| Hepatocellular carcinoma(HCC) is one of the most common malignancies worldwide, with over 782,000 newly diagnosed cases per year. As the second most frequent cause of cancer deaths, HCC is an extremely lethal disease, causing 746,000 cancer deaths annually, half of which occur in China. Although it is well known that HBV infection is the most important risk factor for HCC in China, a lack of sensitive and specific biomarkers for the early detection of this disease has greatly impeded the development and effectiveness of therapeutic strategies. Currently, the most commonly used circulating marker for HCC is alpha-fetoprotein(AFP), but its value in the early diagnosis of this disease is very limited. Therefore, there is an urgent need to identify novel biomarkers to improve the early detection of HCC before it becomes clinically detectable and has progressed to lethal, advanced stages.Micro RNAs(mi RNAs) are a sort of small non-coding RNAs and those small non-coding RNAs can not only repress translation but also promote degradation of target m RNAs by the complementary sequences. Recnet studies shown that a mi RNA may influence hundreds of target genes by change thier expression, besides more and more researches consistently verify the roles that mi RNAs played in cancer development pathways. Although the clear mechanisms of mi RNAs are limited understood in the process of carcinogenesis, the expression lever of mi RNA still can indicate the associated gene expression lever. A number of studies have reported that the altered expression of circulating mi RNAs is associated with many diseases, including HCC. Binding to argonaute proteins stabilizes circulating mi RNAs in the plasma/serum; thus, they are potentially stable noninvasive biomarkers. However, tissue specificity and early activation are important properties of specific biomarkers for the early detection of cancer. Our previously published prospective study, which used a limited number of incident patients, has suggested that circulating mi RNAs could function as biomarkers for the early detection of lung cancer. The discovery of deregulated mi RNAs as biomarkers in the plasma/serum may represent a useful approach to segregate HCC patients from controls, but whether they are liver-originated or could serve as biomarkers for early detection has not been systematically investigated.Here, we performed a multicenter, three-phase study to screen liver-originated HCC-associated plasma mi RNAs in both plasma and tissue samples. We then evaluated and validated the diagnostic potential and effectiveness of these mi RNAs for the early detection of HCC. Early detection refers to the identification of individuals who have this disease before a clinical diagnosis is made in prospective cohort studies using plasma mi RNAs.Phase 1(Screening set)The first phase was to screen liver-originated HCC-associated plasma mi RNAs, which were found to overlap between Taq Man low-density arrays(TLDA, human micro RNA panel V2.0, Applied Biosystems Inc., CA, USA) screens of plasma and tissue samples. We selected candidate mi RNAs that met the following criteria:(i) having CT values of lower than 35(considered as “detected”);(ii) showing four-fold(│ΔΔCT│> 2) altered expression between any of the comparisons; and(iii) having alterations of the same direction in plasma or tissue samples. The mi RNAs from the plasma TLDA screening that overlapped between the “HCC-associated plasma mi RNAs” and the “potentially liver-originated plasma mi RNAs” were identified, and the “mi RNAs up-regulated after liver transplantation” were then removed. The “HCC-associated plasma mi RNAs” were derived from comparisons of the plasma mi RNA expression levels in four HCC patients(A1-I, A2-I, A3-I, and B1-I) and one cancer-free cirrhosis patient(C1-I), whereas the “potentially liver-originated plasma mi RNAs” were identified from comparisons between four HCC patients before liver transplantation/hepatectomy(A1-I, A2-I, A3-I, and B1-I) and one HCC patient at two weeks after hepatectomy(B1-II). The “mi RNAs up-regulated after liver transplantation” were differentially expressed plasma mi RNAs detected in three HCC patients before liver transplantation(A1-I, A2-I, and A3-I) with similar expression after transplantation(A1-II, A2-II, and A3-II). Twenty-nine mi RNAs were excluded from further analyses.The mi RNAs obtained from the tissue screening were differentially expressed mi RNAs that were derived from comparisons of three patients’ HCC tissues(A1-T, A2-T, and A3-T) with their corresponding para-carcinoma tissues(A1-N, A2-N, and A3-N) or another independent cirrhotic liver tissue sample(C1-N) from a patient who had received a liver transplant.Phase 2(Training and Validation sets)The liver-originated plasma mi RNAs from phase 1 were then tested in the phase 2 samples to evaluate their diagnostic potentials using individual Taq Man probe-based quantitative reverse transcription polymerase chain reaction(q RT-PCR) assays. In the training set, ten candidate mi RNAs(mi R-19a-3p, mi R-19b-3p, mi R-20a-5p, mi R-25-3p, mi R-30a-5p, mi R-92a-3p, mi R-132-3p, mi R-185-5p, mi R-320 a, and mi R-324-3p) were evaluated in a case-control study, of which four in the oncogenic mi R-17-92 cluster(mi R-19a-3p, mi R-19b-3p, mi R-20a-5p, and mi R-92a-3p) were over-expressed, and two(mi R-19a-3p and mi R-19b-3p) were highly correlated with mi R-20a-5p(Spearman correlation value > 0.8 and P-value < 0.05). We chose mi R-20a-5p and mi R-92a-3p for further validation by excluding mi R-19a-3p and mi R-19b-3p. In the validation set, the eight mi RNAs were further evaluated in an independent set of 64 subjects.Phase 3(Prospective Cohort Sets)For phase 3, two prospective cohorts(the Changzhou cohort and Qidong cohort) were used to evaluate the potential of these mi RNAs for the early detection of HCC. During this phase, we also included mi RNAs from previously published studies. A review of published literature revealed two studies of HCC based on genome-wide serum/plasma mi RNA screening and validation.The mi RNAs reported in these previously published studies and those identified in our own TLDA screening were selected for evaluation. For example, mi R-192-5p, mi R-21-5p, and mi R-801 are significantly up-regulated in patients with HBV-related HCC, whereas mi R-122-5p, mi R-223-3p, mi R-26a-5p, and mi R-27a-3p are down-regulated in these patients.Li et al. have demonstrated that mi R-1-3p, mi R-25-3p, mi R-92a-3p, mi R-206, mi R-375, and let-7f-5p can be used to distinguish HCC cases from controls and that all of these mi RNAs are up-regulated in HCC patients.We excluded mi R-801, mi R-1-3p, mi R-206, and let-7f-5p because they were undetectable in some of the samples used in our screening. Considering our screening results and selection criteria, we included mi R-25-3p, mi R-92a-3p, mi R-192-5p, mi R-21-5p, and mi R-375 from previously published studies. We conducted two nested case-control studies and performed q RT-PCR on the plasma samples that were collected at baseline from these cohorts. The average leading time from clinical diagnosis was 34.5 months for the Changzhou cohort and 50.5 months for the Qidong cohort.In this multicenter three-phase study, we first performed screening using both plasma(HCC before and after liver transplantation or liver hepatectomy) and tissue samples(HCC, para-carcinoma and cirrhotic tissues). Then, we evaluated the diagnostic potential of the mi RNAs in two case-control studies(training and validation sets). Finally, we used two prospective cohorts to test the potential of the identified mi RNAs for the early detection of HCC. During the screening phase, we identified ten mi RNAs, eight of which(mi R-20a-5p, mi R-25-3p, mi R-30a-5p, mi R-92a-3p, mi R-132-3p, mi R-185-5p, mi R-320 a, and mi R-324-3p) were significantly over-expressed in the HBV-positive HCC patients compared with the HBV-positive cancer-free controls in both the training and validation sets, with a sensitivity of 0.866 and specificity of 0.646. Furthermore, we assessed the potential for early HCC detection of these eight newly identified mi RNAs and three previously reported mi RNAs(mi R-192-5p, mi R-21-5p, and mi R-375) in two prospective cohorts. Our meta-analysis revealed that four mi RNAs(mi R-20a-5p, mi R-320 a, mi R-324-3p, and mi R-375) could be used as preclinical biomarkers(Pmeta < 0.05) for HCC. The expression profile of the eight-mi RNA panel can be used to discriminate HCC patients from cancer-free controls, and the four-mi RNA panel(alone or combined with AFP) could be a blood-based early detection biomarker for HCC screening. |
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