LINC00520 Regulates The Proliferation,Migration And Epithelial-Mesenchymal Transition Of Esophageal Squamous Cell Carcinoma Through MiR-204-5p

Background and Objective(1)Screening of differentially expressed RNA by bioinformatics analysis of transcriptome of TCGA datain esophageal squamous cell carcinoma(ESCC)tissues,constructing a ceRNA regulatory network associated to LINC00520,and predicting the function of LINC00520.(2)Verify the regulatory relationship of the LINC00520/miR-204-5p/TrkB ceRNA network in vitro in ESCC cells.(3)Analysis of biological effects of LINC00520 and miR-204-5p in ESCC cell lines in vitro,including proliferation,migration and EMT.Materials and Methods(1)Download ESCC-related data from the TCGA database,and use R language to screen the differentially expressed lncRNA,miRNA and mRNA between ESCC cancer and adjacent normal tissues,and construct a ceRNA regulatory network associated to LINC00520.(2)Downstream target genes of LINC00520 were predicted through online database MEM,and were performed GO and KEGG analysis.(3)qRT-PCR were performed to y the expressions of LINC00520,miR-204-5p and TrkB in ESCC tissue samples and cell lines.(4)ESCC cells of EC9706,Kyse450 were transfected with LINC00520 siRNA;miR-204-5p and TrkB mRNA expressions were detected by qPT-PCR,TrkB and EMT-related proteins detected by Western-blot in transfected cells.Scratch test,MTT and colony formation assays were performed to observe the effects of LINC00520 on migration,cell proliferation and colony formation in ESCC cells.(5)After ESCC cell line EC9706 and Kyse450 were transfected with miR-204-5p mimic,qPT-PCR or Western-blot was performed to detect TrkB mRNA or protein expression,respectively.Biological effects of miR-204-5p on ESCC cells were detected by scratch test,MTT,clone formation test.Results(1)A total of 1160 differential lncRNA,2064 mRNA and 69 miRNAs were screened in ESCC from TCGA database.The miRNA and mRNA associated with LINC00520 were selected and successfully constructed a ceRNA regulatory network of lncRNA/miRNA/mRNA.The network includes 3 miRNAs and 13 mRNAs.(2)GO functional enrichment and KEGG pathway enrichment analysis using top300 target mRNAs of LINC00520 showed LINC00520 might be involved in the development and progression of ESCC.(3)Expressions ofLINC00520 and TrkB mRNA increased in esophageal cancer tissue samples and cell lines(p <0.05),and miR-204-5p expression decreased(p <0.05).(4)After silencing of LINC00520,the expression of miR-204-5p gene was increased,and the expressions of TrkB mRNA protein were decreased.LINC00520 silencing could inhibit migration,colony formation,cell proliferation and EMT transformation of ESCC cells.(5)Transfection of miR-204-5p mimics decreased expression of TrkB mRNA and protein,inhibit cell migration,colony formation and cell proliferation in ESCC cells.Conclusions(1)LINC00520 as an oncogene in ESCC promote proliferation,migration,colony formation and EMT transformation of ESCC cells.The expression of miR-204-5p is reduced in ESCC,and it can inhibit proliferation,migration and colony formation of ESCC cells as tumor suppressor genes.(2)LINC00520 up-regulates the expression of TrkB by miR-204-5p,thereby promoting the occurrence and development of ESCC.