The Role And Mechanism Of MiR-1246 In Esophageal Cancer Metastasis

Background Esophageal cancer is one of the most invasive and least studied cancer in the world.It is the third most common cancer and the fourth leading cause of cancer-related mortality in China.According to a report published in CA Cancer J Clin by the National Cancer Research Center,there are estimated about 478,000 incidences and 375,000 deaths in China.More astonishing,48.9% of the global incidence and 49.3% of death cases occurred in China.The research in esophageal cancer is relatively lagged other cancers.However,most of the patients are diagnosed at the advanced stage for lack of effective means of early diagnosis.The overall 5-year survival of esophageal cancer patients ranges from 15% to 25%.So,it is necessary to explore applicable molecular markers and targets of esophageal cancer,to provide clues for the early diagnosis and treatment of esophageal cancer.Micro RNAs(mi RNAs),a class of small,endogenic and evolutionarily conserved,single-stranded non-coding RNAs of 18–25 nucleotides in length,mainly affect the body’s a variety of physiological and pathological processes by inhibiting the translation of target genes.Recently,by detecting the differentially expressed molecules in 20 cases of esophageal squamous cell cancer(ESCC)tissues and the corresponding adjacent tissues,we found mi R-1246 is upregulated in esophageal cancer tissues.However,the role of mi R-1246 esophageal cancer metastasis and its molecular mechanism has not been fully elucidated.Aims 1.To detect the expression of mi R-1246 in ESCC cell lines and the normal esophageal squamous epithelium cell line.2.To construct the esophageal cancer cell lines which can upregulate or downregulate the expression of mi R-1246,and study the effects of mi R-1246 on the biological behaviors of esophageal cancer cells through in vivo and in vitro experiments.3.To investigate the role and mechanisms of mi R-1246 in promoting esophageal cancer metastasis.Methods 1.Realtime-PCR was used to evaluate mi R-1246 expression between esophageal cancer tissues and paired adjacent normal tissues,esophageal cancer cell lines and normal esophageal epithelial cells.2.Establishing the esophageal cancer cell models overexpressing mi R-1246 mimics and negative control.3.Establishing the esophageal cancer cell models with decreasd expression of mi R-1246 and the control cell models by transfecting cells with mi R-1246 inhibitor and inhibitor NC.4.After transfection of mi R-1246 mimics or Inhibitors,transwell invasion assay was applied to the effect of mi R-1246 in esophageal cancer cell metastasis.5.Establishing stable overexpressing mi R-1246 cell models by transfecting cells with mi R-1246 lentivirus vector and observe the effect of mi R-1246 overexpression in metastasis of esophageal cancer in vivo;6.Bioinformatics analysis revealed GSK3β as a candidate target genes of mi R-1246..7.Luciferase activity of the luciferase reporter assay was used to detect the effect of mi R-1246 on wild-type and mutant GSK3β in EC109 cells.8.Western blot was used to evaluate whether mi R-1246 affect protein expression of GSK3β.Results1.The expression of mi R-1246 in esophageal cancer tissues was significantly elevated,compared with paired adjacent normal tissues(P <0.01);mi R-1246 expression level in the esophageal cancer cells was significantly elevated,compared with normal esophageal epithelial cell HET-1A(P <0.01).2.Esophageal cancer cells with increased or decreased expression of mi R-1246 were successfully established.3.Mi R-1246 mimics significantly increased invasion of EC109 cells,compared with the negative control(P <0.05).While mi R-1246 inhibitors significantly inhibited invasion of EC109 cells(P <0.05);4.Stable expression of mi R-1246 in EC109 cells significantly increased lung invasion capacity in vivo.5.GSK3β is screened as a potential mi R-1246 target gene through bioinformatic analysis.6.Luciferase reporter assay showed that mi R-1246 inhibited luciferase activity of GSK3β by targeting its 3’-UTR region.7.Western blot assay showed EC109 cells transfected with mi R-1246 significantly decreased GSK3β expression,compared with the negative control;Western blotting showed that overexpression of mi R-1246 significantly inhibited the expression of GSK3β-wt,but not GSK3β-mut.Conclusion Mi R-1246 promotes esophageal cancer metastasis by targeting GSK3β.Mi R-1246 /GSK3β may be a critical pathway in esophageal cancer metastasis.Inhibition of mi R-1246 expression or GSK3β restoration may be effective for the treatment of esophageal cancer metastasis.