Project description:In order to identify miRNAs involved in tumor development of DICER1 syndrome model mouse, next-generation sequencing analysis of RNA extracted from liver tissue was performed.
Project description:DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome.
Project description:We developed a genetically engineered conditional compound heterozygous Dicer1 mouse strain that fully recapitulates the bi-allelic mutations of DICER1 in DICER1 syndrome-associated cancers. Embryonic activation of bi-allelic Dicer1 mutations, driven by the anti-Müllerian hormone receptor 2 (Amhr2)-driven Cre strain (Amhr2+/cre), drove cancer development from oviduct. Small RNA sequencing was performed to compare the microRNA expression profiles between tumor and normal oviduct.
Project description:We developed a genetically engineered conditional compound heterozygous Dicer1 mouse strain that fully recapitulates the bi-allelic mutations of DICER1 in DICER1 syndrome-associated cancers. Embryonic activation of bi-allelic Dicer1 mutations, driven by the anti-Müllerian hormone receptor 2 (Amhr2)-driven Cre strain (Amhr2+/cre), drove cancer development from oviduct. mRNA sequencing was performed to compare the mRNA expression profiles between tumor and normal oviduct.
Project description:To confirm the lack of expression of most miRNAs in DICER1 knock-out cell lines, we performed miRNA microarray analysis. The purpose of this study is to classify DICER1-dependent Small RNA and independent Small RNA using DICER1 cells using deficient cell lines, and to identify novel small RNA and small RNA processing mechanisms.
Project description:A new mouse mutant was identified at the Munich ENU mutagenesis project due to hyperactivity, head tossing, and circling behaviour. Neurological and gross morphological phenotyping of these mutant mice revealed impairment of the vestibular system. Using whole genome exome sequencing and a custom-made variant calling pipeline, we identified the causative mutation as an A->T substitution on the chromosome 2 at the position 128 in the exon 6 of jagged 1 (Jag1) gene. This introduces a premature termination codon at the position 883 of the cDNA. In humans, mutations in the JAG1 gene are associated with Alagille syndrome (ALGS1 ), a multisystem developmental disorder mainly affecting small bile ducts in the liver, but also heart, skeleton, and eyes, and occasionally also kidney or inner ears. Further examination of the Jag1K295*/+ mutant mouse line disclosed multiorgan deficiencies, such as cardiac liver congestion, bile duct hypoplasia, mild nephropathy, subvalvular hypertrophy of the right ventricle, and mild growth retardation. No skeletal abnormalities could be detected. In summary, we report a novel mouse model for Alagille syndrome, Jag1K295*/+, which resembles most of the features of the mild form of Alagille syndrome observed in patients. Total RNA obtained from liver of 4 male heterozygous Jag1K295*/+ and 4 male wildtype mice
Project description:Co-chaperone Aha1 activates HSP90 ATPase to promote the folding of client proteins. However, the client proteins of Aha1 are largely unknown. By employing ascorbate peroxidase (APEX) based proximity labeling, we identified 32 proximity proteins of HSP90 that are modulated by genetic depletion of Aha1. Among them, Dicer1 is one of the top-ranked proteins, which were further confirmed by streptavidin pull-down followed by Western blot analysis, demonstrating the reliability of the approach. Flag pull-down result showed interactions between endogenous HSP90 and Dicer1 and Aha1. The Dicer1 level is regulated synergistically by Aha1 and HSP90. Maturation-dependent interaction results showed a preferential binding of Aha1 and HSP90 to nascently translated Dicer1. Reconstitution of Aha1-depleted cells with WT Aha1 restored Dicer1 level, while the HSP90-binding-defective E67K mutant exhibited partial restoration. Moreover, knockdown of Aha1 and inhibition of HSP90 can diminish the levels of mature miRNA, let-7b and mir-30a. Overall, our study uncovers, for the first time, Dicer1 and transporter proteins as clients of Aha1 and HSP90.
Project description:DICER1 plays a critical role in microRNA (miRNA) biogenesis. Recurrent somatic “hotspot” mutations at four mental binding sites within the RNase IIIb domain of DICER1, were identified in ovarian sex cord-stromal tumors and have since been described in other pediatric tumors. In this study, we identified and characterized DICER1 hotspot mutations in endometrial cancers derived from The Cancer Genome Atlas (TCGA) and our local tumor bank. DICER1 hotspot mutations are found in ~2% of endometrial tumors. Using Illumina and Sanger targeted resequencing we observed biallelic DICER1 mutations in more than 50% of cases with hotspot mutations and identified an additional recurrent mutation G1809R in 2 cases. Through small RNA deep sequencing and real-time PCR, we demonstrated mutations that add a positively charged side chain to residue 1809 have similar detrimental effects on 5p miRNA production as mutations at metal binding sites. In one case G1809R was compound heterozygous with a germline S839F mutation, which contributes to loss of DICER1 expression by promoting protein degradation. As expected, 5p miRNAs are globally reduced in tumors and cell lines with hotspot mutations. Pathway analysis of gene expression profiles indicated that genes derepressed due to loss of 5p miRNAs are strongly associated with cell cycle related pathways. Using a Dicer null cell line model, we demonstrated that DICER1 hotspot mutants abolished the inhibitory effects of wildtype DICER1 on cell proliferation upon re-expression. Furthermore, targets of let-7 family miRNAs are enriched among the upregulated genes, suggesting loss of let-7 may be impacting downstream pathways.
Project description:Through whole-exome sequencing we identified somatic missense mutations in DICER1 and DROSHA in Wilms tumor, a childhood kidney cancer. DICER1 and DROSHA are key enzymes in the microRNA biogenesis pathway. To determine the effect of these mutations on microRNA expression, we prepared small RNAs from Wilms tumors and used next-generation sequencing to determine the expression levels of microRNAs in the tumors. Comparison of miRNA expression in tumors with and without mutations in DICER1 or DROSHA.