Research On Differentially Expressed Serum Mirnas Between Pre-And Post-Operative Gastric Cancer Patients

ObjectiveTo screen differentially expressed serum miRNAs between pre- and post-operative gastric cancer(GC) patients, these serum miRNAs have the pottential to be biomarkers for evaluating therapeutic effect,monitoring recurrence and predicting prognosis of GC.MethodsHigh-throughput microarray was adopted to determine the expression of serum miRNAs in 6 pre- and post-operative GC patients after equivalent volume mixture, then we performed a biological repeat.Those miRNAs with high amplification efficiency, apparent and stable expressed difference were selected for further analysis.Those selected differentially expressed serum miRNAs were further validated by qRT-PCR in other 55 paired GC serum samples to analyze their stability and repeatability for screening remarkable differentially expressed serum miRNA again.QRT-PCR was used to validate the selected miRNA in other serumsamples including healthy volunteers and some common tumors for observing its levels among these samples and verify the possibility as diagnostic biomarker of GC.To selected miRNA, we also analyzed its therapeutic effect and relationship with clinicopathological characterictic of GC. In addition, we followed up these 55 GC patients to observe the efficiency of the selected miRNA in monitoring recurrence and predicting prognosis.Bioinformatics softwares were adopted to predict the target genes and analyzed their related signal pathways of selected serum miRNA,illuminated related molecular mechanism of gastric cancer development and progression.ResultsIn High-throughput microarray results, we found 22 serum miRNAs with significantly difference and high amplification efficiency in preand post-operative GC patients.Verifying these 22 miRNAs in 15 paired before and after treatment GC serum samples by using qRT-PCR, we obtained 12 miRNAs with high reliability and repeatability, combined with statistical analysis,results shown that hsa-miR-20a-5p(miR-20a), hsa-let-7d-5p(let-7d) and hsa-let-7g-5p(let-7g) had significantly difference between pre- and post-operative serum samples.Continuing to validate these 3 miRNAs in the rest of 40 pairedgastric cancer serum samples, results shown that they all had statistically significance. We found that miR-20 a could be easily detected and changed obviously after treatment, which makd it meet the requirement as serum miRNA marker for evaluating therapeutic effect, monitoring recurrence and predicting prognosis of gastric carcinoma.We performed qRT-PCR to test serum miR-20 a levels in healthy volunteers group, GC, nasopharyngeal cancer(NPC), colorectal cancer(CRC), and breast cancer(BC). Results shown that miR-20 a levels in GC,NPC, CRC and BC serum samples were significantly higher than those in healthy volunteers group(P<0.05).Analyzing the relationship between miR-20 a levels and pathological parameters and prognosis of GC patients, we found the levels of miR-20 a were related to age, tumor stage, differentiation degree and lymph node metastasis(P<0.05). Kaplan-Meier survival analysis shown that low miR-20 a levels group has higher survival rate than high miR-20 a levels group(P<0.05).Targetscan, miRanda and Pic Tar software were applied to predict the target genes of miR-20 a, we predict ZNF367, ARID4 B, CFL2 as potential targets of miR-20 a involved in gastric carcinogenesis. Signaling pathways involved to these 3 target genes mainly include MAPK, Calcium and Toll-like receptor.ConclusionsSerum miR-20 a levels are significantly down regulated in post-operative GC patients.Serum miR-20 a levels in GC patients are higher than healthy volunteers, and its concentrations are related to age, tumor stage,differentiation degree and lymph node metastasis.Low serum miR-20 a levels group has higher survival rate than high serum miR-20 a leves group.