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.
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 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.
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 AMPKa-mediated autophagy. Specifically, Tim-4 promoted AMPKa phosphorylation by interacting with LKB1 and AMPKa. In addition, PS binding motif was responsible for Tim-4-mediated AMPKa 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/AMPKa-mediated autophagy, thereby ameliorating the release of IL-1b 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.
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.
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