| Objective: To observe the value of combined detection of the level of microRNA(MIR)-92 a,MIR-100 and MIR-143 in serum of bladder cancer.Methods:Serum of 50 patients with bladder cancer and 10 eases of healthy people was collected.Blood(10ml)was drawn on ethylenediaminetetraaceticacid from an antecubital vein in BC patients or healthycontrol subjects using standard percutaneous venipunctur.Plasma was separated by centrifugation at 3000 rpm for10min at 4℃,and then re-centrifuged at 5000 rpm for 5minto obtain cell-free plasma,which was stored at 80 ℃ untiluse.Only non-haemolysed plasma samples were used in ourstudy,as haemolysis affects MIRNA levels.Haemolysis wasdetected by spectral analysis at 541 nm.Then the plasma were removed and placed in a water bath at 37.0℃ immediately.Total RNA with preserved MIRNAswas extracted from the plasma with Trizolccording to the manufacturer’s instructions for isolation of total RNA,and eluted with 30μl of RNase-free water.The following conditions: 16℃ for 30 min,at 42℃ for 30 min,85℃ for 30 min.Total RNA was reversetranscribedusing the miRNeasy serum/plasma ReverseTranscription Kit accordingto the manufacturer’s instructions with 5 μl of total RNAper reaction.Real-time PCRreactions were performed using an Applied Biosystems 7500 Real Time PCR System with the following conditions: 95℃ for 15 min,followed by 40 cycles at94℃ for 15 s,55℃ for 30 s and 70℃ for 34 s.The expression of MIRNAs was reported as the ΔCtvalue,which was calculated by subtracting the CT values ofMIRNA U6 snRNA from the CT values of the target MIRNAs.And real—time reverse transcriptase-polymerase chain reaction(RT—q PCR)was used to detect the level of MIR-92 a,MIR-100 and MIR-143.The receiver operating characteristic(ROC)curve was used to calculate the cut—off value of MIR-92 a,MIR-100 and MIR-143.and the value of combined detection of the level of MIR-92 a and MIR-143 in serum of bladder cancer was assessed.Results:(1)Plasma levels of MIR-92 a,MIR-100 and MIR-143 were significantly lower in BC patients than in control group(50 persons).However,only MIR-143 expression level differed significantly in theNMIBC(35 persons)and the MIBC groups(15persons).(2)Receiver operator characteristic analysis revealed that the sensitivity and specificity values of MIR-92 a were 96.0% and 74.0%,respectively,with a cut-off value of 0.590.The sensitivity and specificity values of MIR-100 were86.0% and 65.0%,respectively,with a cut-off value of 0.654.The sensitivity and specificity values of MIR-143 were 74.0% and 90.0%,respectively,with a cut-off value of 0.380.(3)The best combination of the plasma markers MIR-92 a and MIR-143,revealed in our plasma quantitative assay,makes a very promising and specific BC screening testpossible.Conclusions:(1)Plasma levels of MIR-92 a,MIR-100 and MIR-143 were significantly lower in BC patients than in control group.However,only MIR-143 expression level differed significantly in theNMIBC and the MIBC groups(P<0.05).(2)In our study,we showed that this study explores the existence of specific plasma MIRs as early diagnostic biomarkers for BC in China patients;and these findings suggest that plasma MIR-92 a,MIR-100 and MIR-143 could be promising novel circulating biomarkers in clinical detection of BC.(3)We employed the ROC curve to analyse the diagnosticvalue of plasma MIR-92 a,MIR-100 and MIR-143 levels inBC patients.As a result,ROC demonstrated that MIR-92 aand MIR-143 had a high diagnostic value for BC with 92.0% sensitivity and 92.0% specificity.(4)There is a high diagnostic value of combed detection of MIR-92 a with MIR-143 to bladder cancer. |
