HNF1A-AS1 Targeting Oncostatin M Expression Inhibits Invasion In Gastroenteropancreatic Neuroendocrine Neoplasms

Objective: To investigate the role of long non-coding RNA(lncRNA)hepatocyte nuclear factor 1 alpha-antisense 1(HNF1A-AS1)on the proliferation,migration,invasion and epithelial-mesenchymal transition(EMT)of gastroenteropancreatic neuroendocrine neoplasms(GEP-NENs),and to explore the mechanism by which HNF1A-AS1 inhibits the invasion of GEP-NENs.Methods: We utilized Human Transcriptome Array 2.0 to identify the abnormal lncRNA expression profiles between three gastric neuroendocrine neoplasms(G-NENs)tissues and their paired adjacent normal gastric tissues.The expression of HNF1A-AS1 was further confirmed by qRT-PCR.Subcellular fractionation location of HNF1A-AS1 was carried out by the separation of nuclear and cytosolic fractions of GEP-NENs tumor cells.We altered the HNF1A-AS1 expression by transfection of the plasmid bearing HNF1A-AS1 and small interference RNAs(siRNAs).We detected the effects of HNF1A-AS1 on the cell proliferation through the colony formation assay and immunofluorescence microscopy analysis.We used the nude mice tumor formation assay to assess whether HNF1A-AS1 affected GEP-NENs tumorigenesis in vivo.The Transwell assay and wound healing assay were conducted to detect the effect of HNF1A-AS1 on tumor cell migration and invasion.We assessed the expression of EMT related markers in LCC-18 cells by qRT-PCR and Western blot.We screened out possible target gene of HNF1A-AS1 from differentially expressed coding genes based on Transcriptome Array.Transwell assaywas used to explore the effects of Oncostatin M(OSM)and TGFβ sigaling on the HNF1A-AS1-mediated migration and invasion.Results: Transcriptome array indicated that HNF1A-AS1 was significantly downregulated in all of the three G-NEN tissues compared with adjacent normal gastric tissues,which was further validated by qRT-PCR.HNF1A-AS1 was mainly localized in the nucleus of GEP-NENs tumor cells.HNF1A-AS1 overexpression remarkably decreased the capacity of proliferation,migration and invasion of LCC-18 and BON-1 cells,while down-regulation of HNF1A-AS1 promoted the proliferation,migration and invasion of GEP-NENs tumor cells.The growth inhibition effect of HNF1A-AS1 was further confirmed in vivo.qRT-PCR and Western blot revealed that HNF1A-AS1 overexpression in LCC-18 cells led to downregulation of mesenchymal marker β-catenin and upregulation of epithelial marker E-cadherin at mRNA and protein level,while down-regulation of HNF1A-AS1 led to the opposite phenomena.Furthermore,we found that HNF1A-AS1 could inhibit OSM expression in LCC-18 and BON-1 cells.Exogenous OSM treatment significantly abolished the HNF1A-AS1-induced decreases in migration and invasion ability of GEP-NENs tumor cells.GEP-NENs tumor cell migration and invasion induced by HNF1A-AS1 knockdown would be inhibited after TGFβ signaling pathway blockage.Conclusions: HNF1A-AS1 was significantly decreased in GEP-NENs tissues compared to adjacent normal tissues.HNF1A-AS1 targeting OSM expression acted as a suppressor in GEP-NENs tumor cell migration,invasion and EMT process,which was TGFβ signaling dependent.HNF1A-AS1 might be a potential target for GEP-NENs diagnosis and therapy.