Study On The Effect And Mechanism Of MiR-28-5p On The Growth Of Esophageal Cancer By Targeting MTSS1

Objective: Esophageal Cancer is one of the most common malignant tumors of digestive tract with the morbidity and mortality near the top of the malignant tumors worldwide.China is one of the most serious regions in terms of the incidence of esophageal cancer.Esophageal cancer clinically presents two kinds of pathological tissue types: namely Esophageal Adenocarcinoma and Esophageal Squamous cell carcinoma,with the latter being more common clinically and accounting for about 80% of all Esophageal cancer cases.Metastasis of esophageal cancer is always hard to detect.The treatment methods are mainly surgery,radiation treatment and chemotherapy,but the efficacy is not ideal.Moreover,the survival rate of patients is low and the recurrence rate is high,thus posing a serious threat to public health.Therefore,it is of great public health significance to study the pathogenesis of Esophageal Cancer.MicroRNA(miRNA)are a class of endogenous,non-coding small RNA molecules that can be involved in the growth,differentiation,metastasis and other aspects of a variety of tumors by regulating downstream target genes,and play a very important role in the occurrence and development of tumors.miR-28-5p is a microRNA with multiple biological functions,which can regulate the proliferation,invasion and migration of renal and colon cancer cells,and is closely related to the development of liver and ovarian cancer.However,the expression and mechanism of miR-28-5p in esophageal cancer remain unclear.This paper intends to investigate the expression of miR-28-5p in esophageal cancer and explore the role and related mechanism of miR-28-5p and its target gene tumor suppressor 1(MTSS1)in the occurrence and development of esophageal cancer.Methods: 1.30 cases of esophageal cancer tissues and tumor adjacent tissues were collected,and the expression level of miR-28-5p in these tissues was detected by qRT-PCR.qRT-PCR was also used to detect the expression levels of miR-28-5p in two types of esophageal cancer cells.By transfection with miR-28-5p mimics or inhibitor,the effects of miR-28-5p mimics on cell proliferation,cell cycle and apoptosis were detected by CCK-8 assay,flow cytometry and western blotting.2.The target gene of mi R-28-5p were predicted and identified by bioinformatics,dual-luciferase reporter gene system,qRT-PCR and western blotting.3.The expression level of MTSS1 in esophageal cancer tissues was detected.The MTSS1 overexpression plasmid was co-transfected with mi R-28-5p mimics into TE-1 cells with the subsequent co-transfection with miR-28-5p inhibitor and MTSS1 si RNA.Their effects on cell proliferation and cell cycle were detected by CCK-8 assay and flow cytometry,respectively.4.Transplanted tumor model of nude mice was established by subcutaneous injection of TE-1 human esophageal cancer cells.After miR-28-5p antagomir treatment,changes in tumor volume,histopathology and MTSS1 expression in nude mice were observed.The expression of cell cycle related proteins(including cyclin A,cyclin D1,cyclin E and CDK2)and apoptosis related proteins(including caspase-3 and caspase-9)were detected by western blotting.Results: 1.Compared with tumor adjacent tissues,the expression of miR-28-5p in esophageal cancer tissues was significantly increased,and the expression was also upregulated in two esophageal cancer cell lines.Up-regulation of miR-28-5p expression in esophageal cancer cells significantly promotes cell proliferation,transferred cell cycle from G1 phase to S phase and inhibit cell apoptosis.Downregulation of miR-28-5p inhibited cell proliferation,inhibited cell cycle transition from G1 phase to S phase and promoted cell apoptosis.2.The bioinformatics method predicted that MTSS1 was a potential target of mi R-28-5p,and the dual luciferase reporter gene system,qRT-PCR and western blotting results showed that MTSS1 was the direct target gene of miR-28-5p,and mi R-28-5p bound complementarily to the 3'-UTR region of MTSS1.mRNA and protein expression levels of MTSS1 were significantly decreased after transfection with miR-28-5p mimics.mRNA and protein expression levels of MTSS1 were significantly increased after transfection with miR-28-5p inhibitor.3.Compared with tumor adjacent tissues,the expression of MTSS1 in esophageal cancer tissues was significantly decreased.After co-transfection with MTSS1 overexpressed plasmid and mi R-28-5p mimics into TE-1 cells,cell proliferation was significantly reduced,indicating that MTSS1 overexpression could reverse the effect of miR-28-5p on cell proliferation and promote apoptosis.In addition,MTSS1 knockdown could reverse the inhibitory effect of miR-28-5p inhibitor on cancer cell growth and inhibit apoptosis.4.Compared with the control group,injection of miR-28-5p antagomir significantly reduced the tumor volume of nude mice bearing esophageal cancer,shrank the tumor nucleus,and inhibited the proliferation of cancer cells.In addition,miR-28-5p antagomir significantly up-regulated the expression of MTSS1 protein in tumor-bearing mice and reduced the expression level of ki-67.miR-28-5p antagomir therapy down-regulated the expression of cyclin A,cyclin D1,cyclin E and CDK2 and up-regulated the expression of caspase3 and caspase9,which resulted in G1 cell cycle arrest and ultimately promoted the apoptosis of tumor cells.Conclusion: The expression of miR-28-5p is up-regulated in esophageal cancer,and MTSS1 is its target gene.The expression of miR-28-5p is negatively correlated with the expression of MTSS1,and miR-28-5p promotes cell proliferation and tumor growth of esophageal cancer by inhibiting the expression of MTSS1.