The Identification Of Circular RNA Expression Profile In Gastric Cancer And Its Diagnostic Value

Objective:Circular RNAs?circRNAs?have been emerged as a kind of abundant non-coding RNA in mammalian cells.Importantly,a growing number of studies have demonstrated that circRNAs play a significant role in the pathogenesis of cancers.The high stability of circRNAs conferred by their circular structure is beneficial for being a novel type of plasma biomarker.Gastric cancer is the second-leading cause of cancer-related deaths worldwide.Though advances in surgical techniques and combined chemotherapy strategies,the 5-year overall survival of this disease in most countries remains less than 30%.This is due to the low early diagnostic rate.A non-inasive,sensitive and specific biomarker is in urgent need.However,the diagnostic values of peripheral blood circRNAs for gastric cancer remain largely uncovered.In this study,we focused on the expression profile of gastric caner-related circRNAs in tissues and plasma through molecular biological technologise and bioinformatics.Then,using fluorochrome-based reverse transcription-droplet digital polymerase chain reaction?RT-ddPCR?to quantify the target circRNAs and assess the value of plasma circRNAs as gastric cancer-specific biomarkers.Methods:1.The differences of circRNA expression profiles between gastric cancer patients' plasma and tissues and healthy controls were assessed by circRNA microarray.Then,the interactions between circRNAs and miRNAs were predicted by TargetScan and miRanda software.Finaly,circRNAs coordinately expressing between tissues and plasma from patients with gastric cancer were screened.2.The pre-operation fasting plasma,gastric cancer tissues and the para-cacinoma tissues during the operation and the post-operation fasting plasma from gastric cancer patients?n=121?,and the dysplasia tissues and normal gastric epithelial tissues through endoscopic submucosal dissection?n=36?were collected.The fasting plasma from healthy person?n=121?were used as normal controls.3.Modulated the primers proportion to make the EvaGreen based RT-ddPCR a highly flexible method for interrogating triplex RNAs?hsa_circ₀001017,hsa_circ₀061276 and GAPDH mRNA?simultaneously.Compared the different quantification approaches of reverse transcription-polymerase chain reaction?qRT-PCR?and RT-ddPCR to confirm the most appropriate calculation method for tissue and plasma circRNAs.4.The qRT-PCR and RT-ddPCR was used to detect the expression levels of circRNAs in tissues and plasma,repecitvely.5.Through the investigation of relationship between circRNA expression levels in tissues and plasma and cell supernatant experiments to explore the origin of circulating circRNAs.Quantified the expression levels of circRNAs and their relative host genes in gastric cancer cells.6.Using combinative receiver operating characteristic?ROC?analysis to assess the effciency of hsa_circ₀001017 and hsa_circ₀061276 in differentiating gastric patients from normal controls.Univariate analysis and further analysis in a multivariate Cox proportional hazards model were used to evaluate the abilities of hsa_circ₀001017 and hsa_circ₀061276 as independent prognostic indicators.The relationships between clinicopathologic factors of patients with gastric cancer and hsa_circ₀001017 and hsa_circ₀061276 in gastric tissues and plasma were further analyzed,respectively.7.Constructed hsa_circ₀001017 overexprssion vector and detected its efficiency.Results:1.We identified 343 circRNA candidates that were differentially expressing in plasma of gastric cancer patients compared with healthy persons,including 172 up-regulated and 171 down-regulated.In gastric cancer tissue circRNA expression profile,there were 107 regulated and 201 downregulated circRNAs.And 3 and 14 circRNAs were elevated and reduced in patients' plasma through intersection analysis based on the differential expression profiles of circRNAs between gastric cancer tissues/adjacent non-tumorous tissues and patients' plasma/healthy controls.2.The single-color multiplex strategy in RT-ddPCR for the plasma circRNA detection was able to efficiently distinguish the droplets population of negative,GAPDH mRNA,hsa_circ₀061276,hsa_circ₀001017 and double positive droplets encapluated GAPDH and hsa_circ₀061276.The RT-ddPCR showed high accuracy and repeatability in circRNA detection of tissue and plasma from gastric cancer patients.3.Hsa_circ₀001017 and hsa_circ₀061276 were both significantly reduced in gastric cancer cells,gastric cancer tissues and paired plasma samples from patients with gastric cancer.Both hsa_circ₀001017 and hsa_circ₀061276 copy numbers in plasma recovered to normal levels after operation.Besides,hsa_circ₀001017 expression levels in gastric dysplasia tissues were lower than those in gastric cancer tissues.4.The gastric cancer cells might release circRNAs.5.Hsa_circ₀001017 and hsa_circ₀061276 exprssions were singnificantly associated with major clinicopathological factors of patients with gastric cancer.6.The area under ROC curve?AUC?of cell-free hsa_circ₀001017 and hsa_circ₀061276 in plasma reached to 0.851 and 0.849,respectively.It's mentionable that AUC of the quadruple combination was up to 0.966;and the sensitivity and specificity increased to 0.901 and 0.867,respectively?NPV=95.5% and PPV=95.7%?.When combined using of two plasma biomarkers,hsa_circ₀001017 and hsa_circ₀061276,in a non-invasion way,the AUC was 0.912,with the sensitivity and specificity up to 0.847 and 0.966.Additionally,The AUC of hsa_circ₀001017 reached to 0.871 in the cohort of differentiating patients with precancerous lesion from healthy controls.7.No matter in all clinical stages or advanced stages,patients with low plasma hsa_circ₀001017 or hsa_circ₀061276 levels had a much shorter overall survival?OS?than those in the high expression group.What's more,the gastric cancer patients with a recovery circRNA levels had a longer disease-free survival?DFS?time,whether in all clinical stages or in the advanced stages.Hsa_circ₀001017 expression levels in plasma and its recovery situation were both independent prognostic indicators of survival in patients with gastric cancer.8.The circRNA upregulating vector significantly elevated the expression levels of hsa_circ₀001017 in gastric cancer cells.Conclusions:Our study first revealed the cell-free circRNAs profile in plasma of patients with gastric cancer.Further,we found that hsa_circ₀001017 and hsa_circ₀061276 were both decreased in gastric cancer tissues and patients' plasma.Moreover,both circRNAs in plasma elevated to the normal levels after operation.As a result,hsa_circ₀001017 and hsa_circ₀061276 may be valuable blood-based biomarkers,either alone or in combination,to screen gastric cancer and estimate prognosis.Moreover,we particularly conducted a strategy which was able to efficiently distinguish triple RNA by the EvaGreen based RT-ddPCR.