| ObjectiveThrough bioinformatics analysis of TCGA gastric cancer transcriptome and miRNA expression data,and then using public online database so as to construct lncRNA-miRNA-mRNA ceRNA regulatory network,lncRNA PVT1 is one of key gene in the ceRNA regulatory network.PVT1 is up-regulated in gastric cancer cell lines and gastric cancer tissues,but the molecular mechanism of PVT1 in gastric cancer is unclear.This study focused on PVT1 to explore the molecular mechanism of PVT1 in gastric cancer.Methods1.Downloading the gastric cancer TCGA transcriptome RNA-seq and miRNA-seq data,conducting differential gene analysis,combining with an online database to construct an lncRNA-miRNA-mRNA ceRNA regulatory network,and find key genes and subnetworks.2.Real-time quantitative PCR method was used to detect the expression of lncRNA PVT1 in gastric cancer cell lines and tissues,and to analyze the correlation between PVT1 and clinical pathology of gastric cancer.3.Using transient transfection technology to construct PVT1 over-expressed and down-regulated models of gastric cancer cells.CCK8,EdU cell proliferation experiment and Transwell invasion experiment were used to analyze the effect of over-expression and down-regulation of PVT1 on gastric cancer cell proliferation and invasion.4.Screening downstream miRNA and target genes of PVT1 based on ceRNA regulatory network and univariate survival analysis.qRT-PCR was used to detect the expression changes of miR-145-5p after over-and down-regulation of PVT1 in gastric cancer cells and the expression changes of PVT1 after over-expression of miR-145-5p;qRT-PCR was used to detect the target gene STAT1 expression levels after up-regulation of miR-145-5p and the expression change of miR-145-5p afterover-expression of STAT1 in gastric cancer cells;The effect of reducing PVT1 and blocking miR-145-5p on proliferation and migration of gastric cancer cells was clearly determined by cell function recovery experiments.The dual luciferase reporter gene experiment verified that PVT1 and the downstream miR-145-5p,miR-145-5p and target gene STAT1 binding site.Western blot was used to detect the expression of key proteins and phosphorylated proteins in PI3K-Akt/JAK-STAT1 signaling pathway.Results1.Gastric cancer TCGA transcriptome and miRNA differential genes(2684differentially expressed lncRNA,240 differentially expressed miRNA,4285 differentially expressed mRNA)combined with an online database to construct an lncRNA-miRNA-mRNA regulatory network,the network has a total of 1626 nodes,2704 pairs Interaction relationship.The enrichment analysis of the target gene KEGG signaling pathway regulated by the LncRNA-miRNA-mRNA regulatory network showed significant enrichment in signaling pathways such as PI3K-Akt,stimulation of neural ligand-receptor interaction,and cytokine-cytokine receptor interaction.In addition,lncRNA ADAMTS9-AS2 and PVT1 were found to be important key node genes of lncRNA-miRNA-mRNA regulatory network.2.The expression of LncRNA PVT1 is up-regulated in gastric cancer cell lines and gastric cancer tissues,and its expression level is positively correlated with the tumor size,microvessel and lymph vessel tumor thrombus,the positive rate of surgically resected lymph node metastasis,lymph node metastasis,and tumor TNM staging.3.The results of CCK-8,EdU cell proliferation detection,scratch test,and Transwell invasion test show that up-regulation of PVT1 promote the proliferation,migration,and invasion of gastric cancer cells,while down-regulation of PVT1 inhibit the proliferation,migration,and invasion of gastric cancer cells.PVT1 can promote the proliferation and invasion of gastric cancer cells in vitro.Subcutaneous tumorigenesis experiments in nude mice show that interfering with PVT1 can inhibit the growth of gastric cancer cells in vivo.4.Through the constructed ceRNA regulatory network and survival analysis,the downstream target gene of PVT1 was selected as miR-145-5p,and STAT1 was predict as the downstream target gene of miR-145-5p by using the online target database.STAT1 expression in SGC-7901 and MKN-45 cells decreased significantly after over-expression of miR-145-5p,whereas miR-145-5p expression in SGC-7901 and MKN-45 cells decreased significantly after over-expression of STAT1.After down-regulating the expression level of PVT1,the mRNA expression of STAT1 transcript level was suppressed;Down-regulation of PVT1 by sh-PVT1 and miR-145-5p inhibitor to block miR-145-5p,the expression of STAT1 reverted.After down-regulating the expression level of PVT1,the proliferation and migration of gastric cancer SGC-7901 and MKN-45 cells were inhibited;Down-regulation of PVT1 by sh-PVT1 and miR-145-5p inhibitor to block miR-145-5p,gastric cancer SGC-7901 and MKN-45 cell proliferation,migration function reverted.After over-expression of PVT1,phosphorylated pAkt and pSTAT1 were up-regulated,indicating that PI3K-Akt/JAK-STAT1 signaling pathway was activated,and the addition of Akt kinase inhibitor MK-2206 significantly inhibited SGC-7901 and MKN-45 cells growth promoted by PVT1 over-expression.Conclusions1.We use gastric cancer TCGA transcriptome and miRNA differential genes(2684 differentially expressed lncRNA,240 differentially expressed miRNA,4285 differentially expressed mRNA),and build lncRNA-miRNA-mRNA ceRNA regulatory network with the help of a public online database,the network has a total of 1626 nodes,and 2704 pairs of interactions.LncRNA PVT1 is a key lncRNA of gastric cancer ceRNA regulatory network.2.The expression of PVT1 is up-regulated in gastric cancer,and its expression level is positively correlated with the tumor size,microvessel and lymphatic vessel tumor thrombus,the positive rate of lymph node metastasis after surgery,lymph node metastasis and TNM stage of gastric cancer patients.3.PVT1 has a positive regulation effect on the biological behavior of gastric cancer cell proliferation and migration,and plays the biological function of oncogene in gastric cancer.Interference with PVT1 expression can inhibit the growth of gastric cancer cells in vivo.4.PVT1 negatively regulates miR-145-5p through the ceRNA mechanism and promotes STAT1 expression.Up-regulation of the PVT1/miR-145-4p/STAT1 axis can activate the PI3K-Akt/JAK-STAT1 signaling pathway to promote gastric cancer cell proliferation and invasion. |
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