| Background and objectiveHepatocellular carcinoma(HCC) is the second cancer killer in China and there are about 55% of HCC associated deaths happened in China.Despite substantial progresses have been made in HCC clinical and basic research,the overall prognosis of HCC remains dismal because of the late diagnosis and low resection rate.A1though alpha fetoprotein(AFP) remains the best HCC biomarker,the specificity and sensitivity of which are still not satisfied.Thus,there is an urgent need for additional better diagnostic markers for HCC。Recently, the discovery of small non-protein-coding RNAs, so-called microRNAs (miRNAs) that play important roles in oncogenesis, has opened new opportunities of a non-invasive test for the early diagnosis of cancer. Studies have shown that profiles of miRNA expression differ between normal tissue and tumour tissues and vary among tumour types. Evidence suggests that miRNA expression profiles can cluster similar tumour types together more accurately. Furthermore, miRNA expression signatures have been used to predict prognosis. More recently, several reports also suggest that cell-free circulating miRNAs existed in serum and plasma. Accordingly, it raises the possibility of using miRNAs as novel non-invasive molecular markers for cancer detection. In the present study, we evaluated the feasibility of using serum miRNAs as a non-invasive diagnostic test for HCC.Methods and ResultsThis study was divided into three phases: phase I, marker discovery; phase II, marker selection and validation; and phase III, large-scale validation.Phase I: marker discoveryIn this phase, serum from ten patients with HCC and ten healthy controll were collected. The serum of each team were pooled together to generate a HCC serum pool and a healthy control serum pool. Then total RNA were isolated from each serum pool followed LNATM microarray scaning. By comparing miRNA probe intensity from the two pools,upregulated miRNAs in HCC serum were identified for further analysis in phase II. We found six miRNAs matching the criterior. Phase II: marker selection and validationSerum samples were collected from 18 patients with HCC before any therapy. Plasma from 18 healthy subjects and 12 patients with HBV infection was collected as control. SYBR green qRT-PCR assay was used for miRNA quantification in serum samples. We final selected miR-1915 as the HCC specific tumour marker.Phase III: large-scale validationSerum was collected from an independent group of 72 patients with HCC before any therapy. Serum from a set of 20 healthy subjects,15 patients with benign liver tumour,31 patients with other malignant liver tumour,23 patients with HBV induced liver cirrhosis was collected as the control. Serum from another 10 patients with HCC was collected before and 7 days after surgical resection. SYBR green qRT-PCR assay was used for miRNA quantification in serum samples .The miR-1915 expession level of each group was campared by△Ct method. The levels of miR-1915 were significantly high than the other groups. Furthermore,we also found that the levels of miR-1915 were significantly reduced in the postoperative samples when compared to the pre-operative samples.ROC curve analyses revealed that the serum levels of miR-1915 were useful biomarkers for differentiating patients with HCC from controls with ROC curve areas of 0.81(95% CI = 0.747–0.879).At the cut-off value of 2.22 for miR-1915 (relative expression in comparison to RNU6B snRNA), the sensitivity was 63.9% and the specificity was 86.5%.ConclusionsIn conclusion, differentially expressed miRNAs in serum of patients with HCC have been tested in this study.1,Some miRNAs express steadly in serum of HCC patients and healthy controls.2,By using microarray scaning, we found that the expression of certain miRNAs in serum was significantly different between HCC group and control group.3,Serum miRNA-1915 may serve as a potential biomarker for HCC detection.
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