| Gastric cancer (GC) is currently the third most common malignancy and the second leading cause of cancer death in China. Early GC is typically small and asymptomatic, and the high mortality from GC is mainly due to late presentation. Therefore, early detection and treatment is an important way to reduce death from GC. Several tests have been applied in population screening, such as barium meal and endoscopy, especially in Japan, with an ultimate goal of reducing mortality associated with GC. However, these approaches may be not eligible for mass screening and early detection of GC in most countries including China for the reasons such as acceptance, availability and cost. In addition, non-invasive serological tests, such as serum pepsinogen and gastrin-17, however, have no sufficient sensitivity and specificity to facilitate the early detection. Therefore, efforts should be invested in the discovery of novel, reliable and non-invasive biomarkers for mass screening and early detection of GC.MicroRNAs (miRNAs) are a class of small non-coding RNAs that can repress translation or promote degradation of target mRNAs of complementary sequences. A single miRNA can influence the expression of hundreds of target genes, and considerable researches consistently establish their central nodal roles in cancer development pathways. Although the molecular mechanisms of miRNAs are incompletely understood in the process of carcinogenesis, specific patterns of miRNA expression have been associated with almost every type of tumour, suggesting diagnostic and prognostic potentials for miRNAs.Since the identification of circulating miRNAs in2008, considerable researches consistently found that circulating miRNAs are not only stable but degradation-resistant which laid the foundation for ideal biomarkers. We and other groups have demonstrated that circulating miRNAs had the potential to be new biomarkers in patients with solid tumours for either early detection or evaluation of therapeutic efficiency. In recent years, several studies have reported a series of circulating miRNAs differentially expressed in patients with GC, and most of these studies focussed on individual miRNAs selected from previous analyses on tissue samples and few with systematic miRNA profiling. However, the reported circulating miRNAs are not consistent among studies. Liu et al performed serum miRNA profiling in20GC patients and20normal controls using Solexa sequencing and identified a five-miRNA signature (miR-1, miR-20a, miR-27a, miR-34and miR-423-5p) for GC diagnosis with the area under the receiver operating characteristic (ROC) curve (AUC) of>0.800. Thereafter, another group analysed genome-wide serum miRNAs by miRNA microarray, and reported three miRNAs (miR-187*, miR-371-5p and miR-378) significantly elevated in GC patients with the best AUC of0.861for miR-378. These two studies reported distinct serum miRNA profiles of GC. The inconsistency may be resulted from inclusion of different gastric tumour subtypes. For example, gastric non-cardia adenocarcinoma (GNCA) and cardia adenocarcinoma (GCA) have distinct clinical and epidemiologic features and each form might be characterized by a distinct expression profile of miRNA. In addition, identifying differential circulating miRNAs by contrasting controls with GC patients of all tumour stages as a whole might lead to the risk of overrepresenting the aberrant miRNAs from the early-stage GC patients. Therefore, large-scale, well-designed studies are needed to verify the clinical utility of circulating miRNAs for early detection of GC.Herein, we stringently designed a four-phase study to identify a panel of circulating miRNAs as early diagnostic biomarkers of GC and evaluated their clinical usefulness in independent samples. In the discovery phase, we systematically screened the miRNA expression profile in plasma of40GNCA patients (stage Ⅰ) and40matched controls by TaqMan low density array (TLDA) chips. Differentially expressed miRNAs were further validated in individual sample in the training phase using qRT-PCR assay. Subsequently, in an independent validation phase, the identified miRNAs were evaluated in additional samples, including48GNCA patients (stage Ⅰ),54subjects with precancerous lesions of GC (PLGC) and48healthy subjects. Finally, the expression levels of the identified miRNAs were assessed in an external validation phase, including18stage Ⅰ and18stage Ⅲ/Ⅳ GNCA patients, and18stage Ⅰ and18stage Ⅲ/Ⅳ GC A patients. The differences of miRNAs among groups were tested using the Mann-Whitney unpaired test.With an effort to identify stably, differentially expressed miRNAs in plasma, we screened a total of667human miRNAs by TLDA chips in pooled samples between the GNCA patients and healthy controls (40subjects for each pool). We further selected candidate miRNAs that had a CT value of o30in at least one pool and showed more than four-fold altered expression between the two pooled samples. As a result, five upregulated plasma miRNAs, including miR-16, miR-25, miR-92a, miR-451and miR-486-5p, were subjected to individual qRT-PCR confirmation in the training and validation phase. Consistent with the results of the discovery phase, all of the five miRNAs had a significantly higher expression level in the GNCA patients as compared with controls. In the external validation, all of the five miRNAs were consistently overexpressed in the advanced GNCA patients and GCA patients.To evaluate the performance of the miRNAs in discriminating the GC patients from cancer-free subjects, the discriminatory parameters were first developed in the training phase and were further independently assessed in the validation phase. The optimal cutoff value for individual miRNA was determined according to the ROC curve for each miRNA in the training phase. Based on the cutoff values developed in the training phase, the value of AUCs in the independent validation phase varied from0.694to0.790, and value of AUCs in both of the independent and external validation phase varied from0.733to0.871. To assess the diagnostic utility of this five-miRNA panel, a linear combination of the risk score of the five miRNAs weighted by the regression coefficient was used to calculate an RSF of the five-miRNA panel for each subject. The RSF was calculated as follows:RSF=(5.142×risk score of miR-16)+(5.609×risk score of miR-25)+(5.398×risk score of miR-92a)+(4.043×risk score of miR-451)+(4.181×risk score of miR-486-5p), where the regression coefficients were derived from subjects in the training phase. Similar to but better than the individual miRNA, in the independent phase, the AUC of the five-miRNA panel was0.812and the diagnostic sensitivity and specificity were68.8%and90.2%, respectively. In both of the independent and external validation phase, the miRNA panel also showed relatively high ability in discriminating the GC patients from the cancer-free controls with an AUC of0.909(sensitivity=84.2%and specificity=91.7%).In addition, we determined whether the identified miRNAs (miR-16, miR-25, miR-92a, miR-451and miR-486-5p) act as secretory miRNAs and are excreted into the culture media by BGC823and MGC803cell lines. We found that miR-16, miR-25and miR-92a levels in media of BGC823and MGC803increased with increasing cell counts (0.5-2×106cells/well) and incubation time (24and48hours), whereas miR-451and miR-486-5p levels did not change in either cell line. These data suggest that miR-16, miR-25and miR-92a but not miR-451or miR-486-5p may be secretory miRNAs.In summary, we conducted a comprehensive assessment of the diagnostic usefulness of a miRNA panel in plasma for GC in a tailored investigation with independent validation. The sensitivity and specificity were high and the discriminative accuracy was satisfactory for the diagnosis of the early-stage GC. Our findings highlight that circulating miRNAs may be a class of novel, noninvasive diagnostic biomarkers with sufficient diagnostic accuracy, and a specified miRNA-panel can be of great value in mass screening or early detection of GC. Our work will serve as a basis for further large-scale validation before circulating miRNAs enable their introduction into the clinic setting. |
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