The Regulation And Oncogenic Role Of LncRNA ASH1L-AS1 In Gastric Cancer

Background:Gastric cancer(GC)is the fifth most frequently diagnosed malignancy and the fourth leading cause of cancer death worldwide in 2020.Incidence rates of GC are markedly elevated in Eastern Asia including China.The early symptoms of GC are concealed and there is a lack of diagnostic biomarkers with high specificity and sensitivity.At the time of diagnosis,more than 60%of patients have local or distant metastases whose 5-year survival rate is only about 5%.LncRNAs(long non-coding RNAs)are a class of non-coding RNAs with a length of more than 200 nucleotides,accounting for 80%-90%of all non-coding RNA content in cells.LncRNAs play an important role in epigenetic regulation of gene expression.It has been shown that lncRNAs can be used as a potential biomarkers for early diagnosis of malignant tumors,prognostic evaluation and drug targets.LncRNA ASH1L-AS1 is located in human chromosome 1q22,which is a gene locus susceptible to GC in East Asian populations.The role of 1ncRNA ASH1L-AS1 in the development of GC is still unclear.Purpose:In this study,we aim to reveal the biological functions and molecular mechanisms of lncRNA ASH1L-AS1 in GC cells and provide new targets and new ideas for early diagnosis and clinical treatments of GC.Methods:1.We detected the expression levels of lncRNA ASH1L-AS1 in GC tissues and matched normal gastric tissues by qRT-PCR.2.We constructed lentivirus to infect GC cell lines and obtained stably knocking-down or overexpressing IncRNA ASH1L-AS1 GC cell lines.We performed cell counting assays,clone formation assays,and nude mouse subcutaneous xenograft assays to determine the role of IncRNA ASH 1L-AS1 on proliferation of GC cells.We performed wound healing assays and the Transwell assays to reveal the impacts of lncRNA ASH 1L-AS 1 on the migration and invasion capability of GC cells.3.We detected the expression levels of ASH1L mRNA in GC tissues and matched normal gastric tissues by qRT-PCR,and analyzed the correlations between ASH1L mRNA levels and IncRNA ASH 1L-AS1 levels in these tissue samples4.We detected whether the expression levels of ASH1L mRNA is affected by IncRNA ASH 1L-AS1 in GC cell lines by qRT-PCR.5.We performed clone formation assays and Transwell assays to explore the impacts of ASH1L on the malignant phenotypes of GC cells.After overexpressing IncRNA ASH1L-AS1 in GC cells,we knocked-down ASH1L to explore whether the malignant phenotypes of GC cells can be reversed6.Through RNA-pulldown assays,protein profiling analyses,RIP assay and Western blot assays,we identified and verified the protein physically interacting with IncRNA ASH1L-AS1 in GC cells.7.We performed Western blot assays to detect whether the levels of NME1 are affected by IncRNA ASH 1L-AS1 in GC cells.We also used Western blot assays and cellular immunofluorescence assays to detect whether the nucleocytoplasmic distribution of NME1 is regulated by IncRNA ASH 1L-AS1 in GC cells.8.We detected the expression of NME1 mRNA in the GC tissues and matched normal gastric tissues by qRT-PCR,then analyzed the expression correlations between IncRNA ASH 1L-AS1,ASH1L mRNA and NME1 mRNA in the tissue samples9.We knocked-down NME1 or overexpressed NME1-NLS respectively in GC cells to detect the expression levels of IncRNA ASH1L-AS1 and ASHIL mRNA by qRT-PCR.10.We explored the binding capacity of NME1 to ASH1L-AS1 and ASH1L gene promoter regions through ChIP assays.Then we determined whether the H3K4me3 modification levels of ASH1L-AS1 and ASH1L gene promoter regions are consistent with the degree of NME1 enrichment through ChIP-seq.Results:1.The expression of IncRNA ASH1L-AS1 is significantly increased in GC tissues.Knocking-down of IncRNA ASH1L-AS1 inhibited the proliferation and migration of GC cells,while overexpression of IncRNA ASHIL-AS1 enhanced the proliferation and migration of GC cells.Animal assays showed that overexpression of IncRNA ASH1L-AS1 significantly promoted the formation and proliferation of GC xenografts.2.LncRNA ASH1L-AS1 can up-regulate the expression of adjacent coding gene ASH1L.Knockdown of ASH1L can inhibit the malignant phenotypes of GC cells.Knockdown of ASH1L can reverse the malignant phenotypes of GC cells after overexpression of lncRNA ASH 1L-AS1.3.We identified NME1 as one of the binding proteins of lncRNA ASH1L-AS1.Western blot and cellular immunofluorescence assays showed that overexpression of lncRNA ASH 1L-AS 1 can increase the NME1 protein levels in the nucleus.Compared with matched normal gastric tissues,NME1 gene is highly expressed in tumor tissues of GC patients.The levels of lncRNA ASH1L-AS1 and ASH1L mRNA in tissue samples are significantly positively correlated with NME1 mRNA levels.Knockdown of NME1 down-regulated the expressions levels of lncRNA ASH 1L-AS1 and ASH1L in cells,while over-expression of NME1-NLS up-regulated the expression of IncRNA ASH1L-AS1 and ASHL1 in cells.ChIP assays showed that NME1 can bind to the promoter regions of ASH1L-AS1 and ASH1L.Moreover,NME1 is more significantly enriched the promoter regions of ASH1L-AS1 and ASH1L in the IncRNA ASH 1L-AS1 overexpressed group compared with the control groupConclusions:In this study,we found that the expression of lncRNA ASH1L-AS1 is significantly increased in GC tissues compared with normal tissues.LncRNA ASH1L-AS1 binds the NME1 protein,which leading to nucleus partitioning of NME1.The RNA-protein complex of IncRNA ASH1L-AS1 and NME1 up-regulates the levels of ASH1L-AS1 and ASH1L which promotes the malignant progression of GC.In all,IncRNA ASH1L-AS1 might be a novel target for clinical treatments of GC.