Establishment of detection method for serum circulating Bmi-1 mRNA and its application in diagnosis of colorectal cancer
10.3760/cma.j.issn.1009-9158.2014.09.010
- VernacularTitle:血清循环 Bmi-1 mRNA 检测方法的建立及其在结直肠癌诊断中的应用
- Author:
Xin ZHANG
;
Haiyan WANG
;
Guixi ZHENG
;
Lili WANG
;
Peilong LI
;
Tong LIU
;
Yongmei YANG
;
Lutao DU
;
Juan LI
;
Chuanxin WANG
- Publication Type:Journal Article
- Keywords:
Colorectal neoplasms;
Reverse transcriptase polymerase chain reaction;
Sensitivity and specificity
- From:
Chinese Journal of Laboratory Medicine
2014;(9):678-682
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a direct reverse transcription real-time fluorescence quantitative polymerase chain reaction ( RT-qPCR-D ) method for detecting serum circulating B cell-specific moloney murine leukemia virus integration site-1 (Bmi-1) mRNA, and analyze the levels of serum circulating Bmi-1 mRNA in colorectal cancer patients by using of this method for exploring its diagnosis value in colorectal cancer.Methods Methodology establishment.RNA was extracted from colorectal cancer HT 29 cell line, and detection standard curves of Bmi-1, ubiquitin C ( UBC), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) mRNAs were established , then the amplification efficiencies were calculated.Bmi-1 mRNA level was directly detected in serum and preparation buffer mixture , then the specificity of assay was evaluated by melting curve, and detection limit was observed through diluted serum samples.The serum circulating Bmi-1 mRNA levels were detected by ELISA in 158 cases with colorectal cancer , of which there were 26 cases of tumor node metastasis ( TNM)Ⅰstage, 53 cases of TNMⅡ, 47 cases of TNMⅢ, 32 cases of TNMⅣand 53 cases of controls with normal colonoscopy collected from January 2008 to January 2009 in Qilu Hospital of Shandong University.Comparisons of groups were determined by applying Mann-Whitney U test or Kruskal-Wallis test, and receiver operating characteristic ( ROC) curves were established to illustrate the diagnostic performance.Results The log values of Bmi-1, UBC and GAPDH showed good linear correlations with quantification cycle (Cq) values(R2 =0.990, 0.990, 0.991, all P <0.001), and the amplification efficiencies were 0.875, 0.917 and 0.935, respectively.Using the established RT-qPCR-D method, the peak of melting curve of Bmi-1, UBC and GAPDH mRNAs were single, the detection limit was up to 1.25μl.The levels of serum circulating Bmi-1 mRNA detected by RT-qPCR-D were 0.138 ( 0.078-0.228 ) in colorectal cancer stage Ⅰ patients, 0.163(0.067 -0.287) instage Ⅱ patients, 0.217(0.072-0.267) instage Ⅲpatients, 0.273(0.139 -0.419) in stage Ⅳ patients and 0.021(0.008 -0.029) in health controls, a significant difference was found among groups ( H =89.5, P <0.001 ).The levels of serum circulating Bmi-1 mRNA in each stage colorectal cancer were all significantly higher than that in control group(U=58.0, 287, 246, 72.5,all P<0.001).The levels in Ⅳstage patients were significantly higher than those in other stages patients (U=247, 590, 540,P=0.008, 0.020, 0.035), while no significant differences among Ⅰstage,Ⅱstage and Ⅲstage patients(U=633, 514, 1170,all P>0.05).ROC curve analysis showed area under the ROC curve ( AUC) for serum circulating Bmi-1 mRNA was 0.921(95%CI=0.876-0.953), which was significantly superior to the AUC of CEA (0.745, 95%CI=0.680-0.802, Z=4.697, P<0.001 ).When cutoff value was 0.034, the diagnostic sensitivity and specificity was 89.2%(141/158) and 90.6%(48/53), while 41.8%(66/158) and 73.6%(39/53) using CEA.The AUC for combination of circulating Bmi-1 mRNA and CEA was 0.933(95%CI=0.890-0.963 ) , which was no statistical significance when compared with the AUC of circulating Bmi-1 mRNA(Z=4.697, P>0.05).Conclusions The study establishes a higher sensitive, specific for detecting serum circulating Bmi-1 mRNA. Based on this method , serum circulating Bmi-1 mRNA is found to be increased in colorectal cancer , and is superior to traditional tumor marker CEA in diagnosis of colorectal cancer, which may become a potential detection index for early detection of colorectal cancer.
