Serum MicroRNA As Biomarkers For Early Diagnosis Of Colorectal Adenocarcinoma

BackgroundColorectal cancer (CRC) is one of the most digestive tract malignancies. The mortality of CRC ranked the fifth in our country with increased incidence and mortality in the past several decades. Colorectal cancer is a class of typical malignant epithelial tumors which resulting in a transformation from normal mucosa to precancerous lesion and finally to malignant tumor that is a well known normal mucosa-adenoma-adenocarcinoma (NM-A-AC) sequence. Colorectal adenocarcinoma is the main histological type of colorectal cancer and colorectal adenoma is widely considered as important precancerous lesions of colorectal adenocarcinoma. The clinical symptoms of colorectal adenocarcinoma were dormant and difficult to be found at the early stage. Most of colorectal adenocarcinoma patients were diagnosed at the middle and late stages and lost the best operation opportunity. There was no significant improvement of5-year survival rates of colorectal adenocarcinoma patients although clinical medical technology was constantly developing. The lack of effective diagnostic method to diagnosis early stage of colorectal adenocarcinoma and precancerous lesion was one of the main reasons. Removal of early-stage cancer and precancerous lesions would play a pivotal role to reduce the currently high colorectal adenocarcinoma mortality. Several colorectal adenocarcinoma screening tests, including colonoscopy, imageological examination and CEA detection, have been used for years. However, none of these methods has been established as a well-accepted screening tool, particularly for the diagnosis of early-stage colorectal adenocarcinoma. It has become an urgent and tough problem to look for high sensitivity and specificity of biomarkers for colorectal adenocarcinoma.MicroRNAs (miRNAs) are a class of small non-coding RNAs with19-24nucleotides, and they have attracted a great deal of attention in cancer research. Functional studies have indicated that deregulation of miRNAs is involved in the initiation and progression of human cancer. Recently, serum miRNAs have been shown to be rich and stable. The sensitivity and specificity of single miRNA as biomarker is hard to meet current clinical needs which can be solved by the use of tumor specific miRNA profile. The miRNA profile has been considered as effective tool for tumor early diagnosis. It will provide a new efficient approach for early diagnosis if the specific miRNA profile of colorectal adenoacarcinoma was acquired.Quantitative real time polymerase chain reaction (RT-qPCR) is the most frequently used approach for detection of miRNAs due to its accuracy, sensitivity, specificity and reproducibility. Many factors for example the quantity and concentration of RNA might lead to deviation of results in the research of gene expression. The use of reference genes as endogenous control is the most common method for normalizing RT-qPCR data of miRNAs expression. The selection of suitable reference genes play a crucial role in miRNAs research because normalization to unreliable reference genes may lead to incorrect determination of miRNAs of interests. As far as aware, no systematic identification and validation of reference genes for normalizing RT-qPCR analysis of serum miRNAs in colorectal adenocarcinoma and colorectal adenoma patients has been published.Based on the above questions, we firstly characterized the genome-wide miRNA expression profile in serum of patients with colorectal adenocarcinoma, colorectal adenoma and healthy controls using a high-throughput Miseq sequencing screening. A great effort was made to screen candidate reference genes which were undifferentially expressed among three groups and candidate biomarkers which were gradually upregulated or downregulated according to our criteria. Then the reliability of candidate reference genes for normalization was evaluated by RT-qPCR assays to select the most suitable reference genes. The selected reference genes were validated in an independent cohort study. To test for the effect of reference genes, we selected serum miR-92a-3p as target miRNA. Using the above reference genes, the differentially expressed biomarkers were validated by two phases of RT-qPCR. The relationship of candidate biomarkers and clinical parameters were analyzed and receiver operating characteristic (ROC) curve was used to evaluate their diagnostic value. Then, the miRNA panel was developed with a logistic regression model to construct logit(P) equation. The miRNAs panel was validated in huge sample using blind methods and ROC was constructed to evaluate the diagnostic accuracy and differential diagnosis value. PART IIdentification and validation of reference genes for qPCR detection of serum microRNAs in colorectal adenocarcinoma patientsObjectiveThe most frequently used approach for serum miRNAs detection is quantitative real time polymerase chain reaction (qPCR). In order to obtain reliable qPCR data of miRNAs expression, the use of reference genes as endogenous control is undoubtly necessary. The aim of our study is to evaluate and validate reference genes for normalizing qPCR analysis of serum miRNAs in colorectal adenocarcinoma.MethodsWe firstly profiled pooled serum of30patients with colorectal adenocarcinoma,25patients with colorectal adenoma and30healthy controls using Miseq sequencing. The miRNAs were selected as candidate reference genes that showing no differential expression among three groups. RT-qPCR was performed to validate above candidate reference genes and U6SnRNA in45patients with colorectal adenocarcinoma,40patients with colorectal adenoma and40healthy controls. The variable stability of candidate reference gene was further evaluated by two algorithms:geNorm and NormFinder to select the most suitable reference gene that was further validated in an independent cohort. The expression of miR-92a-3p was analyzed in colorectal adenocarcinoma patients using the selected most suitable reference gene.Results1. Selection of candidate reference genes by Miseq sequencing:Of the serum miRNAs that were scanned by sequencing, there were17,32and25miRNAs having more than50copies and showing no differential expression among pooling group of colorectal adenocarcinoma and colorectal adenoma, colorectal adenocarcinoma and healthy controls, colorectal adenoma patients and healthy controls, respectively (P>0.05). According to the criteria described in "Material and Methods", a list of13miRNAs was considered as candidate reference genes.2. Confirmation of candidate reference genes by qPCR:Five miRNAs (miR-151a-3p, miR-4446-3p, miR-221-3p, miR-93-5p and miR-3184-3p) were not detected in all samples that were excluded from further analysis. Using the Cq values, there was no evidence for differential expression of any of the nine candidate reference genes among three groups (P>0.05). The candidate reference genes displayed a wide expression range, with Cq values between23.21and44.68. Expression of miR-197-3p and miR-26a-5p was relatively low, with median Cq more than35that were also excluded from further stability analysis.3. Expression stability analysis of selected reference genes by GeNorm and NormFinder:Variable stability of selected reference genes (miR-103b, miR-484, miR-16-5p, miR-3615, miR-18a-3p, miR-191-5p and U6snRNA) was evaluated using two algorithms:geNorm and NormFinder. GeNorm recommended the use of four (miR-191-5p, miR-16-5p, U6snRNA and miR-18a-3p) of the seven most stable genes for optimal normalization. NormFinder and geNorm both identified miR-191-5p as the most stably expressed reference genes and selected miR-191-5p and U6snRNA as the most stable pair of reference gene.4. Validation of suitable reference genes in a cohort samples:The expression level of miR-191-5p, U6snRNA and combination of miR-191-5p and U6snRNA (mean) in three groups and four stages of colorectal adenocarcinoma group were validated in an independent cohort. Using the Cq values of each validate reference gene, there was no evidence for differential expression among the three groups and four stages of colorectal adenocarcinoma.5. Effect of suitable reference genes on relative quantity of serum miR-92a-3p:Our results indicated that serum miR-92a-3p was significantly higher in colorectal adenocarcinoma patients than colorectal adenoma patients and healthy controls (P<0.001). The difference in miR-92a-3p level remained significant between colorectal adenoma patients and healthy controls (P<0.001). The expression of miR-92a-3p increased with the progress of TNM staging. There was significant difference of serum miR-92a-3p between Stage I and StageⅢ/Ⅳ, between Stage Ⅱ and StageⅢ/Ⅳ, between StageⅢ and Stage Ⅳ. The difference of serum miR-92a-3p between Stage Ⅰ and Stage Ⅱ was not found (P=0.66).ConclusionsWe propose that combination of miR-191-5p and U6snRNA could be used as reference genes for serum microRNAs qPCR data in colorectal adenocarcinoma, colorectal adenoma and healthy controls. PART IIThe establishment and clinical value of serum microRNA panel in colorectal adenocarcinomaObjectiveCurrently, none of the available colorectal adenocarcinoma (CAC) testing has been established as a well-accepted diagnosis tool, particularly for the early stage of CAC. The recent discovery of serum microRNA (miRNA) profile has provided a new auxiliary approach for tumor diagnosis. Our study is the global analysis of serum miRNAs during the normal-colorectal adenoma (CA)-CAC sequence.MethodsWe firstly profiled pooled serum of30patients with colorectal adenocarcinoma,25patients with colorectal adenoma and30healthy controls using Miseq sequencing. Differentially expressed serum miRNAs were validated by two phase of reverse-transcription polymerase chain reaction (RT-qPCR). The miRNA panel was developed with a logistic regression model and validated using an independent cohort. Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic accuracy of the panel.Results1. Discovery of candidate biomarkers by miseq sequencing:Of the740sequenced serum miRNAs,348,303and283miRNAs were detectable (more than10copies) in CAC patients, CA patients and healthy controls, respectively. The miRNAs were selected as candidate biomarkers based on the following criteria:(a) having at least50copies in any group;(b) exhibiting at least10-fold altered expression; and (c) the intersection of CAC vs CA and CA vs healthy controls. Levels of12miRNAs, including miR-195-5p, miR-92a-3p, miR-1290, miR-582-5p, miR-223-3p and miR-136-5p, miR-3074-5p, miR-29a-3p, miR-221-3p, miR-148a-3p, miR-19a-3p and miR-17-3p were significantly higher in CAC than those in CA and healthy controls (fold change=10.78-399.02; P<0.05). In contrast, levels of3miRNAs, including miR-422a, miR-1260a and miR-4502, in the CAC group were significantly lower (fold change=0.002-0.09; P<0.05). In summary, a total of15differentially expressed miRNAs were identified as candidate biomarkers, which should be further tested via RT-qPCR.2. Confirmation of miRNAs by RT-qPCR analysis:We first tested the15candidate miRNAs using an independent cohort of80serum samples with RT-qPCR. Only4miRNAs (miR-19a-3p, miR-223-3p, miR-92a-3p and miR-422a) with a differential expression between the CAC vs CA and CA vs healthy control group were selected using these above-mentioned quality control criteria. These four miRNAs were further evaluated by RT-qPCR using additional independent240serum samples. High expression levels of miR-19a-3p, miR-92a-3p and miR-223-3p as well as the low expression level of miR-422a were detected in CAC patients compared with CA and healthy control group. The diagnostic accuracy of these4miRNAs was measured by ROC, and their corresponding AUCs were0.849,0.871,0.890and0.843, respectively.3. Establishing the predictive miRNAs panel:In the present study, we constructed the miRNA panel for CAC diagnosis using320serum samples as the training data. The predicted probability of diagnosis with CAC from the stepwise logistic regression model was calculated using the equation as follows:logit (P)=0.3313-0.0081*miR-149a-3p-0.0257*miR-92a-3p-0.0406*miR-223-3p+0.1328*mi R-422a. The diagnostic performance of the established miRNA panel was evaluated by the ROC analysis. The AUC of the established4-miRNA panel was0.960.4. Validation of the miRNAs panel:We further validated the diagnostic performance of the established4-miRNA panel in another independent validation phase. The AUC of the4-miRNA panel was0.951(95%CI:0.907to0.978; sensitivity=84.3%, specificity=91.6%), which was greater than CEA detection (AUC:0.667,95%CI:0.593to0.735, P<0.001). Moreover, we further evaluated the diagnostic performance of the established miRNA panel at different TNM stages. The corresponding AUCs for patients with TNM stages Ⅰ, Ⅱ, Ⅲ and Ⅳ were0.942,0.935,0.954and0.983, respectively. In addition, we evaluated the diagnostic accuracy of the established miRNA panel according to the CEA level. In the low CEA level (<5ng/mL) group, the AUC of the established miRNA panel was0.810(95%CI,0.725to0.818; sensitivity=57.14%, specificity=86.67%). In the elevated CEA level (>5ng/mL) group, the AUC of the established miRNA panel was0.918(95%CI,0.861to0.957; sensitivity=76.79%, specificity=85.56%). Finally, we also evaluated the diagnostic performance of the4-miRNA panel in discriminating the CA from CAC and healthy controls group. The analysis demonstrated that the miRNA panel possessed a high accuracy in discriminating CA from CAC (AUC=0.886;95%CI:0.809to0.940) and CA from healthy controls (AUC=0.765;95%CI:0.669to0.845).ConclusionsSerum miR-19a-3p, miR-223-3p, miR-92a-3p and miR-422a were considered as promising biomarkers for colorectal adenocarcinoma. We established a serum4-miRNA panel with considerable clinical value in the early-stage diagnosis of CAC.