MiR-93-5p Modulates Epithelial-mesenchymal Transitions Of Gastric Cancer Cells Through The Wnt Signaling Pathway By Targeting AHNAK

Objective: The incidence rate of gastric cancer is one of the most common digestive tract cancers.In our country,gastric cancer(GC)is a serious threat to people’s health and a serious economic burden.According to the data released by the National Cancer Registry in 2015,the incidence rate of GC is about 679 thousand in China,and the death rate is about 498 thousand.The incidence and mortality rate of GC is second only after lung cancer.Therefore,the prevention and control of GC is still a major health problem in our country.Micro RNA(mi RNA)is an endogenous single stranded noncoding RNA.It is encoded and noncoding protein by higher eukaryote genome,and exists in most eukaryotes.It is about 22 nucleotide RNA fragments in length,which are combined to specific target m RNA through the complementarity of nucleic acid sequence.Mi RNA is mainly untranslated with the 3’ end of target gene m RNA.The target is identified by the specific combination of complementary sequences of region noncoding sequence,and the target m RNA is degraded or the translation process is inhibited by RNA induced silencing complex,so as to achieve the purpose of regulating gene expression.Mi R-93-5p is located on chromosome 11q22.1,which is often used as a carcinogen and participates in the development of a variety of tumors.Previous study has shown that mi R-93-5p as an oncogene directly targets AHNAK to promote the development of various cancers.Based on this,this paper aims to study the expression of mi R-93-5p in GC and explore the mechanism of mi R-93-5p targeting regulation of AHNAK mediated Wnt signaling pathway on the epithelial-mesenchymal transition of GC.Methods:(1)Using GEO database to retrieve GC-related mi RNAs and m RNA expression chip,and analyze the difference.To identify the follow-up study objects,we took the intersection of different mi RNAs.Further,target scan database was used to predict mi RNA target genes,and the predicted results were intersected with the analysis results of m RNA expression chip to confirm the subsequent research genes.(2)95 cases of GC from January 2012 to December 2013 were collected.The expression of mi R-93-5p in GC tissue was detected by q RT-PCR.The overall survival(OS)and disease-free survival(DFS)were analyzed by Kaplan-Meier curve.(3)The target relationship of mi R-93-5p to AHNAK was verified by bio-informatics analysis and fluorescein double report gene system.The migration and invasion of mi R-93-5p were detected by Transwell test.The effects of mi R-93-5p on epithelial-mesenchymal transition of GC cells were observed by cell morphology.Western blot was used to detect the changes of AHNAK,wnt-1,β-catenin,p-β-catenin,Kremen,AXIN2,LRP5 and LRP6,E-cadherin,Vimentin and Snail.(4)E-cadherin,Vimentin and snail were detected by immunofluorescence.The binding of AHNAK and β-catenin was detected by TOP/FOP luciferase assay.Results:(1)The results of mi RNA microarray analysis of GC were collected and confirmed that the follow-up study object was mi R-93-5p.(2)The prediction results of the mi RNA in the Target Scan database and the genes significantly down regulated in the m RNA expression chip were analyzed by Venn.Finally,two potential target genes were finally obtained.Further analysis of the two genes showed that the expression level of AHNAK in GC was significantly down regulated.The high expression of mi R-93-5p in GC is related to the poor survival of OS and DFS.(3)Prediction of bioinformatics website and fluorescein double report gene experiment were performed to verify the relationship of AHNAK with mi R-93-5p.Inhibition of mi R-93-5p can up regulate the expression of AHNAK.The migration and invasion experiments showed that down-regulation of mi R-93-5p and over-expression of AHNAK could inhibit the migration and invasion of GC cells,while over expression of AHNAK could reverse the enhancement of migration and invasion ability of GC cells induced by mi R-93-5p mimic.(4)Mi R-93-5p regulates AHNAK to promote epithelial-mesenchymal transition of GC cells.Mi R-93-5p regulates Wnt signaling pathway by targeting AHNAK.Conclusion: Our study provides evidence that down-regulation of mi R-93-5p can inhibit the epithelial-mesenchymal transition of GC cells through Wnt signaling pathway by targeting AHNAK.Mi R-93-5p may be a promoter for GC,while AHNAK may be a cancer suppressive factor.Both mi R-93-5p and AHNAK may be used as important molecular targets for the diagnosis and treatment of GC.