Study On The Mechanism Of Exosomes-derived MiR-103a-2-5p In Facilitating Proliferation And Migration Of Esophageal Cancer Cells

Objectives: Esophageal cancer(EC)ranked seventh cancer incidence and sixth in mortality worldwide in 2018.The prognosis for patients with ESCC remains grim despite improvements in diagnostic methods and multidisciplinary treatment.As a consequence,the study of the molecular mechanism related to the occurrence and development of esophageal cancer is helpful to discover potential diagnostic and prognostic biomarker in the early diagnosis of ESCC.MicroRNAs(miRNAs)are a class of small noncoding RNAs that regulate gene expression at post-transcriptional level.Numerous studies have established that miRNAs function as crucial regulators of differentiation,proliferation,apoptosis,and metabolism in cancer cells.This study aimed to identify the different levels of miRNAs in the plasma derived exosomes of patients with esophageal squamous cell carcinoma(ESCC).The level and mechanism of exosomal miR-103a-2-5p in esophageal cancer are not investigated up to now.Methods:1.miRNA sequencing was performed to screen differential levels of micro RNAs in plasma exosomes between patients with ESCC and controls.Het-1A(normal human esophageal epithelial-1 cell),TE-1(high differentiation),and KYSE-150(low differentiation)esophageal carcinoma cell lines were used to verify the expression of the selected miRNAs.2.After TE-1 cell lines were transfected with mi R-103a-2-5p mimics and KYSE-150 cell lines were transfected with miR-103a-2-5p inhibitors,CCK-8,Transwell assay and wound scratch assay were used to detect the proliferation,migration of two ESCC cells.3.Exosomes from cells transfected with miR-103a-2-5p mimics and miR-103a-2-5p mimics-NC were respectively extracted.TE-1 and KYSE-150 cells were co-cultured with the two types of purified exosomes,respectively,for 24 h.CCK-8,Transwell assay and wound scratch assay were used to detect the proliferation,migration of two ESCC cells.4.Predicted target genes of miR-103a-2-5p were screened by databases and confirmed by q-PCR.Results:1.mi RNA sequencing was performed to explore the levels of miRNAs in exosomes extracted from nine ESCC and nine control patients.The levels levels of 61 miRNAs in plasma exosomes derived from ESCC patients were significantly different from those of control patients.10 miRNAs were identified as having fourfold differential levels between groups.Het-1A,TE-1,and KYSE-150 cell lines were used to verify the expression of the 10 selected miRNAs.miR-103a-2-5p was selected for the subsequent functional experiments.2.Overexpression of miR-103a-2-5p promoted proliferation and migration in TE-1 cells.Compared with the control group,down-regulating miR-103a-2-5p significantly inhibited the proliferation and migration in KYSE-150 cells.3.The exosomes extracted from cells transfected with miR-103a-2-5p mimics significantly increased the proliferation and migration of ESCC cell lines.4.Two genes,CDH11 and NR3C1 were identified as predicted targets of miR-103a-2-5p by the bioinformatics tools Target Scan,miranda,mirDIP and q-PCR.The results of q-PCR showed that compared with the control group,the mRNA levels of CDH11 and NR3C1 in TE-1 and KYSE-150 cell lines after co-culture with high levels of miR-103a-2-5p exosomes significantly decreased.Conclusions: In this study,miRNA sequencing was used for the first time to find that the level of mir-103a-2-5p increased in peripheral blood exosomes of esophageal squamous cell carcinoma.Our results shed light on how exosomal miR-103a-2-5p can promote proliferation and migration of ESCC cells and may represent a potential diagnostic or prognostic biomarker for ESCC therapies.