[Construction of a mouse model of cblC type methylmalonic acidemia with W203X mutation based on the CRISPR/Cas9 technology].

OBJECTIVE: To construct a W203X-mutant mouse model of cblC type methylmalonic acidemia based on the CRISPR/Cas9 technology. METHODS: At first, BLAST was used to compare the conservative nature of the cblC gene and protein sequences in humans and mice, and then, the CRISPR/Cas9 technology was used for microinjection of mouse fertilized eggs to obtain heterozygous F1 mice. Hybridization was performed for these mice to obtain homozygous W203X-mutant mice. The blood level of the metabolite propionyl carnitine (C3) was measured for homozygous mutant mice, heterozygous littermates, and wild-type mice. RESULTS: The gene and protein sequences of MMACHC, the pathogenic gene for cblC type methylmalonic acidemia, were highly conserved in humans and mice. The homozygous W203X-mutant mice were successfully obtained by the CRISPR/Cas9 technology, and there was a significant increase in C3 in these mice at 24 hours after birth (P<0.001). CONCLUSIONS: A W203X-mutant mouse model of cblC type methylmalonic acidemia is successfully constructed by the CRISPR/Cas9 technology.

CRISPR/Cas9 Technology Targeting Fas Gene Protects Mice From Concanavalin-A Induced Fulminant Hepatic Failure.

Fulminant hepatic failure is a life-threatening disease which occurs in patients without preexisting liver disease. Nowadays, there is no ideal therapeutic tool in the treatment of fulminant hepatic failure. Recent studies suggested that a novel technology termed CRISPR/Cas9 may be a promising approach for the treatment of fulminant hepatic failure. In this project, we have designed single chimeric guide RNAs specifically targeting the genomic regions of mouse Fas gene. The in vitro and in vivo effects of sgRNAs on the production of Fas protein were examined in cultured mouse cells and in a hydrodynamic injection-based mouse model, respectively. The in vivo delivery of CRISPR/Cas9 could maintain liver homeostasis and protect hepatocytes from Fas-mediated cell apoptosis in the fulminant hepatic failure model. Our study indicates the clinical potential of developing the CRISPR/Cas9 system as a novel therapeutic strategy to rescue Concanavalin-A-induced fulminant hepatic failure in the mouse model. J. Cell. Biochem. 118: 530-536, 2017. © 2016 Wiley Periodicals, Inc.

MiR-25 suppresses 3T3-L1 adipogenesis by directly targeting KLF4 and C/EBPα.

In the past decade, miRNA emerges as a vital player in orchestrating gene regulation and maintaining cellular homeostasis. It is well documented that miRNA influences a variety of biological events, including embryogenesis, cell fate decision, and cellular differentiation. Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes. It has been shown that adipogenesis is tightly modulated by a number of transcription factors such as PPARγ, KLF4, and C/EBPα. However, the molecular mechanisms underlying the missing link between miRNA and adipogenesis-related transcription factors remain elusive. In this study, we unveiled that miR-25, a member of miR-106b-25 cluster, was remarkably downregulated during 3T3-L1 adipogenesis. Restored expression of miR-25 significantly impaired 3T3-L1 adipogenesis and downregulated the expression of serial adipogenesis-related genes. Further experiments presented that ectopic expression of miR-25 did not affect cell proliferation and cell cycle progression. Finally, KLF4 and C/EBPα, two key regulators of adipocyte differentiation, were experimentally identified as bona fide targets for miR-25. These data indicate that miR-25 is a novel negative regulator of adipocyte differentiation and it suppressed 3T3-L1 adipogenesis by targeting KLF4 and C/EBPα, which provides novel insights into the molecular mechanism of miRNA-mediated cellular differentiation.

Translation of the circular RNA circß-catenin promotes liver cancer cell growth through activation of the Wnt pathway.

BACKGROUND: Circular RNAs are a class of regulatory RNA transcripts, which are ubiquitously expressed in eukaryotes. In the current study, we evaluate the function of a novel circRNA derived from the ß-catenin gene locus, circß-catenin. RESULTS: Circß-catenin is predominantly localized in the cytoplasm and displays resistance to RNase-R treatment. We find that circß-catenin is highly expressed in liver cancer tissues when compared to adjacent normal tissues. Silencing of circß-catenin significantly suppresses malignant phenotypes in vitro and in vivo, and knockdown of this circRNA reduces the protein level of ß-catenin without affecting its mRNA level. We show that circß-catenin affects a wide spectrum of Wnt pathway-related genes, and furthermore, circß-catenin produces a novel 370-amino acid ß-catenin isoform that uses the start codon as the linear ß-catenin mRNA transcript and translation is terminated at a new stop codon created by circularization. We find that this novel isoform can stabilize full-length ß-catenin by antagonizing GSK3ß-induced ß-catenin phosphorylation and degradation, leading to activation of the Wnt pathway. CONCLUSIONS: Our findings illustrate a non-canonical function of circRNA in modulating liver cancer cell growth through the Wnt pathway, which can provide novel mechanistic insights into the underlying mechanisms of hepatocellular carcinoma.