| Objective: Gastric cancer is the fifth most common cancer in the world and the third most common cause of cancer death.Although the long-term survival rate of gastric cancer patients has been significantly improved with the improvement of surgical techniques and the application of radiotherapy and chemotherapy,those diagnosed with advanced gastric cancer still have a high mortality rate.The main cause of poor prognosis is metastasis of tumor cells,so understanding the mechanism of invasion and metastasis plays an important role in finding possible treatment for gastric cancer.Long non coding RNA(lncRNA)is a class of RNA molecules that do not encode proteins and participate in intracellular biological processes.There are many characteristics of lncRNA,including tissue and organ specificity and time specificity.In different tissues,the expression of lncRNA is different,and when we are in different growth stages,the expression of lncRNA will also change.At present,it is found that lncRNA can play an important role in the proliferation and differentiation of gastric cancer.It can play an important role in the proliferation and differentiation of gastric cancer.However,there is little research on the invasion and metastasis of gastric cancer.Weighted co-expression analysis(WGCNA)is an efficient and accurate data mining method using weight algorithm.Weighted co-expression analysis aims to construct a gene co-expression module based on phenotype through accurate calculation of gene expression and disease phenotype,so as to explore and study the relationship between interested phenotypes and genes in the module.Module is composed of a group of genes with relative expression quantity.When constructing the weighted co expression network,if the expression changes of some genes in different tissues are always similar,or have similar expression trends,then we can infer that these genes with similar expression have similar functions,and combine them to form a module.Weighted co-expression network is to simplify the complex gene network into relatively simple modules,that is to say,a module is equivalent to a "super gene",and its function is determined by all genes in this module.At present,due to the lack of research on the function of lncRNA,people’s understanding of it is far from the depth of mRNA.Therefore,when we mix the gene mRNA expression and lncRNA expression of patients,we can get the module of lncRNA and mRNA data mixing by using the weighted co-expression network.Through the functional analysis of mRNA,we can approximately understand the function of lncRNA in the same module.Through this method,we can explore the lncRNA and its function that we are interested in,so as to effectively improve the efficiency of scientific research.Epithelial-mesenchymal transition(EMT),an evolutionarily conserved developmental procedure,has been shown to be closely associated with cell carcinogenesis.Studies have shown that EMT endows cancer cells with metastatic properties by enhancing cell invasion,mobility and resistance to apoptotic stimuli.Thus,EMT complex biological processes are considered to be key markers of cancer metastasis,targeting EMT pathways is an attractive strategy in cancer therapy.TWIST family transcription factor 2(twist family b HLH transcription factor 2,TWIST2),as a transcription factor,can exert molecular function directly or indirectly by regulating the transcription of interstitial cells,activate or inhibit downstream target genes,thus affecting the occurrence and development of interstitial cells.The purpose of this study is to search for lncRNA,related to gastric cancer metastasis and to explore the mechanism of its regulation by screening and integrating the data of TCGA database and using WGCNA algorithms.To provide theoretical basis for further exploration of molecular mechanism of gastric cancer.Methods: We used TCGA database to screen lncRNAs with different expression and survival significance,and selected our interest as our main research target.RT-qPCR was used to verify the expression of LINC01235 in GES-1,SGC-7901,MGC-803,AGS and HGC-27 cell lines,and the cell lines suitable for our further experiments were selected.Meanwhile,the expression of LINC01235 in 48 pairs of clinical gastric cancer tissues and adjacent tissues was compared,and the correlation between LINC01235 and clinicopathological factors was analyzed.The TCGA database and GSEA analysis were used to explore the related pathways that LINC01235 may participate in,and the expression of the marker genes in the pathway was further detected by RT-qPCR to verify the effect of LINC01235 on the related pathways.Scratch and Transwell experiments further verified the effect of LINC01235 on invasion and migration of gastric cancer cells.HGC-27 cells were injected into the tail vein of nude mice to observe the pulmonary metastatic nodules.The TCGA database was used to construct a weighted co-expression network,and the modules of LINC01235 in the network were analyzed.Then go and KEGG were used to predict the biological functions of LINC01235 and the genes highly related to LINC01235 in the same module.TCGA database analysis and clinical tissue examination further confirmed the correlation between LINC01235 and related genes.The protein expression was detected by immunohistochemistry,and the correlation between survival curve and expression was analyzed.Jaspar website predicted the target sequence of transcription factors acting on LINC01235,luciferase experiment and chip verified the direct binding site.Lncbase,miRWalk and miRDB were used to predict possible miRNAs.Results: We screened 18 lncRNAs with different expression and significant survival in TCGA database(P < 0.05).The WGCNA model was constructed,and it was found that cyan was highly correlated with T stage of gastric cancer(P = 3.2e × 10-8).LINC01235 is the hub lncRNA of this module.Comparing the expression of LINC01235 in TCGA matched gastric cancer tissues,it was found that LINC01235 was highly expressed in gastric cancer tissues(P < 0.001),and HGC27 was the highest among the gastric cancer cell lines.Go analysis showed that LINC01235 was involved in composition of extracellular matrix,and KEGG analysis showed that LINC01235 pathway was enriched in local adhesion and ECM-receptor interaction.GSEA analysis showed that LINC01235 was involved in EMT related pathways.Compared with the control group,the expression of E-cadherin was increased,and the expression of N-cadherin,vimentin and fibronectin(FN)was decreased in HGC27 cells silenced with LINC01235.Immunofluorescence assay confirmed this result again.Scratch test showed that silencing LINC01235 significantly reduced the mobility of gastric cancer cells.Transwell’s invasion and migration assay also showed that silencing LINC01235 inhibited the invasion and migration of gastric cancer cells.Compared with the control group,HGC-27 /sh LINC01235 had fewer and smaller metastatic nodules in the nude mice with tail vein injection.TCGA correlation analysis showed that LINC01235 was positively correlated with TWIST2 expression.Similar results occurred in 48 pairs of gastric cancer and adjacent tissues(r = 0.63;r = 0.405).RT-qPCR results showed that TWIST2 expression was significantly down regulated in LINC01235 knockdown cells.Western blotting showed that silencing LINC01235 significantly increased the expression of E-cadherin and decreased the expression of vimentin and N-cadherin.However,overexpression of TWIST2 reversed the inhibition of mesenchymal genes and down regulated the expression of epithelial genes.In scratch and Transwell recovery test,it was further confirmed that TWIST2 could reverse the migration and invasion ability of silenced LINC01235 cells.We transfected HGC-27 and SGC-7901 cells with LINC01235 overexpression plasmid.Western blot showed that the expression of TWIST2 was increased,and the expression of THBS2 decreased,which indicated that THBS2 might be a downstream target of LINC01235 – TWIST2 axis.In the recovery experiment,THBS2 overexpression reversed the effect of TWIST2 on EMT.Immunohistochemistry showed that TWIST2 expression was negatively correlated with THBS2 expression(r =-0.506,P < 0.001).Survival analysis showed that high expression of TWIST2 was associated with poor prognosis,while low expression of THBS2 was associated with poor prognosis.In immunofluorescence analysis,we found that the intensity of green fluorescence(TWIST2)was significantly higher than that of red fluorescence(THBS2)in cancer tissues,while in adjacent tissues,we observed the opposite situation.The difference of correlation analysis was statistically significant(r=-0.448,P < 0.001).The overexpression gradient of TWIST2 in HGC-27 and SGC-7901 cells indicated that LINC01235 expression was also increased.We investigated the predicted binding sites of TWIST2 in the genome LINC01235 on Jaspar website,and used luciferase test to verify that the ex vivo TWIST2 expression enhanced firefly luciferase transcription from wildtype LINC01235 promoter.When TWIST2 binding sequence was deleted,luciferase expression was significantly decreased(P < 0.01).We also performed ChIP assay to determine whether TWIST binds to the LINC01235 promoter in cells.As expected,RTqPCR results showed that TWIST2 group was higher than IgG control group(P < 0.001).In order to investigate the regulatory effect of LINC01235 on TWIST2,we predicted and analyzed the target genes in LncBase,miRWalk and miRDB databases.Venn diagram showed that mir-6852-5p may be the key target miRNA of LINC01235 in regulating TWIST2.TCGA database analysis showed that the expression of miR-6852-5p was negatively correlated with LINC01235 and TWIST2,which further confirmed our idea.Conclusions: LINC01235-TWIST2 positive feedback loop mainly affects GC cell migration and invasion,TWIST2 can express positive feedback LINC01235,and THBS2 is the downstream target of the loop.These results suggest that LINC01235 may be a potential target for gastric cancer therapy. |
PDF Full Text Request