The Effect Of LncRNA＿LINC00200 During The Progression Of Gastric Cancer And Its Mechanism

Gastric cancer(GC)is one of the most common malignant tumors worldwide.Around35% of GC cases are diagnosed in China,where GC is the second most common cancer and ranks the second place for the death of cancer patients.Due to the lack of specific clinical manifestations in early stage,GC patients are always diagnosed in advanced stage.GC is highly malignant with poor response to traditional treatment.Recently,the death rate of GC decreases remarkably but the 5-year survival rate for GC patients remains poor.The molecular mechanism of the development of GC remains unclear.Therefore,it is of great value to explore the mechanism of GC tumorigenesis and find effective biological biomarkers for early diagnosis,prognosis and targeted therapy of GC.Long non-coding RNAs(lncRNAs),once considered transcriptional “noise”,are one subtype of nc RNA that are > 200 nucleotides in length.With the intensive study on lncRNA,the role of lncRNA in the occurrence of diseases,especially tumors,is drawing much more attention.Lnc RNAs play a role in crucial biological functions in multiple ways.Dysregulated lncRNAs can function as oncogenes or tumor suppressors to alter cellular pathways.Lnc RNAs have also been demonstrated to promote tumor cell migration and metastasis by inducing the epithelial-mesenchymal transition(EMT).Furthermore,several lncRNAs have significant effects on multidrug resistance,which is responsible for chemotherapy failure.Additionally,multiple studies have suggested that lncRNAs play crucial roles in the modulation of tumor behavior through various complex mechanisms such as epigenetic regulation,transcriptional regulation,and post-transcriptional regulation.The interaction of lncRNA and miRNA play an important role in the occurrence and development of tumor.In 2011,Salmena et al proposed the competitive endogenous RNA(ceRNA)hypothesis that RNA transcripts communicate with and regulate each other by using shared miRNA response elements(MREs).The ceRNA hypothesis reveals a new mechanism for interaction between RNAs.This competition between mRNAs,lncRNAs and pseudogene transcripts regulates their expression using MREs to compete for binding with miRNAs.There is a complex regulatory network between lncRNA and miRNA or lncRNA and mRNA in GC.An intensive study of the network will contribute to further understand the pathogenesis and progression of GC.The Cancer Genome Atlas(TCGA)which aimed to construct a complete atlas of genome change related to cancer,was composed of NCI and NHGTI.It has collected approximately 11000 patient samples across more than 30 cancer types.With the advancement of genomics and bioinformatics tools,lncRNA-miRNA-mRNA ceRNA network has been constructed in a variety of tumors,including lung cancer,colon cancer,bladder cancer,breast cancer and liver cancer.An increasing number of lncRNAs have been identified to function as ceRNAs and expected to be potential targets for tumor diagnosis,treatment and prognosis.In the present study,we used data from 372 tumor tissues and 32 adjacent non-tumor tissues from TCGA,which provides information on RNA sequencing including mRNA,miRNA and lncRNA data.Then,the ceRNA network in GC was constructed.To verify the reliability of these results,five lncRNAs from the ceRNA network were randomly selected and their expression levels and functions in the GC cell line were examined.Experiments in vitro and in vivo were performed to explore the potential mechanism of LINC00200 regulating the biological process of GC,thus providing valuable biological targets for the diagnosis and treatment of GC.Part Ⅰ TCGA dataset-based construction and integrated analysis of aberrantly expressed long non-coding RNA mediated ceRNA network in gastric cancerObjective: The aberrant expression of lncRNAs has been confirmed to play a pivotal role in tumor initiation and development.Lnc RNAs can interact with miRNAs as ceRNAs to regulate the expression of target genes in various cancers.In the present study,the lncRNA-miRNA-mRNA ceRNA network of GC was constructed through the TCGA database and bioinformatics technology.The signal pathways involved in DEmRNA were analyzed and the RNA molecules related to the prognosis of GC patients were identified.Methods: The RNA sequencing profiles of 372 tumor samples and 32 adjacent non-tumor gastric samples were downloaded from TCGA database.The differential expression of RNAs was identified using the “edge R” package in R language software.The Gene Ontology biological processes and the Kyoto Encyclopedia of Genes and Genomes pathways were analyzed for differentially expressed mRNAs.Survival analysis was estimated based on Kaplan-Meier curves.Results: A total of 999 lncRNAs,137 miRNAs and 1629 mRNAs were identified as differentially expressed in GC with log fold change thresholds ≥ 2 and adjusted P values <0.01.The GO results indicated that DEmRNAs were significantly enriched in terms of the biological process,cellular component and molecular function.KEGG analysis indicated that some pathways corresponded to DEmRNAs were considered to be cancer associated,such as transcriptional misregulation in cancer,signaling pathway regulation of stem cells,p53 signaling pathway,PI3K-Akt signaling pathways and micro RNAs in cancer.A ceRNA network was constructed with 65 DElncRNAs,9 DEmiRNAs and 24 DEmRNAs.Of the65 DElncRNAs in the ceRNA network,9 were identified to be significantly associated with overall survival(P < 0.05).Of the 24 DEmRNAs in the ceRNA network,6 were identified to be significantly associated with overall survival(P < 0.05).Conclusion: Based on the analysis of large sample of GC from TCGA database,we identified the DERNAs between GC tissues and adjacent non-tumor gastric tissues.Then,the lncRNA-miRNA-mRNA ceRNA network was constructed.This network demonstrates a new mechanism of mutual regulation between RNA molecules,which is of great significance for the study of the tumorigenesis and progression.Part Ⅱ In vitro verification and functional study of differentially expressed lncRNAs in ceRNA network of gastric cancerObjective: To confirm the credibility of the TCGA database and bioinformatics results,five upregulated lncRNAs from the ceRNA network were randomly selected for in vitro validation and functional exploration.Methods: Real-time PCR was used to examine the expression levels of these 5lncRNAs between the SGC-7901 cell line and the human gastric epithelial mucosa cell line GES-1.To investigate the effects of the 5 lncRNAs on the proliferation,invasion and migration of GC cells,we transfected with corresponding si RNAs in SGC-7901 GC cells.Results: Real-time PCR results revealed that the expression levels of the 4 lncRNAs(ERVMER61,DSCR4-IT1,HULC,LINC00200)were significantly increased in the SGC-7901 cell relative to GES-1,which were consistent with TCGA database and bioinformatics predicted results.The expression levels of the 5 lncRNAs in SGC-7901 cells were evidently downregulated following si RNA transfection.CCK8 proliferation assays demonstrated that knockdown of 4 lncRNAs(ERVMER61,DSCR4-IT1,HULC and LINC00200)significantly inhibited cell proliferation.Reduced cell invasion in SGC-7901 cells was observed after si-ERVMER61,si-DSCR4-IT1,si-HULC and si-LINC00200 transfection.A decrease in cell migration was observed when HULC or LINC00200 were significantly downregulated in a wound healing assay.Conclusion: With the bioinformatics analysis and experiments validation,the TCGA database can effectively provide us with valuable biological targets.LINC00200 plays an important role in the GC progression and will be a potential target for our further study.Part Ⅲ The effect and mechanism of LINC00200 on biological function of gastric cancer cellsObjective: An increasing number of studies have found that lncRNAs play an important role in carcinogenesis and tumor progression,whereas their molecular mechanisms of function remain largely unknown.We explored the biological function,and underlying mechanism of LINC00200 in GC.Methods: q RT-PCR analysis was conducted to examine the LINC00200 expression level in both GC tissues and cell lines.Functional assays were carried out to detect the effect of LINC00200 on GC cell proliferation,invasion and migration.The interaction between miR-143-3p and LINC00200/SERPINE1 was confirmed by luciferase reporter assays.Rescue assays were performed to confirm the influence of LINC00200-miR-143-3p-SERPINE1 axis on GC development.Results: LINC00200 was found to be upregulated in GC tissues and cell lines.Knockdown of LINC00200 suppressed GC cell proliferation,invasion and migration in vitro.Mechanism research indicated that LINC00200 functioned as a ceRNA to sponge for miR-143-3p,thus leading to the disinhibition of its target gene SERPINE1.Conclusion: LINC00200 is significantly overexpressed in GC and accelerates GC progression through regulating miR-143-3p/SERPINE1 axis.Therefore,our results may provide a potential diagnostic biomarker and therapeutic target for the management of GC patients.Part Ⅳ LINC00200 regulates the expression of SERPINE1 and affects the growth of GC in vivoObjective: To investigate whether LINC00200 affects tumor growth in vivo,we performed a xenograft mouse experiment.Methods: SGC-7901 cells were stably transfected with scramble,sh-LINC00200,SERPINE1 or both sh-LINC00200 and SERPINE1 and injected subcutaneously into the nude mice.The tumor volumes and tumor weights were measured.Results: LINC00200 silencing group significantly reduced tumor volumes and weights compared with control group.SERPINE1 overexpression significantly increased tumor volumes and weights.Meanwhile,tumor growth inhibition caused by LINC00200 silencing could be reversed by SERPINE1 overexpression.Conclusion: The results indicate that LINC00200 promotes the growth of GC in vivo by regulating SERPINE1 expression.