The Expression Of RIOK3 In Esophageal Carcinoma And Its Effect On Cellular Biological Behaviors

BackgroundEsophageal cancer is one of the most common malignant tumors of the digestive system.According to WHO statistics,the number of people who die of esophageal cancer in China is about 200,000 a year,accounting for half of the world.Tumor metastasis is the main causes of prognosis and death in patients.RIOK3 is important one of the RIO kinase(Right Open Reading Frame Kinase)family.RIO kinases play an important role in cell signal transduction and the occurrence and development of various tumors.At present,it is found that RIOK3 is closely related to the progression of breast cancer,pancreatic ductal adenocarcinoma,etc.While,the role of RIOK3 in the etiology and pathogenesis of esophageal cancer remains unclear.ObjectivesRIOK3 was detected by bioinformatics technology,IHC,western blot and qRT-PCR in esophageal cancer tissues and cells.We use cell function test to detecte the effects of RIOK3 on proliferation and migration of esophageal cancer cells.The relationship between RIOK3 and the protein related EMT was detected by the method of western blot.Methods1.Oncomine and GEO databases were used to analyze the expression of RIOK3 in esophageal carcinoma.2.The expression of RIOK3 in esophageal carcinoma and adjacent normal esophageal mucosa was detected by IHC.And analyse the relationship between RIOK3 protein expression and clinicopathological parameters.3.We use qRT-PCR and Western blot technology to detecte the expression of RIOK3 Mrna and protein level in different esophageal cancer cell(NEC、KYSE-150、Eca109、EC9706)and normal esophageal epithelial cell(HEEC).4.We use the method of transient plasmid transfection to structure the model of esophageal cancer cell line with overexpression and interference RIOK3,and they were verified by qRT-PCR and Western blot.5.CCK-8 and Plate Clone methods were used to detect the proliferous capacity of esophageal cancer cells overexpression and interference of RIOK3.While we use transwell experiment to observe their migration capacity.6.We use Western blot technology to test the expression of EMT related proteins in esophageal cancer cells after overexpressing and interfering RIOK3.Results1.The results of Oncomine and GEO database analysis shows that the expression of RIOK3 mRNA in esophageal carcinoma is lower than cancerous tissue.2.IHC results shows that the protein expression level of 65.1%(31/48 cases)esophageal carcinoma paraffin samples are higher than match the right paracancerous tissue.The expression level of RIOK3 is no significant differences with sex,age and lymphatic metastasis.It is significant differences with differentiation of esophageal carcinoma.The lower the degree of tumor differentiation,the lower the expression of RIOK3.3.The expression of RIOK3 in EC9706 and ECa109 esophageal carcinoma cell is higher than the cell of HEEC.And the expression of RIOK3 in carcinoma cells of NEC and KYSE-150 is similar to HEEC.4.qRT-PCR and Western blot detection shows that the expression of RIOK3 protein and mRNA in EC9706.R and ECa109.R is evidently higher than ECa109.vec and EC9706.vec.The expression of RIOK3 protein and mRNA in NEC.shR2 is observably lower than NEC.scr.5.CCK-8 experiment shows that growth rate of RIOK3 overexpression of esophagealcancer cells is faster than the control group.After interfering with RIOK3,the growth rate of esophageal cancer cells was faster than that of control group.6.Plate clone experiment shows that esophageal cancer cells overexpressed RIOK3 formed fewer clones than controls,while cells interfering with RIOK3 were significantly more numerous than controls.7.Transwell experimental results shows that the number of esophageal cancer cells that overexpressed RIOK3 significantly decreased compared with the control group,while the number of cells that interfered with the RIOK3 was significantly higher than that of the control group.8.Western blot experiment shows that the EMT related protein of E-cadherin is significantly up-regulated,however N-cadherin and Vimentin is down regulated in group of expressing RIOK3.While the RIOK3 interference group is contrary.Conclusions1.The expression of RIOK3 was significantly down-regulated in esophageal carcinoma,while was positively correlated with differentiation of esophageal cancer.2.The overexpression of RIOK3 restrains the proliferation and migration ability of esophageal cancer cells,while RIOK3 is interfered with,it can significantly reduce the proliferation and migration ability of esophageal cancer cells.3.RIOK3 is involved in the regulation of epithelial interstitial transformation,positively correlated with E-cadherin and negatively correlated with N-cadherin、Vimentin.