| Objective: Gastric cancer(GC)is the fifth most common tumor in the world and also the fourth leading cause of death from tumors.The prognosis of patients is relatively poor,which seriously threatens human health.Among them,East Asia accounts for the main part of the incidence and death of GC.Nearly half of the new cases and deaths are in China every year.The current gold standard for diagnosis of GC is endoscopy biopsy.The survival rate of patients with early GC exceeds 60%,while the survival rate of advanced GC is less than 24%.However,due to the high cost of diagnosis and poor patient compliance,the proportion of patients diagnosed with early GC in China is less than 10%.The occurrence and progression of GC is the result of multiple factors such as genetics,epigenetics,and environment.Among them,epigenetics has been a hot spot in recent years,including DNA methylation,histone modification,chromatin remodeling and non-coding RNA regulation.The important regulatory role of non-coding RNA in GC and other tumors has been confirmed.Therefore,exploring the regulatory mechanism of non-coding RNA will help to reveal the law of GC occurrence and development and to discover effective biomarkers for GC diagnosis,treatment and prognosis management.Studies have found that only a few genes in the human genome sequence have the function of encoding protein,while most does not and forms non-coding transcripts after transcription.Among them,long non-coding RNA(long non-coding RNA,lncRNA)refers to a long chain RNA molecule with a length of more than 200 nucleotides and no protein coding function.It has been proved to be involved in regulating the proliferation,migration and invasion of tumor cells.The purpose of this study is to discover abnormally expressed lncRNA by analyzing the GC RNA-Seq data from the TCGA(The Cancer Genome Altas)database.Sequencing data found that the expression level of LINC02532 in GC tissues was significantly higher than that in normal tissues adjacent to cancer,and the prognosis of GC patients with high expression of LINC02532 was poor.These findings suggest that LINC02532 may act as a proto-oncogene to promote the occurrence and development of GC.However,the molecular mechanism of LINC02532 in the occurrence and development of GC is still unclear.Therefore,we further confirmed the role of FOXF2/LINC02532/SOX7 axis in the regulation of GC cell migration and invasion through in vivo and in vitro experiments.Methods: Through the analysis of TCGA database RNA-Seq and clinical parameter data,the abnormally high expressed lncRNA LINC02532 with useful clinical significance was selected for in-depth study.Real-time fluorescent quantitative PCR(q RT-PCR)detects the expression of LINC02532 at the level of GC cells and tissues,and analyzes its clinicopathological significance.The method of bioinformatics studies the subcellular localization and non-coding function of LINC02532,and the RNA Fluorescence in situ hybridization confirmed the subcellular distribution of LINC02532.The LINC02532 overexpression plasmid and small interfering RNA(si RNA)were designed,and the effect of LINC02532 overexpression and interference on the migration and invasion of GC cells was detected by Transwell experiment.At the same time,the effect of overexpression of LINC02532 on the tumorigenesis ability of GC cells was observed through the nude mouse tumorigenesis experiment.q RT-PCR and Western blot experiments were performed to observe the regulation of interference LINC02532 on the mRNA and protein levels of genes related to epithelial-mesenchymal transition(EMT).According to the median expression of LINC02532,GC patients from TCGA were divided into two groups.The possible regulation mode of LINC02532 was explored by Gene Set Enrichment Analysis(GSEA).The q RT-PCR and Western blot experiments were used to detect the expression of FOXF2 and SOX7 in GC tissues.Pearson statistically analyzed the correlation between LINC02532 and FOXF2 and SOX7 expression.Bioinformatics tools predict the transcriptional binding sites of FOXF2 and LINC02532 promoter regions and the binding sites of LINC02532 and SOX7 3'UTR regions,and the dual-luciferase reporter assay verify the binding sites.Actinomycin D was used to treat GC cells,and the effect of interfering with LINC02532 on the degradation rate of SOX7 was observed.Then,the Transwell experiment was performed to detect the regulation of the FOXF2/LINC02532 axis and the LINC02532/SOX7 axis on the migration and invasion of GC cells.Result: 1.The TCGA database showed that the expression of LINC02532 in GC tissue was significantly higher than that in normal adjacent tissues(P = 0.001).Kaplan-Meier survival analysis idicated that the prognosis of patients with high expression of LINC02532 was significantly worse than that of patients with low expression(log-rank P = 0.003).Cox regression analysis found that LINC02532 can be used as an independent prognostic factor for GC.The q RT-PCR experiment confirmed that the expression of LINC02532 was abnormally increased in GC cell lines(SGC-7901,MGC-803,AGS,MKN-45)and 52 pairs of GC tissues.Chi-square test analyzed the relationship between the expression of LINC02532 in 52 pairs of GC tissues and clinicopathological parameters.The results showed that the high expression of LINC02532 was associated with high TNM stage(P = 0.008)and low tumor differentiation(P = 0.023).2.LINC02532 expression silencing reduces the migration and invasion ability of GC cells,while overexpression of LINC02532 enhances the migration and invasion ability of GC cells.LINC02532 overexpression in the MGC-803 cell line can promote the enhancement of tumor formation ability in nude mice.LINC02532 expression silencing can significantly increase the mRNA and protein levels of epithelial EMT marker E-cadherin,while the mRNA and protein levels of mesenchymal EMT markers N-cadherin,Vimentin and SLUG are significantly reduced.3.GSEA results show that the high expression of LINC02532 is significantly negatively correlated with the basal transcription factor pathway(P = 0.039).The Jaspar bioinformatics website predicts that the transcription factor FOXF2 can bind to the LINC02532 promoter region.Pearson correlation analysis found that FOXF2 expression in GC tissues was negatively correlated with LINC02532,and interference with FOXF2 could significantly increase the expression of LINC02532.The dual-luciferase reporter assay confirmed the negative regulatory effect of FOXF2 on LINC02532.Cell function experiments show that FOXF2 can negatively regulate LINC02532 to affect the migration and invasion of GC cells.4.The above GSEA results also show that the high expression of LINC02532 is significantly positively correlated with the RNA degradation pathway(P = 0.027).The Inta RNA website predicts that LINC02532 binds to the 3'UTR region of SOX7 mRNA.RNA Fluorescence in situ hybridization experiments show that LINC02532 is mainly located in the cytoplasm,suggesting that LINC02532 mainly functions in the cytoplasm.Pearson correlation analysis indicated that the level of LINC02532 in GC tissue was negatively correlated with SOX7,and interference with LINC02532 could significantly increase the expression of SOX7.The dual-luciferase reporter assay confirmed that LINC02532 binds to the 3'UTR region of SOX7 mRNA.Actinomycin D drug experiment found that SOX7 degradation rate after interfering with LINC02532 was significantly lower than the negative control group.Cell function experiments show that LINC02532 can negatively regulate the ability of SOX7 to participate in regulating the migration and invasion of GC cells.Conclusion: 1.The expression of LINC02532 is abnormally increased in GC cell lines and tissues,which can promote the migration and invasion of GC cells,and participate in the regulation of the expression of EMT-related markers.2.The expression of LINC02532 is significantly related to the TNM stage and tumor differentiation of GC.It can be used as an independent prognostic factor for GC and used to predict the 1-year,2-year and 3-year survival rates of GC patients.3.The low expression of FOXF2 attenuates the transcriptional inhibition of LINC02532 and thus causes abnormally high expression of LINC02532.FOXF2 participates in regulating the migration and invasion of GC cells through transcriptional regulation of LINC02532.4.LINC02532 promotes its degradation and expression by binding to the 3'UTR region of SOX7 mRNA,and also participates in the regulation of GC cell migration and invasion. |
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