LncCDCA3L inhibits cell proliferation via a novel RNA structure-based crosstalk with CDCA3 in hepatocellular carcinoma.

BACKGROUND & AIMS: The molecular mechanisms underlying hepatocellular carcinoma (HCC) remain poorly understood. In this study, we investigated cell division cycle-associated 3 (CDCA3) expression status and characterized a CDCA3-related long non-coding RNA (lncRNA) in HCC. METHODS: RT-qPCR and western blot were used to determine CDCA3 expression level in HCC clinical specimens. 5' and 3'-RACE, RNAscope, RNA pull-down, CRISPR/Cas9-based RNA immunoprecipitation (CRIP) and site-directed mutation experiments were used to characterize lncCDCA3L and investigate its function target. Chi-square test and Kaplan-Meier analysis were used to assess lncCDCA3L clinical significance. The effects of lncCDCA3L on HCC development were assessed by overexpression in vitro and in vivo. RESULTS: In this study, we found CDCA3 was a potential oncogenic factor in HCC and characterized the lncCDCA3L, which could inhibit CDCA3. LncCDCA3L is significantly downregulated in HCC and its expression level is associated with tumour size and can act as an independent risk factor affecting postoperative survival time in HCC patients. Mechanistically, lncCDCA3L can repress CDCA3 protein level and inhibit hepatocarcinogenesis by directly binding to CDCA3 mRNA at 1423-1455 region via a novel manner based on a hairpin structure motif. CONCLUSIONS: Our study collectively unveiled the molecular mechanisms of how lncCDCA3L repressed the tumourigenic properties of HCC cells and exhibited a tumour suppressor character in HCC in a CDCA3-dependent manner. The findings here support lncCDCA3L can be used as a candidate prognostic biomarker for HCC patients.

Engineered extracellular vesicles for delivering functional Cas9/gRNA to eliminate hepatitis B virus cccDNA and integration.

The persistence of HBV covalently closed circular DNA (cccDNA) and HBV integration into the host genome in infected hepatocytes pose significant challenges to the cure of chronic HBV infection. Although CRISPR/Cas9-mediated genome editing shows promise for targeted clearance of viral genomes, a safe and efficient delivery method is currently lacking. Here, we developed a novel approach by combining light-induced heterodimerization and protein acylation to enhance the loading efficiency of Cas9 protein into extracellular vesicles (EVs). Moreover, vesicular stomatitis virus-glycoprotein (VSV-G) was incorporated onto the EVs membrane, significantly facilitating the endosomal escape of Cas9 protein and increasing its gene editing activity in recipient cells. Our results demonstrated that engineered EVs containing Cas9/gRNA and VSV-G can effectively reduce viral antigens and cccDNA levels in the HBV-replicating and infected cell models. Notably, we also confirmed the antiviral activity and high safety of the engineered EVs in the HBV-replicating mouse model generated by hydrodynamic injection and the HBV transgenic mouse model. In conclusion, engineered EVs could successfully mediate functional CRISPR/Cas9 delivery both in vitro and in vivo, leading to the clearance of episomal cccDNA and integrated viral DNA fragments, and providing a novel therapeutic approach for curing chronic HBV infection.

Solely HBsAg intrauterine exposure accelerates HBV clearance by promoting HBs-specific immune response in the mouse pups.

Chronic hepatitis B virus (HBV) infection due to perinatal mother-to-infant transmission (MTIT) remains a serious global public health problem. It has been shown that intrauterine exposure to HBV antigens might account for the MTIT-related chronic infection. However, whether hepatitis B surface antigen (HBsAg) intrauterine exposure affected the offspring's immune response against HBV and MTIT of HBV has not been fully clarified. In this study, we investigated the effects and the potential mechanisms of the HBsAg intrauterine exposure on the persistence of HBV replication using a solely HBsAg intrauterine exposure mice model. Our results revealed that solely HBsAg intrauterine exposure significantly accelerated the clearance of HBV when these mice were hydrodynamically injected with pBB4.5-HBV1.2 plasmids after birth, which may be due to the increased number of HBs-specific CD8+ T cells and interferon-gamma secretion in the liver of mice. Mechanismly, HBsAg intrauterine exposure activated antigen-presenting dendritic cells, which led to the generation of antigen-specific cellular immunological memory. Our data provide an important experimental evidence for the activation of neonatal immune response by HBsAg intrauterine exposure.

Yin-Yang 1 and HBx protein activate HBV transcription by mediating the spatial interaction of cccDNA minichromosome with cellular chromosome 19p13.11.

HBV cccDNA stably exists in the nuclei of infected cells as an episomal munichromosome which is responsible for viral persistence and failure of current antiviral treatments. However, the regulatory mechanism of cccDNA transcription by viral and host cellular factors is not well understood. In this study, we investigated whether cccDNA could be recruited into a specific region of the nucleus via specific interaction with a cellular chromatin to regulate its transcription activity. To investigate this hypothesis, we used chromosome conformation capture (3C) technology to search for the potential interaction of cccDNA and cellular chromatin through rcccDNA transfection in hepatoma cells and found that cccDNA is specifically associated with human chromosome 19p13.11 region, which contains a highly active enhancer element. We also confirmed that cellular transcription factor Yin-Yang 1 (YY1) and viral protein HBx mediated the spatial regulation of HBV cccDNA transcription by 19p13.11 enhancer. Thus, These findings indicate that YY1 and HBx mediate the recruitment of HBV cccDNA minichromosomes to 19p13.11 region for transcription activation, and YY1 may present as a novel therapeutic target against HBV infection.