Establishment of detection method for serum circulating Bmi-1 mRNA and its application in diagnosis of colorectal cancer

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 HT29 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）Ⅰ stage, 53 cases of TNM Ⅱ, 47 cases of TNM Ⅲ, 32 cases of TNM Ⅳ and 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 0.05）. 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 0.05）. 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.（Chin J Lab Med,2014,37:678-682） Key words: Colorectal neoplasms; Reverse transcriptase polymerase chain reaction; Sensitivity and specificity