Study On The Differential Expression Of Exosomal MicroRNA In Gastric Cancer

ObjectiveDetect the serum exosomes miRNAs expression profiles from the blood samples of the gastric cancer patients,determine the exosomal molecular difference in different clinical stage of gastric cancer,on this basis,evaluate the siginificance of serum exosomal miRNA as diagnostic markers,investigate the possibility of evaluating metastatic potential of gastric cancer through serum exosomal miRNA,further more analyze the relationship between specific serum exosomal miRNA levels and tumor biological behavior and clinical features.Method:(1)Case selection:we selected newly admitted gastric cancer patients since March 1,2016 according to the inclusion and exclusion criteria based on experimental design requirements.(2)Sampling: peripheral blood was sampled prior to surgery operation with blood taking needle and separation gel coagulation tube at 6:00 a.m,2ml serum was separated with high speed centrifuge and frozen at-80℃,dry ice transportation.(3)Sample detection: serum exsomes were isolated with RiboTM Exosome Isolation Reagent for plasma or serum,from which total RNAs were extracted with Magen assay.Agilent 2200 TapeStation and ND-1000 Nanodrop were used for quality control of the RNA pruducts,quality inspection criteria were A260:A280>1.8 and A260:A230>2.2.(4)Library construction: for qualified samples,we constructed small RNA library through TruSeq? Small RNA Sample Prep Kit with 1ug initiation mass and then used Agilent 2200 TapeStation for library quality control,quality inspection criteria were with an average length of fragments from 140 to 170 bp.(5)Sequencing: for qualified library,we prepared sequencing sample based on the methodology described in HiSeq 2500 User Guide and final sample concentration was 10 pm,conducted sequencing in HiSeq 2500 with Single Read Flow Cell mode and single-end 1×50 standard sequencing procedure.(6)Data analysis: the exsomal miRNA expression profile was established through the bioinformatics analysis of sequencing data.On this basis we found the relationship between exsomal miRNA expression and tumor clinical features with T test and variance analysis using SPSS 18.0.PCA analysis method and software kit(DEseq and EdgeR)were used to identify differential expressional miRNA,and we used miRDB、GO and KEGG to do functional analysis.Result:The groups were not consistent with PCA analysis.We combined the clinical factors with PCA results and 4 late stage cases and 5 early stage cases were analysed by DEseq and EdgeR.55 differential expression miRNA were reported,of which DEseq contributed 39,and EdgeR contributed 52,36 were overlapped.GO functional analysis showed that thetarget gene enriched in RNA transcription regulation、protein metabolism and modification 、 cellskeleton and nucleus function,KEGG pathway enrichment analysis showed that targeted gene enriched in MAPK signaling pathway、signaling pathway regulating pluripotency of stem cell、pathways in cancer.Conclusion:The expression of exosomal miRNA among different stage of gastric cancer varied differently.Exosomal miRNA differential expression played an important rule in the progression of gatric cancer.