Phenformin activates ER stress to promote autophagic cell death via NIBAN1 and DDIT4 in oral squamous cell carcinoma independent of AMPK.

The efficient clinical treatment of oral squamous cell carcinoma (OSCC) is still a challenge that demands the development of effective new drugs. Phenformin has been shown to produce more potent anti-tumor activities than metformin on different tumors, however, not much is known about the influence of phenformin on OSCC cells. We found that phenformin suppresses OSCC cell proliferation, and promotes OSCC cell autophagy and apoptosis to significantly inhibit OSCC cell growth both in vivo and in vitro. RNA-seq analysis revealed that autophagy pathways were the main targets of phenformin and identified two new targets DDIT4 (DNA damage inducible transcript 4) and NIBAN1 (niban apoptosis regulator 1). We found that phenformin significantly induces the expression of both DDIT4 and NIBAN1 to promote OSCC autophagy. Further, the enhanced expression of DDIT4 and NIBAN1 elicited by phenformin was not blocked by the knockdown of AMPK but was suppressed by the knockdown of transcription factor ATF4 (activation transcription factor 4), which was induced by phenformin treatment in OSCC cells. Mechanistically, these results revealed that phenformin triggers endoplasmic reticulum (ER) stress to activate PERK (protein kinase R-like ER kinase), which phosphorylates the transitional initial factor eIF2, and the increased phosphorylation of eIF2 leads to the increased translation of ATF4. In summary, we discovered that phenformin induces its new targets DDIT4 and especially NIBAN1 to promote autophagic and apoptotic cell death to suppress OSCC cell growth. Our study supports the potential clinical utility of phenformin for OSCC treatment in the future.

Periodontal ligament-associated protein-1 promotes osteoclastogenesis in mice by modulating TGF-ß1/Smad1 pathway.

BACKGROUND: Periodontal ligament-associated protein-1 (PLAP-1), an important target molecule of osteoarthritis research, may affect alveolar bone resorption. The aim of our study was to comprehensively and systematically detect the effect of PLAP-1 on alveolar bone resorption and the underlying mechanism in PLAP-1 knockout mouse models. METHODS: We used a PLAP-1 knockout (C57BL/6N-Plap-1-/- ) mouse model to investigate the effect of PLAP-1 on osteoclast differentiation and the underlying mechanism by adding Porphyromonas gingivalis lipopolysaccharide to stimulate bone marrow-derived macrophages. The effect of PLAP-1 on alveolar bone resorption and the underlying mechanism were studied using a ligature periodontitis model, with microcomputed tomography imaging, immunochemistry, and immunofluorescence. RESULTS: The in vitro analysis results demonstrated that PLAP-1 knockout significantly inhibited osteoclast differentiation under both normal and inflammatory conditions. Bioinformatic analysis, immunofluorescence, and co-immunoprecipitation showed colocalization and interaction between PLAP-1 and transforming growth factor beta 1 (TGF-ß1). The phosphorylation of Smad1 was reduced in the PLAP-1 knockout cells compared with that in the cells from wild-type mice. The in vivo analysis results demonstrated that PLAP-1 knockout decreased bone resorption and the levels of osteoclast differentiation markers in experimental periodontitis compared with those in wild-type mice. Immunofluorescence staining confirmed colocalization of PLAP-1 and TGF-ß1 in the experimental periodontitis model. The phosphorylation level of Smad1 was significantly reduced in PLAP-1 knockout mice compared with that in wild-type mice. CONCLUSIONS: This study revealed that the knockout of PLAP-1 inhibits osteoclast differentiation and decreases alveolar bone resorption through the TGF-ß1/Smad1 signaling pathway, which could serve as an innovative target for the prevention and treatment of periodontitis.

Phenformin suppresses angiogenesis through the regulation of exosomal microRNA-1246 and microRNA-205 levels derived from oral squamous cell carcinoma cells.

Exosomes secreted by cancer cells are important components in the tumor microenvironment, enabling cancer cells to communicate with each other and with noncancerous cells to play important roles in tumor progression and metastasis. Phenformin, a biguanide antidiabetic drug, has been reported to have a strong antitumor function in multiple types of cancer cells, however little research has been reported about whether phenformin can regulate the secretion of exosomes by cancer cells to regulate the tumor microenvironment and contribute to its antitumor function. Here we found that exosomes (Phen-Exo) derived from phenformin-treated oral squamous cell carcinoma (OSCC) cells significantly suppress the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. The inhibition of angiogenesis by Phen-Exo was verified in vivo by matrigel plug angiogenesis assays and by chick chorioallantoic membrane assays. Mechanistically, we discovered that the expression of microRNA-1246 (miR-1246) and microRNA-205 (miR-205) was significantly increased in exosomes secreted by OSCC cells treated with phenformin, while high expression levels of miR-1246 or miR-205 in vascular endothelial cells inhibited their angiogenic effects and decreased expression of the angiogenic factor VEGFA. In conclusion, these results reveal that phenformin can inhibit angiogenesis by regulating the levels of miR-1246 and miR-205 in exosomes secreted by OSCC cells, suggesting that phenformin has the potential to alter the tumor microenvironment to antagonize the growth of OSCCs, which provides a theoretical basis for developing new strategies to treat OSCCs in the future.

CUL4B Upregulates RUNX2 to Promote the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Epigenetically Repressing the Expression of miR-320c and miR-372/373-3p.

Mesenchymal stem cells (MSCs) within the periodontal ligament (PDL), termed periodontal ligament stem cells (PDLSCs), have a self-renewing capability and a multidirectional differentiation potential. The molecular mechanisms that regulate multidirectional differentiation, such as the osteogenic differentiation of PDLSCs, remain to be elucidated. Cullin 4B (CUL4B), which assembles the CUL4B-RING ubiquitin ligase (CRL4B) complex, is involved in regulating a variety of developmental and physiological processes including the skeletal development and stemness of cancer stem cells. However, nothing is known about the possible role of CUL4B in the osteogenic differentiation of PDLSCs. Here, we found that knockdown of CUL4B decreased the proliferation, migration, stemness and osteogenic differentiation ability of PDLSCs. Mechanistically, we demonstrate that CUL4B cooperates with the PRC2 complex to repress the expression of miR-320c and miR-372/373-3p, which results in the upregulation of RUNX2, a master transcription factor (TF) that regulates osteogenic differentiation. In brief, the present study reveals the role of CUL4B as a new regulator of osteogenic differentiation in PDLSCs.

Phosphoinositide-Binding Protein TIPE1 Promotes Alternative Activation of Macrophages and Tumor Progression via PIP3/Akt/TGFß Axis.

Macrophages perform key and distinct functions in maintaining tissue homeostasis by finely tuning their activation state. Within the tumor microenvironment, macrophages are reshaped to drive tumor progression. Here we report that tumor necrosis factor α-induced protein 8-like 1 (TIPE1) is highly expressed in macrophages and that depletion of TIPE1 impedes alternative activation of macrophages. TIPE1 enhanced activation of the PI3K/Akt pathway in macrophages by directly binding with and regulating the metabolism of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Accordingly, inhibition of the PI3K/Akt pathway significantly attenuated the effect of TIPE1 on macrophage alternative activation. Tumor-associated macrophages (TAM) in human liver cancer and melanoma tissues showed significantly upregulated TIPE1 expression that negatively correlated with patient survival. In vitro and in vivo, TIPE1 knockdown in macrophages retarded the growth and metastasis of liver cancer and melanoma. Furthermore, blockade or depletion of TGFß signaling in macrophages abrogated the effects of TIPE1 on tumor cell growth and migration. Together, these results highlight that the phosphoinositide-related signaling pathway is involved in reprogramming TAMs to optimize the microenvironment for cancer progression. SIGNIFICANCE: This work provides insight into the fine tuning of macrophage polarization and identifies a potential target for macrophage-based antitumor therapy.

Hepatic Macrophage as a Key Player in Fatty Liver Disease.

Fatty liver disease, characterized by excessive inflammation and lipid deposition, is becoming one of the most prevalent liver metabolic diseases worldwide owing to the increasing global incidence of obesity. However, the underlying mechanisms of fatty liver disease are poorly understood. Accumulating evidence suggests that hepatic macrophages, specifically Kupffer cells (KCs), act as key players in the progression of fatty liver disease. Thus, it is essential to examine the current evidence of the roles of hepatic macrophages (both KCs and monocyte-derived macrophages). In this review, we primarily address the heterogeneities and multiple patterns of hepatic macrophages participating in the pathogenesis of fatty liver disease, including Toll-like receptors (TLRs), NLRP3 inflammasome, lipotoxicity, glucotoxicity, metabolic reprogramming, interaction with surrounding cells in the liver, and iron poisoning. A better understanding of the diverse roles of hepatic macrophages in the development of fatty liver disease may provide a more specific and promising macrophage-targeting therapeutic strategy for inflammatory liver diseases.

Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes.

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-Ras G12V ) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-Ras G12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-Ras G12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-Ras G12V , which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

ATF3 downmodulates its new targets IFI6 and IFI27 to suppress the growth and migration of tongue squamous cell carcinoma cells.

Activating transcription factor 3 (ATF3) is a key transcription factor involved in regulating cellular stress responses, with different expression levels and functions in different tissues. ATF3 has also been shown to play crucial roles in regulating tumor development and progression, however its potential role in oral squamous cell carcinomas has not been fully explored. In this study, we examined biopsies of tongue squamous cell carcinomas (TSCCs) and found that the nuclear expression level of ATF3 correlated negatively with the differentiation status of TSCCs, which was validated by analysis of the ATGC database. By using gain- or loss- of function analyses of ATF3 in four different TSCC cell lines, we demonstrated that ATF3 negatively regulates the growth and migration of human TSCC cells in vitro. RNA-seq analysis identified two new downstream targets of ATF3, interferon alpha inducible proteins 6 (IFI6) and 27 (IFI27), which were upregulated in ATF3-deleted cells and were downregulated in ATF3-overexpressing cells. Chromatin immunoprecipitation assays showed that ATF3 binds the promoter regions of the IFI6 and IFI27 genes. Both IFI6 and IFI27 were highly expressed in TSCC biopsies and knockdown of either IFI6 or IFI27 in TSCC cells blocked the cell growth and migration induced by the deletion of ATF3. Conversely, overexpression of either IFI6 or IFI27 counteracted the inhibition of TSCC cell growth and migration induced by the overexpression of ATF3. Finally, an in vivo study in mice confirmed those in vitro findings. Our study suggests that ATF3 plays an anti-tumor function in TSCCs through the negative regulation of its downstream targets, IFI6 and IFI27.

Early-stage bilayer tissue-engineered skin substitute formed by adult skin progenitor cells produces an improved skin structure in vivo.

BACKGROUND: In recent years, significant progress has been made in developing highly complex tissue-engineered skin substitutes (TESSs) for wound healing. However, the lack of skin appendages, such as hair follicles and sweat glands, and the time required, are two major limitations that hinder its broad application in the clinic. Therefore, it is necessary to develop a competent TESS in a short time to meet the needs for clinical applications. METHODS: Adult scalp dermal progenitor cells and epidermal stem cells together with type I collagen as a scaffold material were used to reconstitute bilayer TESSs in vitro. TESSs at 4 different culture times (5, 9, 14, and 21 days) were collected and then grafted onto full-thickness wounds created in the dorsal skin of athymic nude/nude mice. The skin specimens formed from grafted TESSs were collected 4 and 8 weeks later and then evaluated for their structure, cell organization, differentiation status, vascularization, and formation of appendages by histological analysis, immunohistochemistry, and immunofluorescent staining. RESULTS: Early-stage bilayer TESSs after transplantation had a better efficiency of grafting. A normal structure of stratified epidermis containing multiple differentiated layers of keratinocytes was formed in all grafts from both early-stage and late-stage TESSs, but higher levels of the proliferation marker Ki-67 and the epidermal progenitor marker p63 were found in the epidermis formed from early-stage TESSs. Interestingly, the transplantation of early-stage TESSs produced a thicker dermis that contained more vimentin- and CD31-positive cells, and importantly, hair follicle formation was only observed in the skin grafted from early-stage TESSs. Finally, early-stage TESSs expressed high levels of p63 but had low expression levels of genes involved in the activation of the apoptotic pathway compared to the late-stage TESSs in vitro. CONCLUSIONS: Early-stage bilayer TESSs reconstituted from skin progenitor cells contained more competent cells with less activation of the apoptotic pathway and produced a better skin structure, including hair follicles associated with sebaceous glands, after transplantation, which should potentially provide better wound healing when applied in the clinic in the future.

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

IL-6 promotes metastasis of non-small-cell lung cancer by up-regulating TIM-4 via NF-κB.

OBJECTIVES: Interleukin-6 (IL-6) is critical for the development of non-small-cell lung cancer (NSCLC). Recently, we identified T-cell immunoglobulin domain and mucin domain 4 (TIM-4) as a new pro-growth player in NSCLC progression. However, the role of TIM-4 in IL-6-promoted NSCLC migration, invasion and epithelial-to-mesenchymal transition (EMT) remains unclear. MATERIALS AND METHODS: Expressions of TIM-4 and IL-6 were both evaluated by immunohistochemical staining in NSCLC tissues. Real-time quantitative PCR (qPCR), Western blot, flow cytometry and RT-PCR were performed to detect TIM-4 expression in NSCLC cells with IL-6 stimulation. The roles of TIM-4 in IL-6 promoting migration and invasion of NSCLC were detected by transwell assay. EMT-related markers were analysed by qPCR and Western blot in vitro, and metastasis was evaluated in BALB/c nude mice using lung cancer metastasis mouse model in vivo. RESULTS: High IL-6 expression was identified as an independent predictive factor for TIM-4 expression in NSCLC tissues. NSCLC patients with TIM-4 and IL-6 double high expression showed the worst prognosis. IL-6 promoted TIM-4 expression in NSCLC cells depending on NF-κB signal pathway. Both TIM-4 and IL-6 promoted migration, invasion and EMT of NSCLC cells. Interestingly, TIM-4 knockdown reversed the role of IL-6 in NSCLC and IL-6 promoted metastasis of NSCLC by up-regulating TIM-4 via NF-κB. CONCLUSIONS: TIM-4 involves in IL-6 promoted migration, invasion and EMT of NSCLC.

Tim-4 Inhibits NLRP3 Inflammasome via the LKB1/AMPKα Pathway in Macrophages.

Nonalcoholic fatty liver disease (NAFLD), characterized by excessive inflammation and lipid deposition, is one of the most common metabolic liver diseases. The expression of NLRP3 inflammasome in macrophages is significantly increased in NAFLD, and its activation aggravates NAFLD greatly. Tim-4, as the phosphatidylserine (PS) receptor, is expressed highly in macrophages, and macrophage Tim-4 inhibits inflammation under various conditions of immune activation. However, the precise role of Tim-4 in NLRP3 inflammasome regulation and NAFLD pathogenesis remains completely unknown. Using NAFLD mice models, we confirmed that the expression of Tim-4 was increased in liver tissues by Western blot, real-time PCR, immunohistochemistry, and immunofluorescence, especially higher expression in liver macrophages, and Tim-4 knockout mice displayed more severe liver inflammation and hepatic steatosis than controls in NAFLD mice model. In vitro, we found that Tim-4 could inhibit NLRP3 inflammasome activation, and the inhibition was dependent on PS binding domain in the IgV domain. Mechanistically, Tim-4 induced the degradation of NLRP3 inflammasome components through activating AMPKα-mediated autophagy. Specifically, Tim-4 promoted AMPKα phosphorylation by interacting with LKB1 and AMPKα. In addition, PS binding motif was responsible for Tim-4-mediated AMPKα and LKB1 interaction. In conclusion, NAFLD microenvironments upregulate Tim-4 expression in macrophages, and elevated Tim-4, in turn, suppresses NLRP3 inflammasome activation by activating LKB1/AMPKα-mediated autophagy, thereby ameliorating the release of IL-1ß and IL-18. Collectively, this study unveils the novel function of Tim-4 in suppressing NLRP3 inflammasome, which would shed new lights on intervention of NAFLD or inflammatory liver diseases by targeting Tim-4.

ROCK inhibitor enhances the growth and migration of BRAF-mutant skin melanoma cells.

Rho-associated protein kinase (ROCK) plays crucial roles in the proliferation and migration of different types of cells. ROCK inhibitor Y-27632 was previously reported to inhibit melanoma cell growth, and ROCK signaling was suggested to be a therapeutic target for treating melanoma. However, the negative effect of Y-27632 on melanoma cells was mainly seen in studies on murine B16 melanoma cells. Here, we reported that ROCK inhibitor actually promoted human melanoma cell growth and migration in vitro. Y-27632 increased the growth and migration of BRAF-mutated melanoma cells but had a negative effect on wild-type melanoma cells or primary melanocytes. We discovered that Y-27632 enhanced the growth of BRAF-mutated melanoma cells through increased ATK and ERK activity. The in vivo study further confirmed the in vitro finding. These data suggested that the effect of ROCK inhibitor on melanoma cells is cell-context dependent, and the application of ROCK inhibitor in the treatment of melanoma requires further study.

The ARE-binding protein Tristetraprolin (TTP) is a novel target and mediator of calcineurin tumor suppressing function in the skin.

An increased incidence of skin inflammatory diseases is frequently observed in organtransplanted patients being treated with calcineurin inhibitor-based immunosuppressive agents. The mechanism of increased skin inflammation in this context has however not yet been clarified. Here we report an increased inflammation following inhibition of calcineurin signaling seen in both chemically induced mouse skin tumors and in tumors grafted from H-rasV12 expressing primary human keratinocytes (HKCs). Following UVB or TPA treatment, we specifically found that deletion of the calcineurin gene in mouse keratinocytes (MKCs) resulted in increased inflammation, and this was accompanied by the enhanced production of pro-inflammatory cytokines, such as TNFα, IL-8 and CXCL1. Furthermore, expression of the RNA-binding protein, tristetraprolin (TTP) was down-regulated in response to calcineurin inhibition, wherein TTP was shown to negatively regulate the production of pro-inflammatory cytokines in keratinocytes. The induction of TTP following TPA or UVB treatment was attenuated by calcineurin inhibition in keratinocytes, and correspondingly, disruption of calcineurin signaling down-regulated the amounts of TTP in both clinical and H-rasV12-transformed keratinocyte tumor models. Our results further demonstrated that calcineurin positively controls the stabilization of TTP in keratinocytes through a proteasome-dependent mechanism. Reducing the expression of TTP functionally promoted tumor growth of H-rasV12 expressing HKCs, while stabilizing TTP expression counteracted the tumor-promoting effects of calcineurin inhibition. Collectively these results suggest that calcineurin signaling, acting through TTP protein level stabilization, suppresses keratinocyte tumors by downregulating skin inflammation.

Hepatitis B core protein promotes liver cancer metastasis through miR-382-5p/DLC-1 axis.

The hepatitis B virus core protein (HBc), also named core antigen, is well-known for its key role in viral capsid formation and virus replication. Recently, studies showed that HBc has the potential to control cell biology activity by regulating host gene expression. Here, we utilized miRNA microarray to identify 24 upregulated miRNAs and 21 downregulated miRNAs in HBc-expressed HCC cells, which were involved in multiple biological processes, including cell motility. Consistently, the in vitro transwell assay and the in vivo tail-vein injection model showed HBc promotion on HCC metastasis. Further, the miRNA-target gene network analysis displayed that the deleted in liver cancer (DLC-1) gene, an important negative regulator for cell motility, was potentially targeted by several differentially expressed miRNAs in HBc-introduced cells. Introduction of miRNAs mimics or inhibitors and 3'UTR luciferase activity assay proved that miR-382-5p efficiently suppressed DLC-1 expression and its 3'-UTR luciferase reporter activity. Importantly, cotransfection of miR-382-5p mimics/inhibitors and the DLC-1 expression vector almost abrogated HBc promotion on cell motility, indicating that the miR-382-5p/DLC-1 axis is important for mediating HBc-enhanced HCC motility. Clinical HCC samples also showed a negative correlation between miR-382-5p and DLC-1 expression level. Furthermore, HBc-positive HCC tissues showed high miR-382-5p level and reduced DLC-1 expression. In conclusion, our findings revealed that HBc promoted HCC motility by regulating the miR-382-5p/DLC-1 axis, which might provide a novel target for clinical diagnosis and treatment.

Design, synthesis and evaluation of pyrazole derivatives as non-nucleoside hepatitis B virus inhibitors.

In continuation of our efforts toward the discovery of potent non-nucleoside hepatitis B virus (HBV) inhibitors with novel structures, we have employed bioisosterism and hybrid pharmacophore-based strategy to explore the chemically diverse space of bioactive compounds. In this article, the original thiazole platform was replaced with pyrazole scaffold to yield the optimal pharmacophore moieties in order to generate novel non-nucleoside HBV inhibitors with desirable potency. Some of the new compounds were able to inhibit HBV activity in the low micromolar range. In particular, compound 6a3 displayed the most potent activity against the secretion of HBsAg and HBeAg with IC50 of 24.33 µM and 2.22 µM, respectively. The preliminary structure-activity relationship (SAR) of this new series of compounds was investigated, which may help designing more potent molecules.

CUL4A facilitates hepatocarcinogenesis by promoting cell cycle progression and epithelial-mesenchymal transition.

CUL4A, a member of the CULLIN family, functions as a scaffold protein for an E3 ubiquitin ligase. It was reported that the CUL4A gene showed amplification in some human primary hepatocellular carcinomas (HCC). However, the exact role of CUL4A in HCC remains unknown. Here, we aimed to investigate the expression and function of CUL4A in HCC development. Through immunohistochemistry study, we showed increased CUL4A expression in HCC tissues. Statistical analysis disclosed an inverse correlation between CUL4A expression and tumor differentiation grade, and patient survival, but a positive correlation with hepatocyte proliferation as well as lymphatic and venous invasion. CUL4A expression in HCC tissues was associated with HBeAg status in patients and upregulated by HBV in HCC cell lines. Further functional assay showed that CUL4A overexpression significantly promoted growth of H22 tumor homografts in BALB/c mice. Consistently, CUL4A knockdown inhibited the proliferation of established HCC cells, accompanied by S-phase reduction and Cyclin A and Cyclin B1 repression. Furthermore, CUL4A siRNA ameliorated the motility of HCC cell lines with altered expression of epithelial-mesenchymal transition (EMT)-associated molecules. Taken together, our findings indicate that CUL4A plays a pivotal role in HCC progression and may serve as a potential marker for clinical diagnosis and target for therapy.

HBV preS2 promotes the expression of TAZ via miRNA-338-3p to enhance the tumorigenesis of hepatocellular carcinoma.

Transactivators encoded by HBV, including HBx and preS2, play critical role in hepatocellular carcinoma (HCC). YAP, a downstream effector of the Hippo pathway, is involved in hepatocarcinogenesis mediated by HBx. Here, we investigated whether preS2, another transactivator encoded by HBV, regulates the Hippo pathway to promote HCC. We found that preS2 overexpression upregulated TAZ, a downstream effector of the Hippo pathway, at protein level but not at mRNA level. preS2 suppressed miRNA-338-3p expression in HCC cell lines. miRNA-338-3p mimics downregulated TAZ, while miRNA-338-3p inhibitor restored the expression of TAZ, suggesting that TAZ is a direct target of miRNA-338-3p. TAZ overexpression stimulated growth of HCC cell lines. Knockdown of TAZ dampened preS2-promoted HCC proliferation and migration. Thus, preS2 upregulates TAZ expression by repressing miRNA-338-3p. TAZ is necessary for preS2-promoted HCC proliferation and migration.