Effects Of MiR-4746 On Proliferation,Migration And Invasion Of Esophageal Squamous Cell Carcinoma TE-1

Objective:Esophageal cancer is a malignant tumor that occurs in esophageal epithelial tissues.There are mainly two types of pathological types:squamous cell carcinoma and adenocarcinoma.Among them,ESCC is the main pathological type of esophageal cancer in China,while EAC mainly occurs to Europe,America and other western countries.The mortality rate of esophageal cancer in China ranks fourth in malignant tumors.The onset of this disease is hidden,most patients have entered the middle and advanced stages when diagnosed,and the 5-year survival rate is only 20%[1],which seriously threatens the patient’s physical health and life safety.Therefore,screening of biological markers and clarification of signaling pathways for esophageal cancer are of great significance for the diagnosis and treatment of esophageal cancer.MicroRNAs(microRNA,miRNA)are a type of non-coding RNA with a length of 21 to 25 nucleotides.They are regulated by genome transcription,control gene expression of the post-transcription level,and participate in inflammation,immune response,cell proliferation and apoptosis biological processes[2].Current research reports have shown that miRNAs are closely related to the occurrence and development of tumor diseases.In the early stage,the group targeted esophageal squamous cell carcinoma tissues and used gene chip experiments to screen for differentially expressed miRNAs.Among them,miRNA-4746(miR-4746)was up-regulated more than 2-fold in esophageal cancer tissues and analyzed by biological information analysis software.miR-4746 was analyzed in TGF-β/Smads pathway that was significantly enriched.At present,there are few reports on the effects of miR-4746 on the biological characteristics of esophageal cancer cells and related mechanisms.Therefore,in this study,we investigated the effects of miR-4746 on the proliferation,migration,invasion and cell cycle biological characteristics of esophageal cancer cell lines by targeting the regulation of miR-4746 expression.We will further explore the molecular regulatory mechanism of miR-4746 on the biological characteristics of esophageal cancer cells.To provide effective experimental data and necessary theoretical basis for clinical targeted prevention and treatment of esophageal cancer.Methods:1.The expression of miR-4746 in human normal esophageal epithelial cell lines HEEC and esophageal squamous cell carcinoma cell lines TE-1 and ECA-109 were detected by real-time fluorescence quantitative(RT-PCR).2.The effect of miR-4746 on the proliferation ability of esophageal cancer cell line TE-1 was tested by CCK-8 experiment.3.The effect of miR-4746 on the migration ability of esophageal cancer cell line TE-1 was detected by a scratch test.4.The effect of miR-4746 on the invasion ability of esophageal cancer cell line TE-1 was tested by Transwell chamber experiment.5.Flow Cytometrywas used to detect the effect of miR-4746 on the cell cycle of esophageal cancer cell line TE-1.6.RT-PCR was used to detect the changes of miR-4746 on the expression of TGF-β1 mRNA,TβR Ⅱ mRNA,and Smad4 mRNA in esophageal cancer cell line TE-1.7.Western blot(WB)method was used to detect the changes of miR-4746 on TGF-β1,TβRⅡ,and Smad4 in esophageal cancer cell line TE-1.Results:1.Compared with the normal esophageal epithelial cell line HEEC,the expression of miR-4746 in esophageal cancer cell lines TE-1 and ECA-109 was up-regulated.The difference was statistically significant(P<0.001),consistent with the results in tissues.2.CCK-8 experiments showed that after 24 hours,the proliferation of TE-1 cells was significantly enhanced after miR-4746 was up-regulated,and the proliferation of TE-1 cells was significantly decreased after miR-4746 was down-regulated.The difference was statistically significant(P<0.05).3.The scratch test showed that the migrating ability of TE-1 cells was significantly enhanced after miR-4746 was up-regulated,and the migrating ability of TE-1 cells was significantly reduced after miR-4746 was down-regulated,the difference was statistically significant(P<0.01).4.Transwell experiments showed that the invasion ability of TE-1 cells was significantly enhanced after miR-4746 was up-regulated,and the invasion ability of TE-1 cells was significantly decreased after miR-4746 was down-regulated,the difference was statistically significant(P<0.01).5.Flow Cytometry showed that the effect of miR-4746 on TE-1 cell cycles was not statistically significant(P>0.05).6.RT-PCR experiments showed that TGF-β1 mRNA expression of TE-1 cells was up-regulated after miR-4746 expression was up-regulated,and TβRⅡ mRNA and Smad4 mRNA expression were down-regulated after down-regulation of miR-4746;The expression was down-regulated,and the expression of TβRⅡ mRNA and Smad4 mRNA was up-regulated.The differences were statistically significant(P<0.05).7.WB experiments showed that the expression of TGF-β1 protein in TE-1 cells was up-regulated and the expression of Smad4 protein was down-regulated after miR-4746 was up-regulated.The differences were statistically significant(P<0.05).TβRⅡ protein expression showed no significant difference(P>0.05).Conclusion:In the occurrence and development of esophageal cancer,miR-4746 inhibits Smad4 and TGF-β/Smads pathway.TGF-β1 increases feedback.So it may promot the proliferation,migration and invasion of esophageal cancer cells.