The Function And Mechanism Of Hypoxia-induced Long Non-coding RNA MTA2TR In Pancreatic Cancer

Subject:Pancreatic cancer is the seventh leading cause of cancer death worldwide.Despite recent research and treatment advances have been made,its 5-year survival rate is still less than 5%.The lack of effective early detection is one of the most important reasons for the poor survival of patients with this disease.The extensive tumor heterogeneity,as well as the network of interactions between the neoplastic epithelium and the components within the microenvironment,are biological features that determine poor prognosis in pancreatic cancer.Recent studies have shown that long noncoding RNA(lnc RNAs)have multiple biological functions in cell growth,differentiation,proliferation,invasion and metastasis and play a crucial role in many cancers,while the function of lnc RNA in the hypoxic microenvironment of pancreatic cancer is not fully elucidated.The aim of this study was to reveal the regulatory role of lnc RNA-MTA2TR(metastasis associated protein 2 transcriptional regulator RNA,AF083120.1)in pancreatic cancer under hypoxic microenvironment.Methods:Next-generation sequencing was applied to analyze the differentially expressed lnc RNAs in pancreatic cancer(PC)and paired paracancerous tissues.Through analysis,we selected an upregulated lnc RNA-MTA2 TR as the research object,as its gene location was close to metastasis-associated protein 2(MTA2).The expression of lnc RNA-MTA2 TR was assessed by real-time fluorescence quantitative PCR(RT-PCR)in 40 pancreatic cancer and paired adjacent tissue(NP)tissues and pancreatic cancer cell lines.Single molecular RNA fluorescence in situ hybridization(RNA-FISH)and Northern blot were used to identify the nuclear localization and expression of lnc RNA-MTA2 TR in pancreatic cancer cells.Knockdown or overexpression of lnc RNA-MTA2 TR in pancreatic cancer cells by transfection of small interfering RNA(si RNA)or overexpression plasmids in cells.MTT and colony formation assays were used to determine the proliferation ability of cells,and transwell and wound healing assays were used to detect the invasion and metastasis ability of cells.Pancreatic cancer cells transfected with virus,containing the sequence of knockdown lnc RNA-MTA2 TR,were injected into nude mice through a xenograft model to further explore the effects of lnc RNAMTA2 TR on cell proliferation and metastasis.To further explore the mechanism of lnc RNA-MTA2 TR functioning in pancreatic cancer,RT-PCR and western blot were used to detect the m RNA and protein level of MTA2 in lnc RNA-MTA2 TR knockdown or overexpression cells.Luciferase reporter assay was used to validate the effect of lnc RNA-MTA2 TR on MTA2 promoter activity.RNA immunoprecipitation(RIP)and RNA pulldown assays were used to demonstrate the binding between lnc RNA-MTA2 TR and transcription activator 3(ATF3).Chromatin immunoprecipitation(Ch IP)assay was utilized to validate the binding between ATF3 and MTA2 promoter.Immunofluorescence and RNA-FISH assays were applied to determine the binding between lnc RNA-MTA2 TR and ATF3.In addition,RT-PCR and RNA-FISH were used to detect the expression of lnc RNA-MTA2 TR in pancreatic cancer cells under hypoxic conditions.Ch IP assay was used to demonstrate the binding between hypoxia inducible factor-1?(HIF-1?)and the lnc RNA-MTA2 TR promoter.Luciferase reporter assay was applied to detect the effect of HIF-1? on lnc RNA-MTA2 TR promoter activity.Co-Immunoprecipitation(Co-IP)was used to detect the effect of lnc RNAMTA2 TR on the binding between HIF-1? and MTA2.Through Immunofluorescence and RNA-FISH co-staining,we examined the expression of lnc RNA-MTA2 TR and HIF-1? in tissues.Kaplan-Meier analyzed the effect of lnc RNA-MTA2 TR on survival in pancreatic cancer patients.Pearson correlation analysis was used to analyze the relationship between lnc RNA-MTA2 TR and MTA2 expression in pancreatic cancer patients.Results:The results from the Next-generation sequencing and RT-PCR demonstrated that lnc RNA-MTA2 TR was upregulated in pancreatic cancer patient tissues compared to paired paracancerous tissues.The in vitro and in vivo experimental results elucidated that knockdown lnc RNA-MTA2 TR significantly inhibited the proliferation and invasion of pancreatic cancer cells.Mechanistically,lnc RNA-MTA2 TR upregulates MTA2 expression by recruiting the transcription factor ATF3 to the promoter region of MTA2.Additionally,we found that lnc RNA-MTA2 TR expression was upregulated under hypoxia.Further mechanistic studies had found that HIF-1? could bind to the hypoxic response element(HRE)site of the MTA2 TR promoters,thereby enhancing promoter activity and transcription of lnc RNA-MTA2 TR.Our previous studies have shown that MTA2 can activate HIF-1? transcriptional activity by deacetylating HIF-1? protein.We found the binding between MTA2 and HIF-1? and the HIF-1? protein stability were decreased in lnc RNA-MTA2 TR knockdown cells.Our results indicated that lnc RNA-MTA2 TR could promote the stability of HIF-1? via promoting MTA2 expression and increasing the deacetylation of HIF-1?.Furthermore,our clinical samples showed that lnc RNA-MTA2 TR was positively correlated with MTA2 expression,and the upregulation of lnc RNA-MTA2 TR expression was associated with reduced overall survival in pancreatic cancer patients.Conclusions:The hypoxia-induced lnc RNA-MTA2 TR recruits ATF3 to the MTA2 promoter and regulates MTA2 expression,enhancing the stabilization of HIF-1? via MTA2-dependent deacetylation,suggesting a positive feedback loop between lnc RNA-MTA2 TR and HIF-1?.Our study elucidated that the HIF-1?/lnc RNAMTA2 TR loop can be used as a new tumor marker and therapeutic target in pancreatic cancer.