Role And Mechanism Of LncRNA HMGA2-AS1 In The Development Of Gastric Cancer

Background and ObjectiveGastric cancer(GC)is one of the most common and deadly malignancies worldwide,and is the fourth leading cause of cancer-related deaths across the globe.Despite advances in the diagnosis and systemic treatment of gastric cancer over the past several decades,there have been no breakthroughs and the overall prognosis for patients with gastric cancer is poor,with five-year survival rates remaining low,primarily due to the high incidence of metastasis and recurrence of gastric cancer.However,our current understanding of the molecular mechanisms associated with gastric cancer metastasis remains minimal.Long-stranded non-coding RNAs(lnc RNAs),a class of endogenous RNA molecules greater than 200 nucleotides in length,the majority of which do not encode proteins.It participates extensively in the regulation of major life events and biological processes,including stem cell differentiation,gene expression,growth and development,cell proliferation and differentiation.An increasing number of studies have demonstrated that lnc RNAs may play oncogenic or oncogenic functions in malignancies,and that lnc RNAs migth be potential biomarkers and targets for early diagnosis of tumors,assessment of prognosis,treatment and reduction of chemotherapy resistance.lnc RNA HMGA2-AS1,one of the antisense coding RNAs of HMGA2,was significantly correlated with the proliferation and invasive metastasis of gastric cancer.It was frequently upregulated in gastric cancer tissues,and was remarkably relevant to the survival,prognosis and clinical staging of gastric cancer patients.At present,the role and mechanism of HMGA2-AS1 in gastric carcinogenesis and development are still unclear.The objective of this study was to clarify the mechanism of HMGA2-AS1 in the development of gastric cancer,and to explore the regulation of HMGA2 by HMGA2-AS1 with the influence and role played by PI3K/Akt signaling pathway in this regulatory axis.Methods1.The RNA-seq analysis was performed to screen the lnc RNA HMGA2-AS1 and the expression of HMGA2-AS1 in gastric cancer and adjacent normal tissues was predicted by The Cancer Genome Atlas(TCGA)database,while its clinical relevance and prognostic potential in gastric cancer patients and its validity as a gastric cancer biomarker were evaluated.The q RT-PCR method was used to detect the expression of HMGA2-AS1 in clinical gastric cancer tissues and adjacent normal tissues as well as gastric cancer cell lines.2.After stable silencing of HMGA2-AS1 expression in MGC-803 and HGC-27 cells,cell proliferation ability was investigated by Ed U and colony formation assays,apoptotic changes were analyzed by flow cytometry,changes in cell invasion and migration were detected by Transwell assay,and epithelial-mesenchymal transition(EMT)process affected was probed by western blot and double immunofluorescent labeling.In vivo experiment,a subcutaneous transplantation tumor model was established in nude mice to assess the effect of silencing HMGA2-AS1 expression on the growth and malignancy of gastric cancer cell tumors as well as target genes.3.An online bioinformatics analysis tool,UCSC,was utilized to probe HMGA2-AS1 neighboring regulated target genes,and RNA-seq was used to analyze target genes differentially expressed downstream of HMGA2-AS1 after its silencing.The correlation of HMGA2-AS1 with target genes within gastric cancer was predicted by TCGA database.The relevance of HMGA2-AS1 and target gene m RNA was further explored in clinical gastric cancer tissues by q RT-PCR.In MGC-803 and HGC-27 cells with stable silencing of HMGA2-AS1,q RT-PCR and western blot were used to explore the changes of target genes after HMGA2-AS1 silencing.4.To predict the expression of target genes in gastric cancer and adjacent normal tissues by TCGA database,as well as to assess their clinical relevance and prognostic potential in gastric cancer patients and their effectiveness as gastric cancer biomarkers.The expression of target genes in clinical gastric cancer tissues and gastric cancer cell lines was detected by q RT-PCR,western blot and immunohistochemistry.5.The miRNAs that bind to both HMGA2-AS1 and target genes were predicted by bioinformatic analysis,and the interaction between HMGA2-AS1 and target genes with target miRNAs were verified using dual luciferase reporter gene assay,q RT-PCR assay and/or western blot assay,respectively.6.HGC-27 cell lines stably silencing HMGA2-AS1 were selected for high-throughput transcriptome sequencing,and the enrichment of signaling pathways related to HMGA2-AS1 expression was performed using bioinformatics methods.The activation levels of related signaling pathway proteins were measured by western blot.Results1.The expression of HMGA2-AS1 which correlated with pathological grading and staging of tumors,patient prognosis,and survival,was elevated in gastric cancer tissues and gastric cancer cell lines.This indicates that HMGA2-AS1 may be involved in regulating the proliferation and invasive migration of gastric cancer as a pro-carcinogenic factor,and suggests that HMGA2-AS1 might serve as a clinical and prognostic biomarker for gastric cancer,which could have significant clinical and prognostic implications.2.In MGC-803 and HGC-27 cells,silencing HMGA2-AS1 expression inhibited cell proliferation and clone formation as well as promoted apoptosis,while inhibiting cell invasion and migration and the EMT process.In vivo experiments,silencing HMGA2-AS1 expression inhibited the growth and HMGA2 expression of subcutaneous transplanted tumors in nude mice,while tumor malignancy was reduced.3.Bioinformatics,q RT-PCR and western blot demonstrated that HMGA2-AS1 was significantly and positively correlated with HMGA2 in gastric cancer tissues and gastric cancer cells,and HMGA2-AS1 positively regulated the expression of HMGA2 at both m RNA and protein levels.4.The expression of HMGA2 was increased in gastric cancer tissues and gastric cancer cell lines and associated with tumor TNM stage,patient prognosis,and survival.5.Dual luciferase reporter gene assay and q RT-PCR revealed that HMGA2-AS1 could act as a competitive endogenous RNA(ce RNA)regulating miR-204-5p.Meanwhile,dual luciferase reporter gene assay,q RT-PCR and western blot assay showed that miR-204-5p might target HMAG2 in gastric cancer.That is,HMGA2-AS1 plays a ce RNA function sponge adsorption miR-204-5p in gastric cancer cells and regulates the expression of HMGA2.6.In HGC-27 cell lines with stably silenced HMGA2-AS1 expression,GO and KEGG enrichment analysis demonstrated that its differentially expressed genes(DEGs)were mainly enriched in the PI3K/Akt signaling pathway.Silencing HMGA2-AS1 expression promoted protein phosphorylation of Akt and m TOR proteins,while this process could be antagonized by the potent and irreversible PI3K-specific inhibitor Wortmannin.ConclusionThe oncogenic lnc RNA HMGA2-AS1 played an important role in gastric cancer,besides,HMGA2-AS1 could negatively regulate the activation of PI3K/Akt signaling pathway and participate in the facilitation of EMT process in gastric cancer cells through HMGA2-AS1/miR-204-5p/HMGA2 axis,which significantly promoted the proliferation and invasive metastasis of gastric cancer cells.