MIR-25-3p Promotes EC109 Cells Migration And Invasion By Targeting NOTCH1 In Esophageal Squamous Cell Carcinoma

Objective: Micro RNA(miRNA)is a small fragment of non-coding RNA of about 21-25 nucleotides,which is of great significance in the development of many cancers.According to our previous studies,The cytological experiments show that increased miR-25-3p expression in ESCC(esophageal squamous cell carcinoma)in cell lines,enhance the invasion and proliferation of cells,indicating that miR-25-3p may have an important role in regulating the function of the occurrence and development of ESCC.In this study,we hope to verify the mechanism of miR-25-3p regulation by bioinformatics and provide a theoretical basis for further understanding of the role of miR-25-3p in esophageal cancer.Methods: the study is divided into three parts,the first: log into the GEO database of NCBI,enter search keywords "esophageal squamous cell carcinoma" to the search box.The data into the R language design of GEO2 R chip based on the data analysis tool,get the differential expression of ESCC cells in the m RNA list;on the other hand,the use of Target Scan 7 target gene prediction software miR-25-3p forecast all the highly conserved gene list,two genes on the column will get from the intersection with miR-25-3p ESCC.Tissue specific target genes may be high.The target gene was uploaded to the functional annotation software of DAVID gene for enrichment analysis,the cell function was enriched and the GO database was selected,and the KEGG database was selected by path enrichment.The target gene was screened by literature and results analysis.The second part(between miR-25-3p and NOTCH1 targeting to verify)liposome: synthesis of miRNA transfected ESCC cell line EC109,divided into three groups(1 groups(enhanced miR-25-3p expression increased),transfection of miR-25-3p agomir;2(miR-25-3p inhibition group decreased expression of transfected miR-25-3p antagomir;3),the control group,transfected with blank sequence).Fluorescence quantitative real-time PCR(q RT-PCR)technique was used to detect the expression of NOTCH1 in three groups.Luciferase reporter to verify the miR-25-3p and NOTCH1 in the 3 ’non encoding region(Untranslated Regions,UTR)at the target site.The third part(miR-25-3p targeting effect of NOTCH1 on cell proliferation and invasion function)previous studies have been carried out to study on the proliferation and invasion of EC109 cells by transfection of NOTCH1 miR-25-3p,the si RNA NOTCH1 miR-25-3p control NOTCH1 silence,simulation,observation of cell function,divided into two groups(1 groups(silence suppression the expression of NOTCH1 gene transfected NOTCH1 si RNA;2),control group,transfection of si-scrambled).Transwell invasion assay was used to detect the invasion ability of transfected cells,and the proliferation of cells was detected by CCK-8 assay.Results: Target Scan 7.0 was used to predict the target genes with highly conserved binding sites of miR-25-3p,with a total of 1037.GEO2 R was set up in groups,KYSE30 and KYSE180 high mobility cell subsets were set to KYSE D group,parental cells were set to KYSE U group.Through the consistency test of,the consistency between the 8 samples was better,and the difference genes were obtained by symbol.The gene ID of miRNA and Gene gene was not removed by GEO2 R,and finally,19491 differentially expressed genes were obtained.With the predicted target gene intersection,the miR-25-3p may be involved in the regulation of 623 genes in ESCC.KEGG filter: P < 0.05 > 2 and the number of genes,Fox O and c AMP genes mainly enriched in the cytoskeleton,signal pathway;GO filter: P < 0.05 > 2 and the number of genes,GO associated with the invasion and proliferation of NOTCH1,PTEN and TGFB2 gene.Combined with the previous literature,bioinformatics function analysis,and the negative control mechanism between miR-25-3p and target genes,NOTCH1 was selected as the target gene of miR-25-3p to verify and test the cell function.After transfection of miR-25-3p q RT-PCR showed that the expression level of miR-25-3p group increased significantly higher than the control group(P < 0.05),the expression of inhibitor of miR-25-3p group was significantly lower than the control group(P < 0.05),miR-25-3p in ESCC cell line EC109 in high and low expression cell model constructed successfully.QRT-PCR detected the expression of NOTCH1 in each group after transfection,the expression of NOTCH1 in the enhanced group was significantly lower than that in the control group(P < 0.05),and the expression of NOTCH1 in the control group was higher than that in the control group(P < 0.05).Luciferase reporter gene assay system showed that miR-25-3p targeting NOTCH1 3 ’UTR complementary sites,luciferase activity is significantly lower than the control group(P < 0.05),it was indicated that miR-25-3p and NOTCH1 have targeted regulation relationship.EC109 cell si-NOTCH1 after transfection,NOTCH1 expression silencing group was significantly lower than the control group,the construction of NOTCH1 silencing cell model,Transwell invasion experiment results show silent group cell invasion was significantly higher than that in control group(P < 0.05),CCK-8 proliferation assay showed that the proliferation ability of silent group was significantly higher than that in control group,and miR-25-3p the high expression cell model cell function results.Conclusion: miR-25-3p can inhibit the proliferation and invasion of ESCC cell line EC109 by targeting NOTCH1.