Experimental Study On The Screening Of Plasma MicroRNAs As Biomarkers For Non-cardia Gastric Cancer And Its Precancerous Lesions

Aims:To screen the potential plasma miRNAs as candidate biomarker for non-cardia gastric cancer(GC)and its precancerous lesions by plasma miRNA profiling,and further evaluate the differentiation value of them for the non-cardia GC and its precancerous lesions,in order to explore the ideal biomarkers for population screening on non-cardia GC and its precancerous lesions and assisting their diagnosis.Contents:To screen the potential plasma miRNAs as candidate biomarker for non-carida GC and its precancerous lesions by detection and analyses of plasma miRNA profiling using TLDA;to quantitatively detect the expression level of candidate plasma miRNAs using RT-PCR in a larger sample size and analysis the differences among the groups,in order to evaluate the differentiation value of them for the non-cardia GC and its precancerous lesions;on the basis of quantitative detection of candidate plasma miRNAs on validation stage,to analysis the correlations between the candidate plasma miRNAs and essential clinical characteristics of GC cases using statistical methods,and clarify the cooperative effects of clinical characteristics on plasma miRNAs.Methods:Four groups were set up,including control group,precancerous lesions,I stage GC group,IV stage GC group,and 15 cases were selected for each group.A plasma pool sample for each group consisted of 15 plasma samples with 70μl plasma sample for each case.TLDA was used to detect the plasma miRNA profiling.The candidate plasma miRNAs,which were detectable(Ct value<35)and differently expressed among groups,were selected for the next validation stage experiment.The sample size was amplified with 22 cases for control group,20 cases for precancerous lesions group,26 cases for I stage GC group,28 cases IV stage GC group,and 200μl plasma sample was collected for each case.The RT-qPCR was used to detect the plasma miRNA level for each case(Ct value),and the relative expression value(△Ct)of each miRNA was calculated with cel-miR-39 as exogenous marker.The expression difference for each candidate miRNA among groups were analyzed with rank sum test.For the candidate plasma miRNAs with different expression among groups,the ROC was used to evaluate the diagnosis value for GC and its precancerous lesions.The △Ct values for each miRNA were divided into 2 groups based on the median of △Ct values:low expression group and high expression group.The clinical information of cases in present study were collected,including age,gender,tumor differentiation,survival time of each GC case and terminal event.The statistical relationships between miRNAs expressions and age,gender,tumor differentiation,survival and prognosis were analyzed with Pearson correlation analysis,rank sum test,Kaplan-Meier and Cox regression survival analysis,χ2 test,respectively.Results:The age,gender and lesions location in stomach for the subjects of four groups were all matched on both screening and validation stage.The plasma miRNA profiling result showed that 25 candidate plasma miRNAs were selected,which were detectable and significantly different almong groups,and plasma expression for 12 of them increased gradually with abnormality of stomach mucosa progressing,while 13 of them decreased.The result of validation stage showed that the plasma expression for 16 out of 25 candidate plasma miRNAs were significantly different at least between 2 groups,while the other 9 miRNAs showed no significant difference among 4 groups.Of the 16 plasma miRNAs with different expression,the plasma expressions in I stage GC group and Ⅳ stage GC group were significantly lower than those of control group and precancerous lesions group for let-7d、let-7e、miR-15b*、miR-26a-1*、miR-100、miR-106b*、miR-142-3p、miR-335*、miR-340*、miR-379、miR-485-3p、miR-652、miR-654-3p,while there showed no statistical difference for them between I stage GC group and IV stage GC group,control group and precancerous lesions group,respectively.The plasma expressions of miR-1274A、miR-1290、miR-1291 in IV stage GC group were significantly higher than those of control group,precancerous lesions group and I stage GC group,and the latter 3 groups showed no statistical difference for these 3 miRNAs.Statistical analysis showed the plasma expressions of 12 miRNAs were significantly correlated with age,but none was found to be related to gender and tumor differentiation.In the survival analysis for total GC cases,the tumor stage and differentiation showed significant correlation with survival time with Kaplan-Meier analysis,and the cases of I stage GC and moderate differentiation GC showed longer survival time than IV stage GC and poor differentiation GC,respectively.There was a significant correlation between plasma miR-1274A expression and the prognosis of GC cases,and the GC cases with high expression of plasma miR-1274A showed poor prognosis.The Cox analysis showed that plasma miR-31 was correlated with GC survival time using tumor stage and differentiation as co-variables,and the cases with high miR-31 expression showed better prognosis.In the stratification survival analysis for Ⅳstage GC cases,plasma miR-31 was correlated with survival time with Kaplan-Meier analysis,while plasma miR-1274A showed the correlation with survival time with χ2 test.Conclusions:The experiment results confirmed that miRNAs were to exist and detectable in the peripheral blood using TLDA and RT-qPCR.The potential plasma miRNAs biomarkers for non-cardia GC and its precancerous lesions could be screened using TLDA,which indicated that TLDA was a useful tool for whole genomic miRNAs screening without an a-priori hypothesis.Our study found that 13 miRNAs could be potential biomarkers for population screening of early stage non-cardia GC,and 3 for distant metastasis or relapse of non-cardia GC.Twelve plasma miRNAs expressions were found to be related to age.Tumor stage was further verified to be an independent factor for GC prognosis.The plasma expressions of miR-1274A and miR-31 was first found to be correlated with survival time of non-cardia GC or Ⅳ stage non-cardia GC cases,so they could become the biomarkers for non-cardia GC prognosis evaluation.For the limitation in present study,such as inclusion criteria and sample size,the diagnostic values of these plasma miRNAs for GC screening need to be further validated and evaluated in a larger sample size.