaplan-Meier plot suggested that patients with the high HNF1A-AS1 expression subgroup experienced worse overall survival compared to the low expression subgroup.Moreover,HNF1A-AS1 was determined to promote tumor proliferation and metastasis,both in vitro and in vivo,by regulating cyclin D1,E-cadherin,N-cadherin and β-catenin expression.In addition,the binding of HNF1A-AS1 to DNMT1 may explain its regulation of E-cadherin.Conclusion:we demonstrated that increased HNF1A-AS1 expression could regulate cell proliferation and metastasis and identified it as a poor prognostic biomarker in lung adenocarcinoma.Part Ⅱ Downregulation of the long noncoding RNA GAS5-AS1 contributes to tumor metastasis in non-small cell lung cancerAim:Long noncoding RNA(lncRNA)plays pivotal roles in cancer initiation and progression.It involves in all hallmarks of cancer that define the malignant phenotype of tumor cells.To date,only a small number of IncRNAs have been characterized at the functional level.The aim of this study is to examine the expression pattern of GAS5-AS1 in NSCLC and its clinical significance as well as its biological role in tumor progression.Methods and Materials:Real-time quantitative PCR was performed to detect the relative expression of GAS5-AS1 in lung adenocarcinoma tissues.Gain of function approaches was performed to investigate the biological functions of GAS5-AS1.The effect of GAS5-AS1 on proliferation was evaluated by MTT,cell-cycle analysis and apoptosis.And transwell assays was used to study the invasion and metastasis of NSCLC cells.qRT-PCR was used to explore the reason of GAS5-AS1 abnormal expression.Western blot assays were used to investigate the underlying targets of GAS5-AS1 in NSCLC cells.Results differences were tested for significance using two-tailed student’s t-tests.Results:We discovered IncRNA GAS5-AS1 as a tumor suppressor in NSCLC.The expression levels of GAS5-AS1 in NSCLC tumors were much lower than that in the adjacent normal lung tissues.The reduced GAS5-AS1 was significantly correlated with larger tumors,higher TNM stages,and lymph node metastasis in NSCLC patients.While ectopic expression of GAS5-AS1 had no effect on proliferation,cell cycle progression,and apoptosis,it dramatically reduced NSCLC cell migration and invasion.Overexpression of GAS5-AS1 in NSCLC cells decreased a cohort of molecules,including ZEB1,N-cadherin,Vimentin,and/or Snail1,critical for epithelial-mesenchymal transition(EMT)and metastasis.Further analysis showed that the DNA demethylating agent decitabine failed to upregulate GAS5-AS1 in NSCLC cells,whereas the pan-HDAC inhibitors panobinostat and SAHA significantly enhanced expression of GAS5-AS1 in a dose-dependent manner.Collectively,these data suggest that histone modifications lead to epigenetic silencing of GAS5-AS1 in NSCLC and subsequently promote tumor metastasis via upregulation of several key EMT markers.Conclusion:Our findings identify the IncRNA GAS5-AS1 as a potential therapeutic target against NSCLC progression.