The Role And Mechanism Of MiR-146a On Esophageal Cancer

Background and ObjectiveEsophagus cancer(EC)is one of the most common digestive tract malignant tumors in the world.China is one of the high incidence countries of esophageal cancer.The morbidity and mortality are the sixth and fourth of malignant tumors in our country respectively.The histological type 90% of EC is Esophageal Squamous Cell Carcinoma(ESCC).Since the early symptoms of esophageal cancer are not typical,more than 80% of the first diagnosed patients were middle and late stage,and the 5-year survival rate is only 15%-25%.Therefore,it is of great significance to study the pathogenesis of esophageal cancer on the molecular level,which is of great important for the prevention and treatment of the disease.microRNAs(miRNAs)are short(approximately 18-25 nucleotides)non-coding RNAs that regulate target mRNAs,mediating the process of cancer,and playing a role of oncogene or anti-oncogene.miR-146 a is a member of the miRNAs family,which is one of the most advanced and widely studied.It is considered as an anti-oncogene or oncogene involved in the process of tumor growth,metastasis and invasion.In this study,we will detect the expression of miR-146 a in ESCC and explore its effect on the biological behavior,and preliminarily explore the biological target of miR-146 and its regulatory mechanism.It is expected to provide a new potential target for the diagnosis and treatment of EC.Methods Real-time quantitative PCR(RT-qPCR)was used to detect the expression of miR-146 a in three esophageal squamous cell carcinoma cell lines(Eca109,KYSE140,KYSE150)and a normal esophageal epithelial cell(HEEC),It is determined that miR-146 a plays the role of oncogene or tumor suppressor gene in the cancer of the esophagus,and the appropriate cell lines are screened as target cell line for follow-up study.miR-146 a mimics was transfected into the target cell line by a Lipofectamine2000 to construct miR-146 a overexpressed cell model.Cell proliferation was detected by CCK8 assay,apoptosis and cycle were detected by flow cytometry,invasion was detected by Transwell assay and migration was detected by Scratch assay.Using related bioinformatics software to predict the target gene of miR-146 a.The effect of miR-146 a on the target gene was detected by the dual luciferase reporter system.The expression of mRNA and protein of target gene in the target cell line was detected by RT-qPCR and Western blot,respectively,and the relationship between them and the level of miR-146 a expression were futher analyzed? Rusults 1.The relative expression of miR-146 a in three ESCC cancer cell lines(Eca109,KYSE140,KYSE150)and the normal esophageal epithelial cells(HEEC): 0.36±0.05?0.16±0.06?0.09±0.02 and 1±0.05.miR-146 a levels of Eca109,KYSE140 and KYSE150 are lower than the HEEC cells(P <0.01).2.The OD value was detected by CCK8 assay at 0h,24 h,48h and 72 h.The results showed that the proliferation rate of mimics group was lower than that of NC group and MOCK group(P <0.01).3.The apoptosis of Eca109 was detected by flow cytometry.The results showed that the total apoptosis rate of mimics group was significantly higher than that of NC group MOCK group(P <0.01).4.The cell cycle was detected by flow cytometry.The results showed that the number of G1 phase cells in the mimics group decreased(P <0.05)and G2 / M phase cells increased(P <0.01))compared with NC group and MOCK group,There was no significant difference in S phase cells(P> 0.05)?5.The results of Transwell assay with Matrigel Matrigel showed that the number of Eca109 cells passing through the cell was significantly decreased compared with NC group and MOCK group(P <0.01)).6.The results of Scratch detected showed that the migration of mimics group was lower than that of NC group and MOCK group(P <0.01).7.The prediction bioinformatics software(Targetscan?MicroRNA.org?miRDB)suggest that miR-146 a sequence can be combined with the 3'UTR region of IRAK1,and IRAK1 may be the target gene of miR-146 a.The results of dual luciferase reporter assay showed that the most significant decrease in luciferase activity was observed in IRAK1 WT + miR-146 a mimics group(P <0.01).8.After transfected with mimics,the expression of IRAK1 mRNA was significantly decreased detected in Eca109(P <0.01),and the expression of IRAK1 protein was also decreased(P <0.01).ConclusionThe expression of miR-146 a is decreased in ESCC,and miR-146 a should play the role of tumor suppressor gene in esophageal cancer.miR-146 a can not only inhibit the proliferation of esophageal squamous cell carcinoma cell line Eca109,promote tumor cell apoptosis,but also inhibit its invasion and migration ability.miR-146 a inhibits the activation of downstream of IRAK-1 by inhibiting the expression of target gene IRAK1,thereby affecting the proliferation,differentiation,apoptosis,invasion,and metastasis of Eca109.The discovery of the miR-146a/IRAK-1 regulatory mechanism has revealed a new mechanism for the development of esophageal cancer and has provided a new potential target for the treatment of esophageal cancer.