Regulation of hepatic microRNA expression by hepatocyte nuclear factor 4 alpha.
ABSTRACT: AIM:To uncover the role of hepatocyte nuclear factor 4 alpha (HNF4?) in regulating hepatic expression of microRNAs. METHODS:Microarray and real-time PCR were used to determine hepatic expression of microRNAs in young-adult mice lacking Hnf4? expression in liver (Hnf4?-LivKO). Integrative genomics viewer software was used to analyze the public chromatin immunoprecipitation-sequencing datasets for DNA-binding of HNF4?, RNA polymerase-II, and histone modifications to loci of microRNAs in mouse liver and human hepatoma cells. Dual-luciferase reporter assay was conducted to determine effects of HNF4? on the promoters of mouse and human microRNAs as well as effects of microRNAs on the untranslated regions (3'UTR) of two genes in human hepatoma cells. RESULTS:Microarray data indicated that most microRNAs remained unaltered by Hnf4? deficiency in Hnf4?-LivKO mice. However, certain liver-predominant microRNAs were down-regulated similarly in young-adult male and female Hnf4?-LivKO mice. The down-regulation of miR-101, miR-192, miR-193a, miR-194, miR-215, miR-802, and miR-122 as well as induction of miR-34 and miR-29 in male Hnf4?-LivKO mice were confirmed by real-time PCR. Analysis of public chromatin immunoprecipitation-sequencing data indicates that HNF4? directly binds to the promoters of miR-101, miR-122, miR-194-2/miR-192 and miR-193, which is associated with histone marks of active transcription. Luciferase reporter assay showed that HNF4? markedly activated the promoters of mouse and human miR-101b/miR-101-2 and the miR-194/miR-192 cluster. Additionally, miR-192 and miR-194 significantly decreased activities of luciferase reporters for the 3'UTR of histone H3F3 and chromodomain helicase DNA binding protein 1 (CHD1), respectively, suggesting that miR-192 and miR-194 might be important in chromosome remodeling through directly targeting H3F3 and CHD1. CONCLUSION:HNF4? is essential for hepatic basal expression of a group of liver-enriched microRNAs, including miR-101, miR-192, miR-193a, miR-194 and miR-802, through which HNF4? may play a major role in the post-transcriptional regulation of gene expression and maintenance of the epigenome in liver.
Project description:Hepatocyte nuclear factor 4α (HNF4α) controls the expression of liver-specific protein-coding genes. However, some microRNAs are also modulated by HNF4α, and it is not known whether they are direct targets of HNF4α and whether they influence hepatic function. In this study, we found that HNF4α regulates microRNAs, indicated by marked down-regulation of miR-194 and miR-192 (miR-194/192) in liver-specific Hnf4a-null (Hnf4aΔH) mice. Transactivation of the shared miR-194/192 promoter was dependent on HNF4α expression, indicating that miR-194/192 is a target gene of HNF4α. Screening of potential mRNAs targeted by miR-194/192 revealed that expression of genes involved in glucose metabolism (glycogenin 1 (Gyg1)), cell adhesion and migration (activated leukocyte cell adhesion molecule (Alcam)), tumorigenesis and tumor progression (Rap2b and epiregulin (Ereg)), protein SUMOylation (Sumo2), epigenetic regulation (Setd5 and Cullin 4B (Cln4b)), and the epithelial-mesenchymal transition (moesin (Msn)) was up-regulated in Hnf4aΔH mice. Moreover, we also found that miR-194/192 binds the 3'-UTR of these mRNAs. siRNA knockdown of HNF4α suppressed miR-194/192 expression in human hepatocellular carcinoma (HCC) cells and resulted in up-regulation of their mRNA targets. Inhibition and overexpression experiments with miR-194/192 revealed that Gyg1, Setd5, Sumo2, Cln4b, and Rap2b are miR-194 targets, whereas Ereg, Alcam, and Msn are miR-192 targets. These findings reveal a novel HNF4α network controlled by miR-194/192 that may play a critical role in maintaining the hepatocyte-differentiated state by inhibiting expression of genes involved in dedifferentiation and tumorigenesis. These insights may contribute to the development of diagnostic markers for early HCC detection, and targeting of the miR-194/192 pathway could be useful for managing HCC.
Project description:Deregulation of microRNAs (miRNAs) is a typical feature of human hepatocellular carcinoma (HCC). However, the in vivo relevance of miRNAs along hepatocarcinogenesis remains largely unknown. Here, we show that liver tumors induced in mice by c-Myc overexpression or AKT/Ras co-expression exhibit distinct miRNA expression profiles. Among the downregulated miRNAs, eight (miR-101, miR-107, miR-122, miR-29, miR-365, miR-375, miR-378, and miR-802) were selected and their tumor suppressor activity was determined by overexpressing each of them together with c-Myc or AKT/Ras oncogenes in mouse livers via hydrodynamic transfection. The tumor suppressor activity of these microRNAs was extremely heterogeneous in c-Myc and AKT/Ras mice: while miR-378 had no tumor suppressor activity, miR-107, mir-122, miR-29, miR-365 and miR-802 exhibited weak to moderate tumor suppressor potential. Noticeably, miR-375 showed limited antineoplastic activity against c-Myc driven tumorigenesis, whereas it strongly inhibited AKT/Ras induced hepatocarcinogenesis. Furthermore, miR-101 significantly suppressed both c-Myc and AKT/Ras liver tumor development. Altogether, the present data demonstrate that different oncogenes induce distinct miRNA patterns, whose modulation differently affects hepatocarcinogenesis depending on the driving oncogenes. Finally, our findings support a strong tumor suppressor activity of miR-101 in liver cancer models regardless of the driver oncogenes involved, thus representing a promising therapeutic target in human HCC.
Project description:Malignant pleural mesothelioma (MPM) is an asbestos-induced cancer with poor prognosis that displays characteristic alterations in microRNA expression. Recently it was reported that the expression of a subset of microRNAs can distinguish between MPM and adenocarcinoma of the lung. However, the functional importance of these changes has yet to be investigated. We compared expression of miR-192, miR-193a-3p and the miR-200 family in normal pleura and MPM tumor specimens and found a statistically significant reduction in the levels of miR-193a-3p (3.1-fold) and miR-192 (2.8-fold) in MPM. Transfection of MPM cells with a miR-193a-3p mimic resulted in inhibition of growth and an induction of apoptosis and necrosis in vitro. The growth inhibitory effects of miR-193a-3p were associated with a decrease in MCL1 expression and were recapitulated by RNAi-mediated MCL1 silencing. Targeted delivery of miR-193a-3p mimic using EDV minicells inhibited MPM xenograft tumour growth, and was associated with increased apoptosis. In conclusion, miR-193a-3p appears to have importance in the biology of MPM and may represent a target for therapeutic intervention.
Project description:We sought to identify circulating microRNAs as biomarkers of prevalent or incident diabetes. In a pilot study of 18 sex- and age-matched patients with metabolic syndrome, nine of whom developed diabetes during 6 years of follow-up, an array of 372 microRNAs discovered significantly elevated serum levels of microRNAs -122, -192, -194, and -215 in patients who developed diabetes mellitus type 2 (T2DM). In two cross-sectional validation studies, one encompassing sex- and age-matched groups of patients with T2DM, impaired fasting glucose (IFG) and euglycemic controls (n?=?43 each) and the other 53 patients with type 1 diabetes and 54 age- and BMI-matched euglycemic controls, serum levels of miR-192, miR-194, and mi215 were significantly higher in diabetic subjects than in probands with euglycemia or IFG. In a longitudinal study of 213 initially diabetes-free patients of whom 35 developed diabetes during 6 years of follow-up, elevated serum levels of microRNAs 192 and 194 were associated with incident T2DM, independently of fasting glucose, HbA1c and other risk factors. Serum levels of miR-192 and miR-194 were also elevated in diabetic Akt2 knockout mice compared to wild type mice. In conclusion, circulating microRNAs -192 and -194 are potential biomarkers for risk of diabetes.
Project description:Identification of circulating microRNAs for the diagnosis of liver injury and as an indicator of underlying pathology has been the subject of recent investigations. While several studies have been conducted, with particular emphasis on miR-122, the timing of miRNA release into the circulation and anchoring to tissue pathology has not been systematically evaluated. In this study, miRNA profiling was conducted over a time course of hepatobiliary injury and repair using alpha-naphthylisothiocyanate (ANIT) and a proprietary compound, FP004BA. ANIT administration (50?mg/kg) to rats caused significant biliary epithelial cell and hepatocellular necrosis between 24 and 72?h, followed by resolution and progression to biliary hyperplasia by 120?h which was associated with miRNA release into the blood. FP004BA (100?mg/kg) was used to confirm associations of miRNA along a time course with similar hepatic pathology to ANIT. Treatment with ANIT or FP004BA resulted in significant alterations of overlapping miRNAs during the early and peak injury phases. In addition to well-characterized liver injury markers miR-122-5p and miR-192-5p, multiple members of the 200 family and the 101 family along with miR-802-5p and miR-30d-5p were consistently elevated during hepatobiliary injury caused by both toxicants, suggesting that these species may be potential biomarker candidates for hepatobiliary injury. After 14 days of dosing with 4BA, miR-182-5p remained elevated-while miR-122-5p and miR-192-5p had returned to baseline-suggesting that miR-182-5p may have added utility to monitor for hepatobiliary injury in the repair phases when there remains histological evidence of ongoing cellular injury.
Project description:MicroRNAs (miRNAs) have been confirmed to participate in liver fibrosis progression and activation of hepatic stellate cells (HSCs). In this study, the role of miR-193a/b-3p in concanavalin A (ConA)-induced liver fibrosis in mice was evaluated. According to the results, the expression of miR-193a/b-3p was down-regulated in liver tissues after exposure to ConA. Lentivirus-mediated overexpression of miR-193a/b-3p reduced ConA-induced liver injury as demonstrated by decreasing ALT and AST levels. Moreover, ConA-induced liver fibrosis was restrained by the up-regulation of miR-193a/b-3 through inhibiting collagen deposition, decreasing desmin and proliferating cell nuclear antigen (PCNA) expression and lessening the content of hydroxyproline, transforming growth factor-β1 (TGF-β1) and activin A in liver tissues. Furthermore, miR-193a/b-3p mimics suppressed the proliferation of human HSCs LX-2 via inducing the apoptosis of LX-2 cells and lowering the levels of cell cycle-related proteins Cyclin D1, Cyclin E1, p-Rb and CAPRIN1. Finally, TGF-β1 and activin A-mediated activation of LX-2 cells was reversed by miR-193a/b-3p mimics via repressing COL1A1 and α-SMA expression, and restraining the activation of TGF-β/Smad2/3 signalling pathway. CAPRIN1 and TGF-β2 were demonstrated to be the direct target genes of miR-193a/b-3p. We conclude that miR-193a/b-3p overexpression attenuates liver fibrosis through suppressing the proliferation and activation of HSCs. Our data suggest that miR-193a-3p and miR-193b-3p may be new therapeutic targets for liver fibrosis.
Project description:MicroRNAs are small non-coding RNAs with a length of 18-25 nucleotides. They can regulate tumor invasion and metastasis by changing the expression and translation of their target mRNAs. Their expression is substantially altered in colorectal cancer cells as well as in the adjacent tumor-associated stroma. Both of these compartments have a mutual influence on tumor progression. In the development of metastases, cancer cells initially interact with the host tissue. Therefore, compartment-specific expression signatures of these three locations-tumor, associated stroma, and host tissue-can provide new insights into the complex tumor biology of colorectal cancer. Frozen tissue samples of colorectal liver (n = 25) and lung metastases (n = 24) were laser microdissected to separate tumor cells and the adjacent tumor-associated stroma cells. Additionally, normal lung and liver tissue was collected from the same patients. We performed a microarray analysis in four randomly selected liver metastases and four randomly selected lung metastases, analyzing a total of 939 human miRNAs. miRNAs with a significant change >2-fold between the tumor, tumor stroma, and host tissue were analyzed in all samples using RT-qPCR (11 miRNAs) and correlated with the clinical data. We found a differential expression of several miRNAs between the tumor, the tumor-associated stroma, and the host tissue compartment. When comparing liver and lung metastases, miR-194 showed a 1.5-fold; miR-125, miR-127, and miR-192 showed a 2.5-fold; miR-19 and miR-215 a 3-fold; miR-145, miR-199-3, and miR-429 a 5-fold; miR-21 a 7-fold; and, finally, miR-199-5 a 12.5-fold downregulation in liver metastases compared to lung metastases. Furthermore miR-19, miR-125, miR-127, miR-192, miR-194, miR-199-5, and miR-215 showed a significant upregulation in the normal liver tissue compared to the normal lung tissue. Univariate analysis identified an association of poor survival with the expression of miR-125 (p = 0.05), miR-127 (p = 0.001), miR-145 (p = 0.005), miR-192 (p = 0.015), miR-194 (0.003), miR-199-5 (p = 0.008), miR-215 (p < 0.001), and miR-429 (p = 0.03) in the host liver tissue of the liver metastases. Colorectal liver and lung metastases have a unique miRNA expression profile. miRNA expression in the host tissue of colorectal liver metastases seems to be able to influence tumor progression and survival. These findings can be used in the development of tailored therapies.
Project description:Different microRNAs are dysregulated in ovarian cancer where some of them have proved to be valid biomarkers. miRNA profiling analyses have shown that the different histotypes of ovarian carcinoma display differential expression of specific miRNAs. In the present study, we used miRNA-sequencing and Real-Time qPCR to detect the expression levels of miRNAs belonging to the miRNA-192/215 family, namely miR-192, miR-194, and miR-215, in different types of ovarian neoplasia, finding that miR-192, miR-194, and miR-215 were upregulated in ovarian carcinomas of the mucinous subtype, but downregulated in other types of carcinoma and in sex cord-stromal tumors. The expression of the said miRNAs was 6-fold higher in mucinous tumors compared to the other histotypes making them candidates for a possible role as diagnostic biomarkers.
Project description:microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.
Project description:BACKGROUND:MicroRNAs can play an important role in tumorigenesis through post-transcriptional regulation of gene expression, and are not well characterized in follicular lymphoma. DESIGN AND METHODS:MicroRNA profiles of enriched follicular lymphoma tumor cells from 16 patients were generated by assaying 851 human microRNAs. Tandem gene expression profiles were obtained for predicting microRNA targets. RESULTS:The expression of 133 microRNAs was significantly different (> 2-fold; P<0.05) between follicular lymphoma and follicular hyperplasia. Forty-four microRNAs in three groups generated a unique follicular lymphoma signature. Of these, ten microRNAs were increased (miR-193a-5p, -193b*, -345, -513b, -574-3p, -584, -663, -1287, -1295, and -1471), 11 microRNAs were decreased (miR-17*, -30a, -33a, -106a*, -141, -202, -205, -222, -301b, -431*, and -570), and 23 microRNAs formed a group that was increased in most cases of follicular lymphoma but showed lower expression in a subset of cases (let-7a, let-7f, miR-7-1*, -9, -9*, -20a, -20b, -30b, -96, -98, -194, -195, -221*, -374a, -374b, -451, -454, -502-3p, -532-3p, -664*, -1274a, -1274b, and -1260). Higher expression of this last group was associated with improved response to chemotherapy. Gene expression analysis revealed increased expression of MAPK1, AKT1, PRKCE, IL4R and DROSHA and decreased expression of CDKN1A/p21, SOCS2, CHEK1, RAD51, KLF4, BLIMP1 and IRF4 in follicular lymphoma. Functional studies indicated that CDKN1A/p21 and SOCS2 expression is directly regulated by miR-20a/-20b and miR-194, respectively. CONCLUSIONS:Follicular lymphoma is characterized by a unique microRNA signature, containing a subset of microRNAs whose expression correlate with response to chemotherapy. miR-20a/b and miR-194 target CDKN1A and SOCS2 in follicular lymphoma, potentially contributing to tumor cell proliferation and survival.