Project description:Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then entailed to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the Resistant-Hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin-19, indicating that several HCC-associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene-target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the NRF2 pathway and up-regulation of the miR-200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR-200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3-induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin-19 positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated to human HCC as well underlines the translational value of our results. Conclusions: this study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target. 20 nodules (10 weeks after initiation with DENA), 4 adenomas (10 months), 5 eHCCs (10 months) and 9 aHCCs (14 months) were dissected. 10 controls also included.
Project description:Studies on gene and/or microRNA (miRNA) dysregulation in the early stages of hepatocarcinogenesis are hampered by the difficulty of diagnosing early lesions in humans. Experimental models recapitulating human hepatocellular carcinoma (HCC) are then entailed to perform this analysis. We performed miRNA and gene expression profiling to characterize the molecular events involved in the multistep process of hepatocarcinogenesis in the Resistant-Hepatocyte rat model. A high percentage of dysregulated miRNAs/genes in HCC were similarly altered in early preneoplastic lesions positive for the stem/progenitor cell marker cytokeratin-19, indicating that several HCC-associated alterations occur from the very beginning of the carcinogenic process. Our analysis also identified miRNA/gene-target networks aberrantly activated at the initial stage of hepatocarcinogenesis. Activation of the NRF2 pathway and up-regulation of the miR-200 family were among the most prominent changes. The relevance of these alterations in the development of HCC was confirmed by the observation that NRF2 silencing impaired while miR-200a overexpression promoted HCC cell proliferation in vitro. Moreover, T3-induced in vivo inhibition of the NRF2 pathway accompanied the regression of cytokeratin-19 positive nodules, suggesting that activation of this transcription factor contributes to the onset and progression of preneoplastic lesions towards malignancy. The finding that 78% of genes and 57% of dysregulated miRNAs in rat HCC have been previously associated to human HCC as well underlines the translational value of our results. Conclusions: this study indicates that most of the molecular changes found in HCC occur in the very early stages of hepatocarcinogenesis. Among these, the NRF2 pathway plays a relevant role and may represent a new therapeutic target.
Project description:Using RNA-seq analysis, we study a DEN-induced HCC rat model during fibrosis progression and HCC development with special focus on liver inflammatory microenvironment. RNA-seq results show that DEN-induced liver tumors in rat model share remarkable molecular characteristics with human HCC, especially with HCC associated with high proliferation. In conclusion, our study provides detailed insight into the hepatocarcinogenesis in a commonly used model of HCC, facilitating the future use of this model for preclinical testing.
Project description:expression profile of conditional knock out of beta-catenin by K19-CRE at E7.5. Tested a wild type with two alleles of beta-catenin, a heterzyote with one deleted allele and the conditional null in the domain on cytokeratin 19 driven CRE expression Keywords: other
Project description:expression profile of conditional knock out of beta-catenin by K19-CRE at E7.5. Tested a wild type with two alleles of beta-catenin, a heterzyote with one deleted allele and the conditional null in the domain on cytokeratin 19 driven CRE expression
Project description:Microarray analysis is a useful methodology to identify target genes modulated by anticancer drugs. Here, celecoxib effect on gene expression profiles was evaluated in the modified resistant hepatocyte model. Animals subjected to carcinogenic treatment were fed with diet containing 1500 ppm of celecoxib. Two schemes of celecoxib administration were designed. In the progression protocol, celecoxib was administrated between days 18 and 25 post-cancer initiation, a total of 8 celecoxib treatment days, when well established preneoplastic lesions starts to appear. In the initiation protocol, celecoxib was administrated from one week before until 25 days after of cancer initiation, a total of 32 celecoxib treatment days. A rat group was subjected only to the carcinogenic treatment as cancer positive control. Gene expression profiles of all groups were compared to a negative untreated control. The evaluation of gene expression profiles permitted us to identify new target genes that are modulated by celecoxib treatment. A possible mechanism of celecoxib chemoprevention of hepatocarcinogenesis is proposed. 9 weeks old Sprague Dawley rats, 4 rats per group, were subjected to Semple-Roberts' modified hepatocarcinogenesis protocol and sacrificed 25 days post-initiation. In celecoxib-treated groups, celecoxib was administrated mixed in diet at 1500 ppm. Negative and positive cancer progression controls were included to compare gene expression profiles. Microarray analysis was performed on liver samples, one replicate per rat, using dye swap.
Project description:Predisposition to hepatocarcinogenesis is under polygenic control. We analyzed gene expression patterns of dysplastic liver nodules (DN) and hepatocellular carcinoma (HCC) chemically-induced in F344 and BN rats, respectively susceptible and resistant to hepatocarcinogenesis, to evaluate effector mechanisms of predisposition genes, and commonalties between transcription signatures of rat and human liver lesions. Gene expression profiles were determined by microarray analysis and validated by quantitative RT-PCR and Western blot. Cluster analysis revealed two distinctive gene expression patterns, the first of which included normal liver of F344 and BN rats and BN rat nodules, and the second one F344 nodules and HCC of both strains. Expression of 91 and 55 genes of DN and HCC, respectively, showed significant interstrain differences. Considering genes mostly associated with cell proliferation we identified a signature predicting DN evolution to HCC. Notably, expression of oncosuppressors Csmd1, Dmbt, Dusp1, and Gnmt ,in DN, and Bhmt, Dmbt1, Dusp1, Gadd45g, Gnmt, Napsa, Pp2ca, and Ptpn13, in HCC, was higher in BN than F344 rats. Comparative genomics approach, using gene expression data from rat lesions, and human HCC with different prognosis (based on survival length), revealed expression patterns of BN and F344 lesions close to those of human HCCs with better and poorer prognosis, respectively, suggesting that similar molecular events contribute to create a phenotype prone to progression in rats and human lesions. In conclusion, we identified a molecular signature of DN prone to progress to HCC and suggest that oncosuppressor genes are determinants of the genetically transmitted resistance to hepatocarcinogenesis.
Project description:Many clinical risk factors for severe COVID-19, such as diabetes, hypertension, and high body mass index have been reported. However, searching for additional risk factors should be continued to predict the progression of severe COVID-19 more accurately. We suppose that clonal hematopoiesis of indeterminate potential (CHIP) can also be regarded as one of risk factors. To identify the influence of CHIP in COVID-19 pathogenesis, we performed single-cell RNA-seq using peripheral blood mononuclear cells (PBMCs) obtained from severe COVID-19 patient with CHIP and integrate the data with other published COVID-19 scRNA seq data (GSE149689). After clustering and annotating cell types, we compare the expression profiles between CHIP vs non-CHIP COVID-19 severe patient.