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CKLF1 is highly expressed in HCC tissues and cell lines.

Knockdown of CKLF1 in HCCLM3 cells result in significantly smaller tumor volume compared to the positive control.

The proliferation rate of HCC cells is enhanced by CKLF1.

Supplementary materials, methods and supplementary figure legends.

CKLF1 regulates the activation of IL-6/STAT3 signaling pathway.

CKLF1 promotes the malignant transformation in HepG2 and Bel7402 cells.

Purpose:Hepatocellular carcinoma (HCC), one of the most common and deadliest malignancies worldwide, has a poor prognosis, owing to its high potential for vascular invasion and metastasis and the lack of biomarkers for early diagnosis. Thus, it must be a crucial factor for investigating therapeutic strategies for HCC to identify the functional molecular targets. Here, we reported a novel chemokine, CKLF1, that might act as a pivotal modulator in the invasion and metastasis of HCC and could serve as an attractive target for cancer therapy.Experimental Design:Bioinformatics analysis, PCR, Western blotting, and IHC were performed to detect the expression of CKLF1 in HCC. The function of CKLF1 was demonstrated by a series of in vitro and in vivo experiments. Pharmacologic treatment, flow cytometry, and Western blotting were carried out to demonstrate the potential mechanisms of CKLF1.Results:We proved that CKLF1 was overexpressed in HCC tissues and was related to tumor stage, vascular invasion, and patient survival. Then, functional assays showed that CKLF1 promoted hepatocellular carcinogenesis and metastatic potential. Finally, the IL6/STAT3 signaling pathway was involved in the mechanistic investigation. The results demonstrated that CKLF1 enhanced the progression of HCC and prevented doxorubicin-induced apoptosis through activating the IL6/STAT3 pathway.Conclusions:These data showed that CKLF1 inhibited apoptosis and promoted malignant transformation through the IL6/STAT3 pathway, and ultimately enhanced the development and metastasis of HCC. Thus, our work revealed that CKLF1 was a significant prognostic factor of HCC and might be a potential molecular therapeutic target for HCC.

Human endogenous retrovirus W family envelope protein (HERV-W env) is associated with several neurological and psychiatric disorders, including multiple sclerosis (MS) and schizophrenia. Clinical studies have demonstrated a common link between inflammatory abnormalities and HERV-W env in neuropsychiatric diseases. Nonetheless, the molecular mechanisms by which HERV-W env mediates neuroinflammation are still unclear. In this study, we found that HERV-W env significantly increased the mRNA and protein levels of TNF-α and IL-10 in U251 and A172 cells. HERV-W env also induced a notable increase in Toll-like receptor 4 (TLR4). Knockdown of TLR4 impaired the expressions of TNF-α and IL-10 induced by HERV-W env. Overexpression of HERV-W env led to the upregulation of MyD88 but caused a decrease in MyD88s. MyD88s overexpression suppressed the expressions of TNF-α and IL-10 induced by HERV-W env. These findings indicate that HERV-W env upregulates the expressions of IL-10 and TNF-α by inhibiting the production of MyD88s in glial cells. This work sheds light on the immune pathogenesis of HERV-W env in neuropsychiatric disorders.

The human endogenous retroviruses type W family envelope (HERV-W env) gene is located on chromosome 7q21-22. Our previous studies show that HERV-W env is elevated in schizophrenia and HERV-W env can increase calcium influx. Additionally, the 5-HTergic system and particularly 5-hydroxytryptamine (5-HT) receptors play a prominent role in the pathogenesis and treatment of schizophrenia. 5-hydroxytryptamine receptor 4 (5-HT4R) agonist can block calcium channels. However, the underlying relationship between HERV-W env and 5-HT4R in the etiology of schizophrenia has not been revealed. Here, we used enzyme-linked immunosorbent assay to detect the concentration of HERV-W env and 5-HT4R in the plasma of patients with schizophrenia and we found that there were decreased levels of 5-HT4R and a negative correlation between 5-HT4R and HERV-W env in schizophrenia. Overexpression of HERV-W env decreased the transcription and protein levels of 5-HT4R but increased small conductance Ca

Purpose: Hepatocellular carcinoma (HCC), one of the most common and deadliest malignancies worldwide, has a poor prognosis, owing to its high potential for vascular invasion and metastasis and the lack of biomarkers for early diagnosis. Thus, it must be a crucial factor for investigating therapeutic strategies for HCC to identify the functional molecular targets. Here, we reported a novel chemokine, CKLF1, that might act as a pivotal modulator in the invasion and metastasis of HCC and could serve as an attractive target for cancer therapy. Experimental Design: Bioinformatics analysis, PCR, Western blotting, and IHC were performed to detect the expression of CKLF1 in HCC. The function of CKLF1 was demonstrated by a series of in vitro and in vivo experiments. Pharmacologic treatment, flow cytometry, and Western blotting were carried out to demonstrate the potential mechanisms of CKLF1. Results: We proved that CKLF1 was overexpressed in HCC tissues and was related to tumor stage, vascular invasion, and patient survival. Then, functional assays showed that CKLF1 promoted hepatocellular carcinogenesis and metastatic potential. Finally, the IL6/STAT3 signaling pathway was involved in the mechanistic investigation. The results demonstrated that CKLF1 enhanced the progression of HCC and prevented doxorubicin-induced apoptosis through activating the IL6/STAT3 pathway. Conclusions: These data showed that CKLF1 inhibited apoptosis and promoted malignant transformation through the IL6/STAT3 pathway, and ultimately enhanced the development and metastasis of HCC. Thus, our work revealed that CKLF1 was a significant prognostic factor of HCC and might be a potential molecular therapeutic target for HCC.

Human endogenous retrovirus (HERVs), originating from exogenous retroviral infections of germ cells millions of years ago, have the potential for human diseases. Syncytin-1, an envelope protein encoded by the HERV W family, participates in the contexts of schizophrenia, multiple sclerosis, diabetes, and several types of cancers. Nevertheless, there is no report on the expression pattern and potential mechanism of Syncytin-1 in HCC. Here we found Syncytin-1 expression was up-regulated in HCC compared to adjacent non-tumorous tissues, especially in advanced HCC. Syncytin-1 was an independent risk factor to predict vascular invasion, metastasis, larger tumor size, and poor prognosis in HCC patients. Further analysis discovered that Syncytin-1 overexpression positively associated with HCC patients with serum HBsAg positive. Functional experiments in vitro and in vivo demonstrated that Syncytin-1 enhanced cell proliferation, metastasis, and tumorigenicity in HCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the mitogen-activated protein kinase (MEK)/extracellular signal-regulated protein kinase (ERK) pathway was involved in HCC. Our clinical data indicated that the levels of phosphorylation MEK1/2 and ERK1/2 were increased in HCC comparing with adjacent non-tumorous tissues. It showed the linear correlation between Syncytin-1 expression and upregulated MEK1/2 and ERK1/2 phosphorylation levels in HCC. Furthermore, Syncytin-1 activated MEK/ERK pathway in HCC cells. In-depth research showed that the inflammation-activated MEK/ERK pathway was essential in Syncytin-1 promoted hepatocarcinogenesis. Syncytin-1 suppressed doxorubicin-induced apoptosis via MEK/ERK cascade. In conclusion, Syncytin-1 promoted HCC progression and doxorubicin resistance via the inflammation-activated MEK/ERK pathway. Our findings revealed that Syncytin-1 was a potential prognostic biomarker and therapeutic target for HCC.

Human endogenous retrovirus W family envelope protein (HERV-W env) is associated with several neurological and psychiatric disorders, including multiple sclerosis (MS) and schizophrenia. Clinical studies have demonstrated a common link between inflammatory abnormalities and HERV-W env in neuropsychiatric diseases. Nonetheless, the molecular mechanisms by which HERV-W env mediates neuroinflammation are still unclear. In this study, we found that HERV-W env significantly increased the mRNA and protein levels of TNF-α and IL-10 in U251 and A172 cells. HERV-W env also induced a notable increase in Toll-like receptor 4 (TLR4). Knockdown of TLR4 impaired the expressions of TNF-α and IL-10 induced by HERV-W env. Overexpression of HERV-W env led to the upregulation of MyD88 but caused a decrease in MyD88s. MyD88s overexpression suppressed the expressions of TNF-α and IL-10 induced by HERV-W env. These findings indicate that HERV-W env upregulates the expressions of IL-10 and TNF-α by inhibiting the production of MyD88s in glial cells. This work sheds light on the immune pathogenesis of HERV-W env in neuropsychiatric disorders.

Human endogenous retrovirus W family (HERV-W) envelope (env) is known to be associated with neurological and psychiatric disorders, such as multiple sclerosis and schizophrenia. Previous studies showed that overexpression of HERV-W env could induce brain-derived neurotrophic factor (BDNF) gene expression. In human and rat cells, BDNF-mediated signal transduction might be modulated by glycogen synthase kinase 3β (GSK3β). Both BDNF and GSK3β are schizophrenia-related genes. In this paper, we investigated whether GSK3β was involved in the HERV-W env-induced expression of BDNF. We found that HERV-W env increased phosphorylation of GSK3β at Ser9 (p-GSK3β (Ser9)) and the ratio of p-GSK3β (Ser9) to total GSK3β (p0.05) in U251 cells. Overexpression of HERV-W env led to a 36.2% reduction in GSK3β activity compared to control (p0.05). The levels of β-catenin, cyclin D1 and TSC2 mRNAs were upregulated (p0.05). These data suggested that overexpression of HERV-W env might activate the GSK3β signaling pathway in U251 cells. Further, knockdown of GSK3β reduced the expression of total GSK3β, p-GSK3β (Ser9), and the ratio of p-GSK3β (Ser9) to total GSK3β by 28.6%, 50.4%, and 30.2%, respectively (p0.05). Levels of β-catenin, cyclin D1 and TSC2 mRNAs were also reduced (p0.05). Interestingly, GSK3β activity increased (p0.05). Knockdown of GSK3β also decreased mRNA and protein expression of BDNF by 49.9% and 48.5% respectively (p0.05). These results indicated that phosphorylation of GSK3β at Ser9 might be involved in HERV-W env-induced BDNF expression, and will hopefully improve our understanding of the role of HERV-W env in neurological and psychiatric diseases (schizophrenia, etc).

The activation and involvement of human endogenous retroviruses W family envelope gene (HERV-W env, also called ERVWE1) have been reported in several neuropsychiatric disorders, including schizophrenia, as well as in multiple sclerosis (MS). Dysregulation of intracellular calcium content is also involved in the pathogenesis of these diseases. Our previous studies showed that HERV-W env overexpression results in activation of small conductance Ca2+-activated K+ channel protein 3 (SK3), a potential risk factor for schizophrenia. In the present study, we aimed to elucidate the relationship between HERV-W env and calcium signaling in schizophrenia. Our results showed that HERV-W env could induce Ca2+ influx in two human neuroblastoma cell lines and upregulate the expression and activation of TRPC3 in cells. The abnormal increase in intracellular Ca2+ concentration was inhibited by addition of the TRPC3 channel blocker pyr3, demonstrating that the Ca2+ influx induced by HERV-W env was TRPC3-dependent. Further experiments showed that HERV-W env overexpression downregulated DISC1, while knockdown of DISC1 promoted activation of TRPC3 without affecting TRPC3 expression. In conclusion, HERV-W env induced Ca2+ influx in human neuroblastoma cells by activating the TRPC3 channel through directly regulating its expression or downregulating DISC1, which could also increase TRPC3 activation without affecting TRPC3 expression. These findings provide new insights into how HERV-W env affects neuronal activity and contributes to the pathogenesis of schizophrenia.

Schizophrenia is a devastating psychiatric disorder that impacts on social functioning and quality of life, and there is accumulating evidence that inflammation is a potential pathogenic mechanism of schizophrenia. However, the mechanism of inflammation possibly occurred in schizophrenia has not been well understood. The endogenous retroviral protein syncytin-1 and inflammatory marker CRP are both abnormally expressed in schizophrenia patients. CRP is one of the markers of bacterial infection generally. Less clear is whether virus or viral protein can trigger the activation of CRP. Here, we detected a robust increase of the levels of syncytin-1 and CRP in schizophrenia patients, and displayed a positive correlation and marked consistency between expressions of syncytin-1 and CRP in schizophrenia patients. Furthermore, overexpression of syncytin-1 significantly elevated the levels of CRP, TLR3, and IL-6 in both human microglia and astrocytes. TLR3 deficiency impaired the expressions of CRP and IL-6 induced by syncytin-1. Importantly, we observed a cellular co-localization and a direct interaction between syncytin-1 and TLR3. Additionally, knockdown of IL-6 inhibited the syncytin-1-induced CRP expression. Thus, the totality of these results showed that viral protein syncytin-1 could trigger the activation of CRP, which might explain the elevated CRP in sterile inflammation and exhibit a novel mechanism for regulation of inflammation by syncytin-1 in schizophrenia.

Human endogenous retrovirus W env (HERV-W env) plays a critical role in many neuropsychological diseases such as schizophrenia and multiple sclerosis (MS). These diseases are accompanied by immunological reactions in the central nervous system (CNS). Microglia are important immunocytes in brain inflammation that can produce a gasotransmitter – nitric oxide (NO). NO not only plays a role in the function of neuronal cells but also participates in the pathogenesis of various neuropsychological diseases. In this study, we reported increased NO production in CHME-5 microglia cells after they were transfected with HERV-W env. Moreover, HERV-W env increased the expression and function of human inducible nitric oxide synthase (hiNOS) and enhanced the promoter activity of hiNOS. Microglial migration was also enhanced. These data revealed that HERV-W env might contribute to increase NO production and microglial migration ability in neuropsychological disorders by regulating the expression of inducible NOS. Results from this study might lead to the identification of novel targets for the treatment of neuropsychological diseases, including neuroinflammatory diseases, stroke, and neurodegenerative diseases.

The intestinal metabolites of ellagic acid (EA), urolithins are known to effectively inhibit cancer cell proliferation. This study investigates antiproliferative and antioxidant effects of urolithin A (UA) on cell survival of the HepG2 hepatic carcinomas cell line. The antiproliferative effects of UA (0-500 μM) on HepG2 cells were determined using a CCK assay following 12-36 h exposure. Effects on β-catenin and other factors of expression were assessed by using real-time PCR and Western blot. We found that UA showed potent antiproliferative activity on HepG2 cells. When cell death was induced by UA, it was found that the expression of β-catenin, c-Myc and Cyclin D1 were decreased and TCF/LEF transcriptional activation was notably down-regulated. UA also increased protein expression of p53, p38-MAPK and caspase-3, but suppressed expression of NF-κB p65 and other inflammatory mediators. Furthermore, the antioxidant assay afforded by UA and EA treatments was associated with decreases in intracellular ROS levels, and increases in intracellular SOD and GSH-Px activity. These results suggested that UA could inhibit cell proliferation and reduce oxidative stress status in liver cancer, thus acting as a viably effective constituent for HCC prevention and treatment.