A Screening Of Plasma IncRNAs As Diagnostic Biomarkers Of Gastric Cancer

Background:China has always been a country with high morbidity of gastric cancer(GC),and the number of incidence and deaths each year accounted for about 50%of the world's total.Stomach cancer has bro?ght heavy burden to Chinese people,which should be the key point of tumor prevention and control.Gastric cancer is usually diagnosed at advanced stage because of its slight and nonspecific symptoms.The prognosis of advanced GC is poor,the five-year survival rate of which is less than 30%.However,the five-year survival rate of early GC could be more than 90%.So it is very important to search for effective biomarkers for early detection of GC.Long non-coding RNAs(IncRANs)are a class of RNA transcripts more than 200 nucleotides with limited protein-encoding potential.It has been well described that ectopic expression of IncRANs is responsible for the development and progression of malignancies.Recently,accumulating evidence has demonstrated that IncRANs could be detected stably in circulation and have the capacity of distinguishing tumor sufferers from healthy individuals.Therefore,the circulating IncRANs show us the potential to be biomarkers for early diagnosis of cancers.However,the research on circulating IncRANs is just in its early phase,and the mechanism underlying the secretion and the roles of plasma IncRANs have not been systematically investigated.Methods:The Arraystar Human IncRNA/mRNA v3.0 microarray was conducted to detect the plasma IncRANs between 5 GC samples and 5 healthy controls matched to age and sex.The top 10 of highly expressed IncRANs in stomach cancer patients was validated in two-stage plasma sample(the first stage contains 50 gastric cancer plasmas and 50 cancer-free plasmas,the second stage contains 173 gastric cancer plasmas and 173 cancer-free plasmas)throggh real-time quantitative PCR.Receive operating charactreristic curve(ROC)analysis and area under the curve(AUC)were conducted to evaluate the diagnostic value of selected IncRANs for gastric cancer.Furthermore,the real-time PCR was used to detected the stability of the three IncRANs after the plasma sample were incubated at room temperature or frozen and thawn repeatly.Then we explore the origin of plasma IncRANs through GC cells and plasma sample before and after radical operation.At last,we analyzed the roles of RP11-47304.5 in malignant phenotype of GC cells.Results:From the IncRNA microarray detection,we found a total of 3,878 IncRANs are differentially expressed in the plasma of GC patients(603 IncRANs up-regulated and 3,275 IncRANs down-regulated).Ten candidate plasma IncRANs were quantified with real-time PCR in the next two-stage validation sample,and three of them(RP11-47304.5,GUSBP11 and CTD-2303H24.2)were significantly upregulated in the patient group.The expression levels of the three IncRANs in plasma had no significant change when treated with multiple freeze-thaw cycles or stored at room temperature.The ROC analysis indicated that CTD-2303H24.2 provides the best diagnostic efficiency of the three IncRANs in distinguishing GC patients from healthy controls.Moreover,the combination of the three IncRANs achieved higher diagnostic power than any single of them with an AUC of 0.818,the sensitivity of 77.5%,and the specificity of 73.9%.The expression of the three IncRANs could be detected in the cell culture supernatant of different GC cells,and the three IncRANs significantly decreased in patients' plasma after surgery,which indicated that the plasma IncRANs are at least partly derived from tumor tissue.In the cell biology study,the over-expression of RP11-47304.5 were found to increase the ability of proliferation and invasion of GC cells,and the knock-down of RP11-47304.5 inhibited the proliferation and invasion.Conclusion:We identified three novel gastric cancer-related plasma IncRANs(RP11-47304.5,GUSBP11 and CTD-2303H24.2)in this study,which stably existed in human's plasma,and significantly upregulated in individuals' plasma suffering from gastric cancer.So the three plasma IncRANs showed the potential to be diagnostic biomarkers for distinguishing GC patients from healthy controls in the future.The cell biology study found that RP 11-47304.5 was related to the proliferation and invasion of GC cells.