It is well accepted that HBx plays the major role in hepatocarcinogenesis associated with hepatitis B virus (HBV) infections. However, little was known about its role in regulating long noncoding RNAs (lncRNAs), a large group of transcripts regulating a variety of biological processes including carcinogenesis in mammalian cells. Here we report that HBx upregulates UCA1 genes and downregulates p27 genes in hepatic LO2 cells. Further studies show that the upregulated UCA1 promotes cell growth by facilitating G1/S transition through CDK2 in both hepatic and hepatoma cells. Knock down of UCA1 in HBx-expressing hepatic and hepatoma cells resulted in markedly increased apoptotic cells by elevating the cleaved caspase-3 and caspase-8. More importantly, UCA1 is found to be physically associated with enhancer of zeste homolog 2 (EZH2), which suppresses p27Kip1 through histone methylation (H3K27me3) on p27Kip1 promoter. We also show that knockdown of UCA1 in hepatoma cells inhibits tumorigenesis in nude mice. In a clinic study, UCA1 is found to be frequently up-regulated in HBx positive group tissues in comparison with the HBx negative group, and exhibits an inverse correlation between UCA1 and p27Kip1 levels. Our findings demonstrate an important mechanism of hepatocarcinogenesis through the signaling of HBx-UCA1/EZH2-p27Kip1 axis, and a potential target of HCC.

Rationale: Bovine milk constitutes an essential part of human diet, especially for children, due to its enrichment of various nutrients. We recently developed an effective protocol for the isolation of extracellular vesicles from milk (mEVs) and discovered that mEVs contained large amounts of immune-active proteins and modulated the gut immunity and microbiota in healthy mice. Here, we aimed to explore the therapeutic effects of mEVs on inflammatory bowel disease. Methods: MicroRNAs and protein content in mEVs were analyzed by RNA sequencing and proteomics, respectively, followed by functional annotation. Ulcerative colitis (UC) was induced by feeding mice with dextran sulfate sodium. Intestinal immune cell populations were phenotyped by flow cytometry, and the gut microbiota was analyzed via 16S rRNA sequencing. Results: We showed that abundant proteins and microRNAs in mEVs were involved in the regulation of immune and inflammatory pathways and that oral administration of mEVs prevented colon shortening, reduced intestinal epithelium disruption, inhibited infiltration of inflammatory cells and tissue fibrosis in a mouse UC model. Mechanistically, mEVs attenuated inflammatory response via inhibiting TLR4-NF-κB signaling pathway and NLRP3 inflammasome activation. Furthermore, mEVs were able to correct cytokine production disorder and restore the balance between T helper type 17 (Th17) cells and interleukin-10 + Foxp3 + regulatory T (Treg) cells in the inflamed colon. The disturbed gut microbiota in UC was also partially recovered upon treatment with mEVs. The correlation between the gut microbiota and cytokines suggests that mEVs may modulate intestinal immunity via influencing the gut microbiota. Conclusions: These findings reveal that mEVs alleviate colitis by regulating intestinal immune homeostasis via inhibiting TLR4-NF-κB and NLRP3 signaling pathways, restoring Treg/Th17 cell balance, and reshaping the gut microbiota.

Probiotics plays an important role in regulating gut microbiota and maintaining intestinal homeostasis. Extracellular vesicles (EVs) derived from probiotics have emerged as potential mediators of host immune response and anti-inflammatory effect. However, the anti-inflammatory effect and mechanism of probiotics derived EVs on inflammatory bowel disease (IBD) remains unclear. In this study, the effect of Lactobacillus plantarum Q7-derived extracellular vesicles (Q7-EVs) on gut microbiota and intestinal inflammation was investigated in C57BL/6J mice. The results showed that Q7-EVs alleviated DSS-induced colitis symptoms, including colon shortening, bleeding, and body weight loss. Consumption of Q7-EVs reduced the degree of histological damage. DSS-upregulated proinflammatory cytokine levels including IL-6, IL-1β, IL-2 and TNF-α were reduced significantly by Q7-EVs (p < 0.05). 16S rRNA sequencing results showed that Q7-EVs improved the dysregulation of gut microbiota and promoted the diversity of gut microbiota. It was observed that the pro-inflammatory bacteria (Proteobacteria) were reduced and the anti-inflammatory bacteria (Bifidobacteria and Muribaculaceae) were increased. These findings indicated that Q7-EVs might alleviate DSS-induced ulcerative colitis by regulating the gut microbiota.

The de-regulation of the miR-29 family and DNA methyltransferase 3A (DNMT3A) is associated with gastric cancer (GC). While increasing evidence indicates miR-29b/c could regulate DNA methylation by targeting DNMT3A, it is currently unknown if epigenetic silencing of miR-29b/c via promoter hypermethylation in GC is caused by abnormal expression of DNMT3A. Thus, we aimed to evaluate whether cross-talk regulation exists between miR-29b/c and DNMT3A and whether it is associated with a malignant phenotype in GC. First, wound healing and Transwell assays revealed that miR-29b/c suppresses tumor metastasis in GC. A luciferase reporter assay demonstrated that DNMT3A is a direct target of miR-29b/c. We used bisulfite genomic sequencing to analyze the DNA methylation status of miR-29b/c. The percentage of methylated CpGs was significantly decreased in DNMT3A-depleted cells compared to the controls. Furthermore, the involvement of DNMT3A in promoting GC cell migration was associated with the promoter methylation-mediated repression of CDH1. In 50 paired clinical GC tissue specimens, decreased miR-29b/c was significantly correlated with the degree of differentiation and invasion of the cells and was negatively correlated with DNMT3A expression. Together, our preliminary results suggest that the following process may be involved in GC tumorigenesis. miR-29b/c suppresses the downstream gene DNMT3A, and in turn, miR-29b/c is suppressed by DNMT3A in a DNA methylation-dependent manner. The de-regulation of both of miR-29b/c and DNMT3A leads to the epigenetic silencing of CDH1 and contributes to the metastasis phenotype in GC. This finding reveals that DNA methylation-associated silencing of miR-29b/c is critical for GC development and thus may be a therapeutic target.

Increasing studies showed that long-noncoding RNAs (lncRNAs) play important roles in the biological processes, including cancer initiation and progression. However, little is known about the exact role and regulation mechanism of lncRNA UCA1 during the progression of gastric cancer (GC). In this study, we found that UCA1 was aberrantly elevated in gastric cancer tissues, and was significantly associated with lymph node metastasis and TNM stage. In vivo and in vitro, enforced UCA1 level promoted cell migration and invasion of GC cell. Depleted UCA1 expression level attenuated the ability of cell migration and invasion in GC. And then, we detected that expression level of ZEB2, a transcription factor related to tumor metastasis, was regulated by UCA1 in GC cells. miR-203 targets and suppresses to ZEB2 expression. Furthermore, we found that UCA1 could directly interact with miR-203 and lead to the release of miR-203-targeted transcripts ZEB2. Herein, we revealed the novel mechanism of UCA1 on regulating metastasis-related gene by sponge regulatory axis during GC metastasis. Our findings indicated that UCA1 plays a critical role in metastatic GC by mediating sponge regulatory axis miR-203/ZEB2. To explore function of UCA1-miR-203-ZEB2 axis may provide an informative biomarker of malignancy and a highly selective anti-GC therapeutic target.