Recombinant adeno-associated virus 8-mediated inhibition of microRNA let-7a ameliorates sclerosing cholangitis in a clinically relevant mouse model

BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.

Inhibition of GLUD1 mediated by LASP1 and SYVN1 contributes to hepatitis B virus X protein-induced hepatocarcinogenesis

Glutamate dehydrogenase 1(GLUD1)is implicated in oncogenesis.However,little is known about the relationship between GLUD1 and hepatocellular carcinoma(HCC).In the present study,we demonstrated that the expression levels of GLUD1 significantly decreased in tumors,which was relevant to the poor prognosis of HCC.Functionally,GLUD1 silencing enhanced the growth and migration of HCC cells.Mechanistically,the upregulation of interleukin-32 through AKT activation contributes to GLUD1 silencing-facilitated hepatocarcinogenesis.The interaction between GLUD1 and AKT,as well asα-ketoglutarate regulated by GLUD1,can suppress AKT activation.In addition,LIM and SH3 protein 1(LASP1)interacts with GLUD1 and induces GLUD1 degradation via the ubiquitin–proteasome pathway,which relies on the E3 ubiquitin ligase synoviolin(SYVN1),whose interaction with GLUD1 is enhanced by LASP1.In hepatitis B virus(HBV)-related HCC,the HBV X protein(HBX)can suppress GLUD1 with the participation of LASP1 and SYVN1.Collectively,our data suggest that GLUD1 silencing is significantly associated with HCC development,and LASP1 and SYVN1 mediate the inhibition of GLUD1 in HCC,especially in HBV-related tumors.

Deregulation of tumor suppressive ASXL1−PTEN/AKT axis in myeloid malignancies

Mutations of epigenetic regulators are pervasive in human tumors.ASXL1 is frequently mutated in myeloid malignancies.We previously found that ASXL1 forms together with BAP1 a complex that can deubiquitinylate mono-ubiquitinylated lysine 119 on histone H2A(H2AK119ub1),a Polycomb repressive mark.However,a complete mechanistic understanding of ASXL1 in transcriptional regulation and tumor suppression remains to be defined.Here,we find that depletion of Asxl1 confers murine 32D cells to IL3-independent growth at least partly due to sustained activation of PI3K/AKT signaling.Consistently,Asxl1 is critical for the transcriptional activation of Pten,a key negative regulator of AKT activity.Then we confirm that Asxl1 is specifically enriched and required for H2AK119 deubiquitylation at the Pten promoter.Interestingly,ASXL1 and PTEN expression levels are positively correlated in human blood cells and ASXL1 mutations are associated with lower expression levels of PTEN in human myeloid malignancies.Furthermore,malignant cells with ASXL1 downregulation or mutations exhibit higher sensitivity to the AKT inhibitor MK2206.Collectively,this study has linked the PTEN/AKT signaling axis to deregulated epigenetic changes in myeloid malignancies.It also provides a rationale for mechanism-based therapy for patients with ASXL1 mutations.