The Preliminary Analysis Of Exosomal LncRNA Expression Profile In Lymph Node Metastasis Of Esophageal Squamous Cell Carcinoma

Objective To find out exosome lncRNA tumor biomarkers of esophageal squamous cell carcinoma?ESCC?patients with lymph node metastasis.Investigating lncRNA expression in ESCC and exploring its correlation with clinicopathological features.Furthermore,to determine whether the plasm exosomal level of lncRNA corresponded to the tissue expression and provide the clues for molecular mechanism of ESCC.Methods1.Patients with ESCC who underwent surgical resection in Fujian Cancer Hospital from December 2014 to August 2017 were enrolled and carried out epidemiological investigation and clinical data collection.Four subjects with node_negative ESCC add four normal person who had benign disease or without systemic inflammation as control group,were matched with four patients with node_positive ESCC.Three compared groups,which is preoperative and postoperative status in node_positive ESCC patients,node_positive ESCC and node_negative ESCC patients,node_positive ESCC and control group,were performed to identify differentially expressed lncRNA using a genome-wide analysis of lncRNA microarray and GeneSpring GX software?FC>2,P<0.05?.2.Venny online analysis software was used to obtain the intersection results of the three comparison groups.Then,expression profile of differentially expressed exosomal lncRNA in ESCC metastasis was constructed.Pearson correlation coefficient was used to analyze differential expressed genes,while Cytoscape software was applied to draw gene co-expression network diagram.Next,we used clusterProfile package in R software for GO and KEGG pathway enrichment analysis,to infer that lncRNA,which were related to tumor metastasis,may be involved in GO biological functions and KEGG pathways.3.Besides,the method of quantitative real-time polymerase chain reaction?qPCR?was used to measure exosomal lncRNA expression in node_positive and node_negative ESCC subjects and the association between lncRNA expression in specimen and clinicopathological features was analyzed.Furthermore,we evaluated diagnosis value of plasm exosomal lncRNA,and the tissue lncRNA expression level in samples from node_positive and node_negative ESCC patients were also detected by qPCR.Results1.The microarray results showed that 55 lncRNA were differentially expressed,of which 35 lncRNA were up-regulated and 20 lncRNA were down-regulated,while 66 mRNA were differentially expressed,among them,34 genes were up-regulated and 32 genes were down-regulated.The results of GO analysis mainly include: negative regulation of smooth muscle cell proliferation,regulation of humoral immune response,regulation of smooth muscle cell proliferation,microtubule polymerization or depolymerization,et al.The results of KEGG pathway enrichment analysis include: Cytokine-cytokine receptor interaction and Circadian rhythm.2.We screened out 4 up-regulated lncRNA,which had the minimum significance Pvalue?Top5?and biggest fold change?Top5?of differentially expression plus row signal value greater than 30,to detect expression level by qPCR: ENST00000424487.1,NR₀26954.1,uc009 vwu.1 and ENST00000608144.1,respectively.3.The expression level of ENST00000424487.1 and NR₀26954.1 in the node_positive ESCC group was significantly higher than the node_negative ESCC group?P<0.05?,whereas that of uc009 vwu.1 and ENST00000608144.1 showed no significance.ENST00000424487.1 had no relationship with the clinicopathological factors such as sex,age,tumor location,tumor size,degree of differentiation,invasion depth and TNM stage?P>0.05?.However,the expression of NR₀26954.1 of well differentiation patients was significantly lower than moderate differentiation subjects?adjusted P=0.028?.4.The AUC of ENST00000424487.1 and NR₀26954.1 diagnosing ESCC metastasis was 0.628 and 0.655,respectively.The sensitivity was 69.4% and 81.6% while the specificity was 60% and 48.9%,respectively.The sensitivity and specificity of combining ENST00000424487.1 and NR₀26954.1 were 98% and 37.8%,respectively.5.The expression level of NR₀26954.1 in the node_positive ESCC group were lower than those in the node_negative ESCC group?P=0.034?.ConclusionENST00000424487.1 and NR₀26954.1 were up-regulated in the plasma exosomal of node_positive ESCC patients.ENST00000424487.1 in parallel to NR₀26954.1 could screen out more node_positive ESCC patients as much as possible and could be a certain auxiliary diagnostic significance for differentiating node_positive and node_negative ESCC patients.