Deubiquitinating enzyme Josephin-2 stabilizes PHGDH to promote a cancer stem cell phenotype in hepatocellular carcinoma.

BACKGROUND: Deubiquitinating enzymes (DUBs) have been shown to be possible targets for hepatocellular carcinoma (HCC) treatment. OBJECTIVE: This study was designed to reveal the effect and underlying mechanism of Josephin-2, a relatively newly defined DUB, in HCC progression. METHODS: SNU-387 and PLC/PRF/5 cells were used for in vitro functional assays. The levels of Josephin-2 and phosphoglycerate dehydrogenase (PHGDH) were determined using RT-qPCR and western blotting. Cell proliferation, migration and invasion were assessed by CCK-8, colony formation and Transwell. Spheroid-forming assay was performed to assess the cancer stem cell (CSC)-phenotype of HCC cells. A xenograft mice model was applied to verify the effect of Josephin-2 on HCC cell growth in vivo. RESULTS: Herein, we showed that Josephin-2 expression was negatively correlated with HCC patient survival in data from the online database. Cell experiments indicated that knockdown of Josephin-2 attenuated HCC cell malignant biological behaviors. Besides, Josephin-2 silencing also decreased the spheroid-formation while inhibited the expression of CSC biomarkers (CD133, OCT4, SOX2 and EpCAM) in HCC cells. Mechanistically, Josephin-2 had a deubiquitinating activity towards the regulation of PHGDH protein, the rate-limiting enzyme in the first step of serine biosynthesis pathway. Depletion of Josephin-2 enhanced the ubiquitination degradation of PHGDH and ultimately inhibited the proliferation and CSC-phenotype of HCC in vitro and in vivo. CONCLUSION: Our work uncovered the regulatory effects of Josephin-2 on PHGDH protein stability and profiled its contribution in HCC malignant progression, which might provide a potential therapeutic target for HCC.

Coordinated regulation of IFITM1, 2 and 3 genes by an IFN-responsive enhancer through long-range chromatin interactions.

Interferon-induced transmembrane protein (IFITM) 1, 2 and 3 genes encode a family of interferon (IFN)-induced transmembrane proteins that block entry of a broad spectrum of pathogens. However, the transcriptional regulation of these genes, especially whether there exist any enhancers and their roles during the IFN induction process remain elusive. Here, through public data mining, episomal luciferase reporter assay and in vivo CRISPR-Cas9 genome editing, we identified an IFN-responsive enhancer located 35kb upstream of IFITM3 gene promoter upregulating the IFN-induced expression of IFITM1, 2 and 3 genes. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA) and luciferase reporter assay demonstrated that signal transducers and activators of transcription (STAT) 1 bound to the enhancer with the treatment of IFN and was indispensable for the enhancer activity. Furthermore, using chromosome conformation capture technique, we revealed that the IFITM1, 2 and 3 genes physically clustered together and constitutively looped to the distal enhancer through long-range interactions in both HEK293 and A549 cells, providing structural basis for coordinated regulation of IFITM1, 2 and 3 by the enhancer. Finally, we showed that in vivo truncation of the enhancer impaired IFN-induced resistance to influenza A virus (IAV) infection. These findings expand our understanding of the mechanisms underlying the transcriptional regulation of IFITM1, 2 and 3 expression and its ability to mediate IFN signaling.

Construction of CTCF degradation cell line by CRISPR/Cas9 mediated genome editing.

The CCCTC-binding factor (CTCF) is the main insulator protein described in vertebrates. It plays fundamental roles during diverse cellular processes. CTCF gene knockout mice led to death during embryonic development. To further explore the functions of CTCF, we employed a CRISPR/Cas9-based genome engineering strategy to in-frame insert the mitosis-special degradation domain (MD) of cyclin B into the upstream open reading frame of CTCF gene. Fusion protein is designed to degrade during mitosis leaded by MD. As a control group, mutation of a single arginine (R42A) within the destruction box inactivates the MD leading to constitutive expression of MD*-CTCF. The homozygous clones were obtained via the screening by puromycin when coexpressed with puromycin resistence gene. The protein level of CTCF in MD-CTCF cell line was about 10% of wild-type cells throughout cell cycles by the analyses of Western blotting and immunofluorescence. There was no significant difference between MD*-CTCF cell line and wild type. Flow cytometry results showed prolonged G1 phase in MD-CTCF cell line. Taken together, we demonstrated the feasibility of efficiently inserting MD domain into genome with the CRISPR/Cas9 technology and reported the first CTCF-specific degradation human cell line.

Involvement of aberrant miR-139/Jun feedback loop in human gastric cancer.

Accumulating evidence indicates that some miRNAs could form feedback loops with their targets to fine-tune tissue homeostasis, while disruption of these loops constitutes an essential step towards human tumorigenesis. In this study, we report the identification of a novel negative feedback loop formed between miR-139 and its oncogenic target Jun. In this loop, miR-139 could inhibit Jun expression by targeting a conserved site on its 3'-UTR, whereas Jun could induce miR-139 expression in a dose dependent manner through a distant upstream regulatory element. Interestingly, aberration in this loop was found in human gastric cancer, where miR-139 was down-regulated and inversely correlated with Jun expression. Further functional analysis showed that restored expression of miR-139 in gastric cancer cells significantly induces apoptosis, and inhibits cell migration and proliferation as well as tumour growth through targeting Jun. Thus, our data strongly suggests a role of aberrant miR-139/Jun negative feedback loop in the development of human gastric cancer and miR-139 as a potential therapeutic target for gastric cancer. Given that miR-139 and Jun are deregulated in many cancers, our findings here might have broader implication in other types of human cancers.

Functional screening for miRNAs targeting Smad4 identified miR-199a as a negative regulator of TGF-ß signalling pathway.

The transforming growth factor-ß (TGF-ß) signalling pathway participates in various biological processes. Dysregulation of Smad4, a central cellular transducer of TGF-ß signalling, is implicated in a wide range of human diseases and developmental disorders. However, the mechanisms underlying Smad4 dysregulation are not fully understood. Using a functional screening approach based on luciferase reporter assays, we identified 39 microRNAs (miRNAs) as potential regulators of Smad4 from an expression library of 388 human miRNAs. The screening was supported by bioinformatic analysis, as 24 of 39 identified miRNAs were also predicted to target Smad4. MiR-199a, one of the identified miRNAs, was inversely correlated with Smad4 expression in various human cancer cell lines and gastric cancer tissues, and repressed Smad4 expression and blocked canonical TGF-ß transcriptional responses in cell lines. These effects were dependent on the presence of a conserved, but not perfect seed paired, miR-199a-binding site in the Smad4 3'-untranslated region (UTR). Overexpression of miR-199a significantly inhibited the ability of TGF-ß to induce gastric cancer cell growth arrest and apoptosis in vitro, and promoted anchorage-independent growth in soft agar, suggesting that miR-199a plays an oncogenic role in human gastric tumourigenesis. In conclusion, our functional screening uncovers multiple miRNAs that regulate the cellular responsiveness to TGF-ß signalling and reveals important roles of miR-199a in gastric cancer by directly targeting Smad4.