The Preliminary Study Of MiRNAs As Biomarkers In Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the fifth most common malignant cancer and the third leading cause of death of cancer-related death. Although most cases occur in Asia and Africa, the incidence has been steadily increasing in the west over the last20years. The major etiologies of HCC include hepatitis B or C virus infection, aflatoxin or water pollution and hereditary factor. It has been known that the hepatocarcinogenesis involves a complex, multistep process, which is linked tightly to chronic liver damage. A variety of molecular alterations that can result in the loss of cell-cycle checkpoints, resistance to apoptosis or activation of oncogenic pathways occurred at the initiation stage and in the development of liver cancer. However, neither of these molecular alterations has steadily been revealed in hepatocellular carcinomas. Recent studies suggested that miRNA expression alteration could play an important role in pathogenesis of HCC.MiRNAs are small non-coding RNAs of20-22nucleotides, which control a wide range of biological processes including developmental timing, cell proliferation, differentiation, apoptosis, and metabolism, etc. MiRNAs interact with target mRNAs at specific sites within their3’-untranslated regions (UTRs) to inhibit protein translation or induce the target mRNAs cleavage. More than60%of human protein-coding genes are predicted to contain miRNAs binding sites and therefore subject to miRNAs regulation. Furthermore, aberrant expression of miRNAs has been associated with various human diseases. A considerable body of evidence support that miRNAs can control the regulation of cancer development, and also represented in the environmental pollutant-and toxicant-induced carcinogenesis. All of the investigated human cancers, including HCC, are characterized by globally abnormal miRNA expression patterns. Moreover, the association demonstrated between the characteristic miRNA expression patterns and cancer types suggests that different response of miRNAs is observed among cancers from different tissues of origin. The special changes of miRNAs expression in cells can stimulate a cascade of cancer development. Therefore, the assay of miRNAs expression alteration in early stages of cancer promises an insight into the underlying mechanisms of carcinogenesis.In the present study, we sought to explore the altered expression of miRNAs underscored in HCC patients and the MC-LR-induced hepatocarcinogenesis. We consider that these results will promise to identify HCC and MC-LR-related miRNA alterations in tumorigenesis, which could be taken as biomarkers for using diagnosis in HCC or in water environmental monitoring, even for the development of miRNA-based prevention and treatment of hepatocellular carcinoma.Part Ⅰ miRNA profiling in hepatocellular carcinoma is associated with clinical featuresObjectiveIn this present study, we examined the expression profiles of miRNA in4pairs of HCC tumorous tissue and adjacent non-tumorous tissue by microRNA microarray method and then identified differentially expressed miR-491-5p and miR-875-5p in85patients to analysis the correlation between HCC and their clinic phenotype. Different miRNA expression could have relevance to the clinical behavior HCC and the miRNA expression may prognosticate disease outcome in HCC.Methods1. MicroRNA microarray experimentation including labeling, hybridization, scanning, normalization and data analysis was conducted by Exiqon A/S Technology Platform (Vedbaeck, Denmark). 2. TaqMan microRNA real-time quantitative-PCR analysis85HCC tumorous tissue and adjacent non-tumorous tissue for miR-491-3p and miR-875-3p.3. The recombinant plasmids expressing miR-491-3p and miR-875-3p were obtained by transfection of pEGFP-C1-miR-491and pEGFP-C1-miR-875into HepG2cells. We used PI staining to determine the cell cycle.Result1. The first aim of the present study was to investigate whether aberrantly expressed miRNAs between HCC tissues (T) to their corresponding normal tissues (NT). We identified22miRNAs were significantly upregulated expressed and20miRNAs were significantly downregulated expressed in the HCC tissues (mean fold change>2or<0.5).2. MiRNA specific real-time RT-PCR was used to test miR-491-3p and miR-875-3p expression lever in85HCC tissues to their corresponding normal tissues. The relative expression lever of these miRNAs significantly differed in T and NT. MiR-491-3p (41.2%) and miR-875-3p (60%)(Wilcoxon signed-rank test) were down-regulated in T.3. Of all variables tested, the statistical analysis only revealed that the miR-491-3p and miR-875-3p expression lever is lower in non-cirrhosis compared with cirrhosis (p=0.017and p<0.001) and in poorly differentiation (p<0.001and p<0.001).4. The transfection of pEGFP-C1-miR-491cells represented a significant enhanced proportion of apoptosis in cell cycle analysis, and the transfection of pEGFP-C1-miR-875cells had a significant enhanced proportion of G0/G1phase and a reduced of G2/M and S phase.ConclusionIn the present study, we have shown for the first time that the expression of miR-491-3p and miR-875-3p were downregulated in HCC. Results reported here may provide a useful clue for the research into HCC. Further investigation is needed to expound the function of miR-491-3p and miR-875-3p in the HCC, which may lead to finding new methods to diagnose, treat and prevent HCC.Part II Alteration of microRNA Expression Profile Linked to Microcystin-LR-Induced TumorigenicityObjectiveMC-LR has been shown to be a cyclic heptapeptide that acts as a potent hepatotoxin and cancinogen. However, the mechanism for its carcinogenic action remains to be determind. In this work, we used MC-LR to induce the malignant transformation in vitro and in vivo, and analyzed the miRNA expressed alteration in the transformed cells for addressing the miRNA role in the process.Methods1. The cultured WRL-68cells were continuously exposed to low concentration (10μg/L) of MC-LR for25passages (25MC10). We tested for their growth kinetics, resistance to serum-induced terminal, cell cycle and tumorigenicity in nude mice to investigate the transformed cells.2. MicroRNA microarray experimentation including labeling, hybridization, scanning, normalization and data analysis was conducted by Exiqon A/S Technology Platform (Vedbaeck, Denmark).3. The mice were fed on commercial laboratory chow and give MC-LR water. Serum was separated by centrifugation, and serum AST and ALT activities were estimated. Liver sections were taken and fixed in4%neutral-buffered formalin and prepared for examination under a photomicroscope.4. TaqMan microRNA real-time quantitative RT-PCR analysis mice liver for miR-21、 miR-122、 miR-195、 miR-221、 miR-222、 miR-338-5p and miR-491-3p.Result1. The cultured WRL-68cells were continuously exposed to low concentration (lOμg/L) of MC-LR for25passages (25MC10). Compared to the mocked treated parental cells, the induced25MC10cells represented a higher growth rate, a resistance to serum-induced terminal differentiation, and the tumorigenicity phenotype in nude mice xenograft test.2. Array-based miRNA expression profiles showed that78of miRNAs were up-regualted and48were down-regualted respectively in the25MC10cells.3. While treatment of MC-LR80ug/L in9months, serum AST and ALT activities were remarkably increased. The histological pattern was significant increased of dicaryon in hepatoceyte, which showed MC-LR induced malignant transformation in vivo.4. MiRNA specific real-time quantitative RT-PCR was used to test. miR-21、 miR-221and miR-222were upregulated, and miR-122、. miR-338-5p and miR-491-3p were downregulated in MC-LR-treatment livers. miR-195were upregulated in40ug/L group, but downregulated in80ug/L group.ConclusionThese results suggest that chronic exposure to MC-LR can alternate the miRNA expression profile of hepatocarcinogenesis in vitro and in vivo. The characteristic miRNA alterations could be taken as the molecular targets for developing environment molecular inspection techniques.