| Objective1.The growth pattern of gastric cancer(GC)is often related to the malignant degree,invasion,metastasis and other pathological characteristics of tumors.Finding the regulators that influence the growth pattern of GC is helpful to explore the pathogenesis of different classifications of GC.Accurate identification of molecular markers related to the biological characteristics of GC is of great significance for the prevention and treatment of GC.2.To screen and identify miRNAs related to different growth patterns of GC,and explore their main biological pathways and cellular functions.To reveal the regulation mechanisms of these miRNAs and their target genes in the histological classification of GC and other pathological processes,their relationship with clinical pathological characteristics of GC,and to evaluate their clinical value as potential prognostic markers of GC.3.To search for circRNA molecules that can regulate the above-mentioned GC Ming classification related miRNAs,to explore the role of circRNA-miRNAs regulatory network in different growth patterns of GC and its main biological functions,and to identify the clinical significance of circRNA-miNA-mRNA regulatory axis target gene in the evaluation of disease progression and prognosis of GCMethods1.Typical samples of GC with different growth patterns were selected for miRNA microarray screening,and the results were verified by real-time quantitative RT-PCR in GC cells from different Ming classifications.2.Identification of miRNAs family related to different growth patterns of GC,screening of miRNAs pathway and enrichment of target gene function through bioinformatics,and searching for miRNAs family target genes highly related to tissue subtypes of GC by using tumor chip database.3.Molecular biological techniques such as luciferase reporter gene assay,qRT-PCR and Western blotting were used to verify the molecular regulatory mechanism between the Ming classification related miRNAs and their target genes.4.Meta-analysis was used to confirm the expression of the target gene in GC.Enzyme-linked immunosorbent assay and tumor protein histochemical database were used to investigate the exocrine capacity and distribution of the protein of target gene expression in GC cells with different growth patterms.5.The correlation between target gene expression and histopathological subtypes,malignant degree,clinical stage and Helicobacter pylori infection in GC was analyzed.6.Cell migration and invasion experiments were conducted to evaluate the effect of target genes on the invasion ability of GC cells,and to provide evidence for their involvement in the formation of different growth patterns of GC.Logical regression model was used to analyze the value of target genes as markers in the clinical prognosis of GC.7.Literature search and bioinformatics predicted the circRNA molecule binding to miRNA related to different growth patterns of GC and analyzed its subcellular localization.8.The above circRNA-specific siRNA was synthesized and the expression vector was constructed to transfect GC cells,and its regulatory effect on the expression level of related miRNAs was detected.9.To construct a circRNA-miRNA-mRNA regulatory network to screen the pathway and enrich the target gene function,CCK-8 cell proliferation experiment was conducted to verify the biological effect of circRNA-miRNA-mRNA regulatory axis on the growth of GC cells.10.Tumor microarray expression database mining and ROC curve analysis were used to identify the gene expression of circRNA-miRNA-mRNA regulatory axis target in GC and to evaluate its reference value in differentiating cancer tissue from normal tissue.11.The overall survival curve and the first progression curve of two groups of patients with high or low target expression were calculated by Kaplan-Meier method to evaluate the value of their expression levels in the progression of GC.Results1.The expression profiles of miRNAs related to different growth patterms of GC were screened.RT-qPCR confirmed that miRNAs were significantly correlated with the gastric carcinogenesis and Ming classification.2.Bioinformatics reveals that the biological function of miR-29s is most closely related to the pathway of ECM-receptor interaction.Functional enrichment of the interaction network between the ECM-receptor interaction pathway and the target gene of miR-29s reveals that ECM structural assembly,cell movement and cell adhesion are the main functional categories of target genes in this pathway.3.The above target genes and high-risk genes of diffuse GC in Oncomine database were screened by set operation.It was confirmed that COL4A1 gene may play an important role in miR-29s biological function.4.The high expression of COL4A1 in GC was confirmed in large TCGA samples.miR-29s inhibited its translation by binding to the 3’-UTR region of COL4A1 gene.5.Meta-analysis confirmed that the high expression of COL4A1 gene promotes the development of GC.Infltrative type GC cells secrete higher level of COL4A1 protein than expanding type GC cells.6.The expression of COL4A1 gene in GC is correlated with clinicopathological features such as tumor subtype,malignant degree,disease stage and HP infection,suggesting that COL4A1 gene can promote the gastric carcinogenesis and accelerate pathological progress.7.Downregulation of COL4A1 expression significantly inhibited the migration and invasion of GC cells.Survival analysis revealed that patients with high COL4A1 expression had poor prognosis.8.It was found that circHIPK3 was mainly distributed in the cytoplasm and could regulate their expression by adsorbing miR-124 and miR-29b.9.The circHIPK3-miR-124/miR-29b regulatory axis is closely related to Pathways in cancer,and its main biological function is to influence cell growth.10.CCK-8 proliferation experiment showed that high expression of circHIPK3 could promote the proliferation of GC cells,while low expression of circHIPK3 inhibited the proliferation of GC cells.11.The common target genes(COL1A1,COL4A1 and CDK6)of circHIPK3-miR-124/miR-29b regulatory axis were highly expressed in GC,and were regulated by circHIPK3.ROC curve revealed that the expression of COL1A1 and COL4A1 genes could differentiate GC from normal gastric tissue to some extent.12.The expression levels of COL1A1,COL4A1 and CDK6 in GC tissues can be used as prognostic biomarkers for survival of patients.The prognosis of patients with low expression is better than those with high expression.ConclusionDifferent growth patterns of GC are closely related to the regulation of miRNAs,which affect the histological classification and pathological progress of GC by regulating the function of target genes.The circRNA-miRNA-mRNA regulatory axis also participates in the development of different growth patterns of GC.The transcriptional regulation mechanism mediated by circRNA regulatory network broadens the understanding of the pathogenesis of GC.The target gene expression level in the regulatory network has important clinical value in eva12uating the disease progression of GC. |
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