| Objective: Using droplet digital PCR(ddPCR)to absolute quantification of miR-155,miR-99 b and let-7a in plasma of patients with primary colorectal cancer and colorectal livermetastasis.To further investigate the value of three miRNAs consist of miR-155,miR-99 b and let-7a in the early diagnosis of CRLM.Provide a novel thoughtsfor the monitoring of CRLM.Methods: We selected 14 healthy subjects,16 primary colorectal cancer patients and 11 colorectal liver metastasis patients to dectect expression level of miR-155,miR-99 b and let-7a in plasma samples,detecting the effect of TaqMan probe.Then,we used ddPCR to quantify the expression level of plasma miR-155,miR-99 b and let-7a in 30 primary colorectal cancer patients and 34 colorectal liver metastases patients.Analyze the expression level of miR-155,miR-99 b and let-7a in two groups plasma samples and clinical characteristics of the cases.At last,compared the data of qPCR and ddPCR.Results: 1.qPCR:(1)Compared with healthy subjects,the expression level of miR-155 in the patients of primary colorectal cancer and colorectal liver metastasis was significantly up-regulated,whereas miR-99 b and let-7a were down-regulated(P<0.05).(2)The expression level of three miRNAs haven'tsignificant difference between primary colorectal cancer and colorectal liver metastasis.(3)The logisticregression and ROC curve analysis show that compared with CG,the AUC of miR-155,miR-99 b,let-7a and miR-155+miR-99b+let-7a in the plasma of colorectal cancer patients was 0.831,0.867,0.759,and was 0.971,respectively.Compared with CG,the AUC of miR-155,miR-99 b,let-7a and miR-155+miR-99b+let-7a in the plasma of colorectal cancer patients was 0.727,0.838,0.883,0.981,respectively.The diagnostic efficiency of joint index is greater than that of single index.2.ddPCR:(1)Compared with primary colorectal cancer patients,the expression level of plasma miR-155 was detected significantly up-regulated in colorectal liver metastases patients(P<0.05),while miR-99 b was detected significantly down-regulated in colorectal liver metastases patients(P<0.05),let-7a were no significant differencein colorectal liver metastases patients plasma samples(P>0.05).(2)The logistic regression and ROC curve analysis shows that the AUC of miR-155,miR-99 b and CEA in the patients with colorectal liver metastases was 0.801,0.712,0.589 respectively,and AUC of miR-155+miR-99 b was 0.874,and the diagnostic efficiency of the combined index was greater than that of the single index.(3)Comparison of clinical data base: there was no significant difference in sex,age,tumor size and degree of differentiation between the primary colorectal cancer patients and colorectal liver metastasis patients.3.Comparison and analysis of the results of qPCR and ddPCR:(1)The detection results showed that the sensitivity of ddPCR was significantly higher than that of qPCR.(2)Comparison of ddPCR and qPCR test efficiency: the test efficiency of ddPCR is obviously higher than that of qPCR.(3)Correlation analysis between ddPCR and qPCR detection showed that the expression level of miRNA was consistent with that of qPCR in ddPCR.Theexpression of miR-155 between ddPCR test results and qPCR test results correlation coefficient was r=0.83,P<0.05,and the expression of miR-99 b between ddPCR test results and qPCR test results correlation coefficient was r=0.75,P<0.05.Conclusion: The expression levels of miR-155 and miR-99 b were significantly different in the plasma of patients with primary CRC and CRLM,but let-7ano significant differences were observed between the two groups.Compared with qPCR,ddPCR has higher detection sensitivity and is more suitable for detecting miRNA in plasma.Detection of miR-155 and miR-99 b expression by ddPCR may play a significant role in the monitoring of CRLM.This study provides an experimental basis for the development of ddPCR diagnostic kit for miR-155 and miR-99b. |
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