Construction And Validation Of LncRNA AOC4P-based Diagnostic Index For Gastric Cancer And Its Mechanism In Regulating Proliferation And Invasion

Background:Gastric cancer(GC)represents a major health burden worldwide,with the highest incidence rate in Eastern Asia.In China,GC was the second most prevalent cancer and the second cause of cancer-related deaths with an estimate of 679,100 new cases and 498,000 deaths in 2015.No typical signs indicative of GC present until the cancer is advanced,leading to a poor prognosis with a 5-year survival of only 25%.Thus,there is an unmet need to develop a rational approach for early detection of GC,which would greatly facilitate early intervention.Undoubtedly,blood-based tumor biomarkers provide an easily accessible and noninvasive way to detect GC.In current clinical practice,the most widely used are serum tumor markers,including carcinoembryonic antigen(CEA),carbohydrate antigen(CA)19-9,CA72-4 and CA125.However,these markers have been shown to be far from clinically satisfactory for GC detection,with low sensitivity and specificity,even if they are used in combination.Development of reliable biomarkers for GC detection with high accuracy should be a priority.Long non-coding RNAs(lncRNA),a class of transcripts more than 200 nucleotides in length without protein-coding ability,have been reported to play key roles in gene regulation,thereby influencing several facets of cellular homeostasis,including proliferation,apoptosis,metastasis and genomic stability.Interestingly,lncRNA are usually expressed in a highly tissue-and cell type-specific manner.More importantly,tumor-derived lncRNA can be detected in several biological fluids,including urine and blood,which makes IncRNA potentially ideal markers.The present study screened blood-based lncRNA with diagnostic ability,and constructed lncRNA-based diagnostic index for gastric cancer and investigated the mechanic role of IncRNA in regulating proliferation and invasion.Methods:1.Genome-wide IncRNA profiles were produced for both five paired tissue samples and five patient/control plasma samples to identify candidate tumor-derived differentially expressed IncRNAs,and further validated by qRT-PCR.2.Training phase:measuring plasma lncRNA level in 30 GC patients and 30 healthy controls,and constructing AOC4P-based diagnostic index,and comparing it with currently used clinical tumor biomarkers.3.Validation phase:confirming the accuracy of AOC4P-based diagnostic index in another 80 GC patients and 80 healthy controls,and in a cohort of 37 GC patients,28 precancerous lesions and 21 gastrointestinal stromal tumor(GIST)patients.4.Investigating the mechanic role of lncRNA AOC4P in regulating proliferation and invasion.Results:1.Fifty-eight differentially expressed IncRNA were screened by IncRNA array with 20 upregulated and 38 downregulated in tumors.After excluding the reported IncRNA and further validating by qRT-PCR,TINCR,CCAT2,AOC4P,BANCR and LINC00857 were finally identified.2.In the training phase,the combining Index I consists of TINCR,CCAT2,AOC4P,BANCR and LINC00857 had a value of area under the receiver operating curve(AUC)of 0.93[95%(confidence interval,CI),0.87-0.99],better than the AUC value of indexⅡ of 0.71(95%Cl,0.58-0.84)consisting of CEA,CA19-9,CA72-4 and CA125.Compared with preoperatively,values of Index I and Index II decreased on postoperative day(POD)14 without statistically significance(P = 0.057and P = 0.089).3.In the validation phase with a cohort of 80 GC patients and 80 healthy controls,the AUC value of plasma lncRNA-based Index I[0.90,(95%CI,0.86-0.95)]were higher than that of CEA-based Index II[0.69,(95%CI,0.61-0.78)](P<0.001).Compared with preoperatively,the value of Index I decreased significantly on POD 14(P = 0.016).However,there was no difference between preoperative and POD 14 in terms of Index II(P = 0.469).In a cohort of 37 GC patients,28 precancerous lesions and 21 GIST patients,the value of AUC for Index I discriminating GC from precancerous lesions was 0.82(95%CI,0.71-0.92),and that for Index I discriminating GC from GIST was 0.80(95%CI,0.68-0.91).4.AOC4Pwas upregulated in GC tissues and high expression levels of AOC4P was correlated with poorer overall survival(OS)and disease-free survival(DFS).In vitro,knockdown of AOC4P inhibited tumor cell proliferation,migration,and invasion,and promoted apoptosis of MGC-803 and BGC-823 cells.In vivo,BGC-823 cells transfected with AOC4P siRNA formed smaller and lighter tumors than BGC-823 cells transfected with negative control siRNA in severe combined immunodeficiency mice.Mechanistically,knockdown of AOC4P increased the expression of Vimentin and MMP9,while reducing the expression of E-cadherin in MGC-803 and BGC-823 cell lines.Conclusion:Our findings demonstrate that the plasma IncRNA AOC4P-based diagnostic panel could detect GC with relatively high accuracy and could monitor tumor dynamics;AOC4P may regulate GC proliferation and invasion through epithelial-mesenchymal transition;AOC4P may serve as prognostic biomarker in clinical settings.