The Expression Level Of MiR-483-5p In Serum And Tissue And Its Regulation Mechanism In Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma(ESCC)is one of the most common types of malignacies in China.Most ESCC patients are diagnosed at middle to late stages with poor prognosis due to the lack of an effective method for early diagnosis.Therefore,it is needed to find an effective method for early diagnosis.MiRNAs are a group of endogenous small non-coding RNAs that can regulate ESCC development and progression by repressing their expression of specific target genes.In this study,firstly,the four miRNAs(miR-25-3p,miR-223-3p,miR-373-3 and miR-483-5p)were selected according to serum miRNAs microarray in some reports;Secondly,the expression levels of four miRNAs and the mRNA related to miR-483-5p were measured by RT-qPCR,and the methylation levels of related genes detected by MSP method.The study was aimed to explore the expression levels of four miRNAs in ESCC and the molecular regulation mechanism of miR-483-5p.The results were as follows:1.The expression levels of miR-25-3p,miR-223-3p,miR-373-3p,miR-483-5p were significantly up-regulated in patients' serum compared with those in healthy volunteers(p?0.01).ROC curve analyses revealed that the AUC value for miR-25-3p,miR-223-3p,miR-373-3p and miR-483-5p was 0.794(95% CI:0.659-0.928;sensitivity=81.0%;specificity=66.7%),0.839(95% CI:0.717-0.961;sensitivity=100.0%;specificity=61.9%),0.873(95% CI: 0.763-0.983;sensitivity=90.5%;specificity=81.0%),and 0.927(95%CI: 0.850-0.1.000;sensitivity=95.2%;specificity=81.0%),respectively.In addition,the expression level of miR-483-5p was correlated with the TNM stage(p<0.05)and lymph node metastasia(p<0.05).As a result,four serum miRNAs were observed to be significantly reduced after surgical removal of the tumors(p<0.01).We have demonstrated that four serum miRNAs which had high sensitivity and specificity can beused for the early prediction of ESCC.PCR products amplified using stem-loop primers from serum and tissue of ESCC patients were right by sequencing.So the method was feasible.The high expression of four miRNAs in cancer tissues and adjacent normal tissues of ESCC revealed that four miRNAs were observed to be markedly elevated in cancer tumors(p<0.01).Therefore,four miRNAs were located in the tissues of ESCC.2.MiR-483-5p showed high sensitivity and specificity with a high diagnostic value for ESCC.Therefore,the regulation mechanism of miR-483-5p was further studied.Bioinformatic analysis showed that miR-483-5p directly targets the 3'UTR of Rho GDI1,Socs3,ALCAM.The expression of Rho GDI1,Socs3,ALCAM by RT-qPCR showed that Rho GDI1,Socs3,ALCAM were down-regulated.These results revealed that miR-483-5p down-regulated the expression of Rho GDI1,Socs3,ALCAM.3.MiR-483-5p was located in the second intron of IGF2 gene,and high expression level of IGF2 was positively correlated with miR-483-5p expression level(r<0.05)by RT-PCR,and miR-483-5p can be coexpressed together with its host gene IGF2.We analyzed the relationship between miR-483-5p and methylation level of CDKN2 A and IGF2,the results showed that CDKN2 A had high methylation with81.82% in ESCC.However,the methylation level IGF2 was only 54.55% when miR-483-5p was high expression.Therefore,high expression of miR-483-5p might be ascribed to low methylation level of IGF2 promoter.In summary,this article explained the function of miR-483-5p in the pathogenesis of ESCC.The hypomethylation of IGF2 could lead to elevated expression of miR-483-5p and IGF2,and further accelerate the degradation of GDI1 Rho,ALCAM and Socs3,so that carcinogenic factor was enhanced,tumor suppressor factor was decreased,and thus led to the occurrence and development of tumor.The changes of Methylation of IGF2 and CDKN2 A showed that epigenetic modification played an importantrole in the pathogenesis of ESCC.This study laid the foundation for further research of the mechanism of miRNAs in ESCC.