Hypermethylation of secreted frizzled related protein 2 gene promoter serves as a noninvasive biomarker for HBV-associated hepatocellular carcinoma.

For patients with hepatocellular carcinoma (HCC), early detection is critical to improve survival. Secreted frizzled-related protein 2 (SFRP2) is a candidate tumor suppressor as Wnt antagonist and SFRP2 promoter has been found hypermethylated in various malignancies. This study aimed to investigate the methylation status of SFRP2 promoter in hepatitis B virus (HBV) associated HCC and estimate its diagnostic value as a non-invasive biomarker. A total of 293 patients, including 132 patients with HBV-associated HCC, 121 with chronic hepatitis B (CHB) and 40 healthy controls (HCs) were enrolled. SFRP2 methylation level in peripheral mononuclear cells (PBMCs) was quantitatively detected by MethyLight. SFRP2 methylation level was significantly higher in patients with HBV-associated HCC than in those with CHB (p < 0.001) and HCs (p < 0.001) while mRNA level of SFRP2 was significantly lower in HCC group than the other two groups (p < 0.05). In HCC subgroup, SFRP2 methylation level markedly increased in patients >50 years old, female, with negative HBeAg, negative HBV-DNA and poor differentiation compared with the remaining groups (P < 0.05). Furthermore, SFRP2 methylation level showed a significantly better diagnostic value than alpha-fetoprotein (AFP) and the combination of AFP and methylation levels of SFRP2 markedly improved the area under the receiver operating characteristic curve (p < 0.05). In conclusion, hypermethylation of SFRP2 promoter exists in HBV-associated HCC. The combination of SFRP2 methylation level in PBMCs and AFP could significantly improve the diagnostic ability of AFP in discriminating HBV-associated HCC from CHB and SFRP2 methylation level had the potential to serve as a non-invasive biomarker for HCC diagnosis.

5-Aza-2'-deoxycytidine may enhance the frequency of T regulatory cells from CD4+ naïve T cells isolated from the peripheral blood of patients with chronic HBV infection.

OBJECTIVES: Methylation pattern of gene modification is essential for the differentiation of T regulatory cells (Tregs) and 5-Aza-2'-deoxycytidine is a common inhibitor of methylation. This study aimed to investigate the potential effects of Treg polarizing conditions and 5-Aza-2'-deoxycytidine treatment in the differentiation of naïve T cells during chronic hepatitis B virus (HBV) infection. METHODS: The frequency of Tregs in peripheral blood was determined by flow cytometry from patients with chronic hepatitis B (CHB) (n = 51), liver cirrhosis (LC) (n = 47), hepatocellular carcinoma (HCC) (n = 40) and healthy controls (HCs) (n = 17). Gene expression were detected by qRT-PCR and DNA methyltransferases (DNMT) Activity was also determined. RESULTS: The frequency of Tregs and Foxp3 expression in peripheral blood from 5-Aza-2'-deoxycytidine-treated groups were higher than that with acetic acid treatment as a control. Foxp3 mRNA and the frequency of Tregs derived from naïve CD4+T cells from peripheral blood of patients with HCC or LC were more pronounced compared with HCs. 5-Aza-2'-deoxycytidine may have induced a more pronounced upward trend of PD-1 expression in HBV patients. CONCLUSIONS: 5-Aza-2'-deoxycytidine mediated demethylation has potential effects on enhancing the differentiation of naïve T cells to Tregs in chronic HBV infection.

Diagnostic Value of the Hypomethylation of the WISP1 Promoter in Patients with Hepatocellular Carcinoma Associated with Hepatitis B Virus.

Wnt1-inducible signaling pathway protein 1 (WISP1) regulates cell proliferation, differentiation, adhesion, migration and survival. Abnormal WISP1 expression is associated with the carcinogenesis of hepatocellular carcinoma (HCC). Aberrant DNA methylation is one of the major epigenetic alterations in HCC. However, the methylation status of the WISP1 promoter is still unclear. We therefore aimed to determine the methylation status of the WISP1 promoter and evaluate its clinical value in HCC. The study enrolled 251 participants, including 123 participants with HCC, 90 participants with chronic hepatitis B (CHB) and 38 healthy controls (HCs). WISP1 methylation status, mRNA levels and plasma soluble WISP1 were detected by methylation-specific polymerase chain reaction (MSP), quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. We found that the methylation frequency of WISP1 in patients with HCC was significantly lower than that in patients with CHB and HCs, while the relative expression levels of WISP1 mRNA were markedly higher in patients with HCC than in patients with CHB and HCs. Furthermore, the plasma soluble WISP1 in patients with HCC was obviously lower than in that in patients with CHB and HCs. Alpha-fetoprotein (AFP) is a widely recognized biomarker to diagnose HCC which lacks enough sensitivity and specificity. WISP1 promoter methylation status combined with AFP significantly improved the diagnostic ability in discriminating HCC from CHB compared with AFP or WISP1 methylation status alone. In conclusion, hypomethylation of the WISP1 gene promoter may serve as a noninvasive biomarker for detecting HBV-associated HCC.

MIR-520f Regulated Itch Expression and Promoted Cell Proliferation in Human Melanoma Cells.

Accumulating evidence suggests that abnormal expression and dysfunction of microRNA is involved in development of cancers. However, the function of miR-520f especially in human melanoma remains elusive. In the current study, the underlying function of miR-520f in human melanoma was investigated. Our study demonstrated that the miR-520f level in human melanoma cell lines and clinical tissues was increased. Overexpression of miR-520f promoted cell proliferation by using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation, anchorage-independent growth assay, and 5-bromo-2-deoxyuridine assays. Furthermore, we revealed that miR-520f could interact with circular RNA Itchy E3 ubiquitin protein ligase (ITCH) 3'-untranslated region and suppress ITCH expression in human melanoma cells. The inhibitory effect of miR-520f-in could be partially restored by knockdown of ITCH in human melanoma cells. In summary, this study provides novel insights into miR-520f act as a crucial role in the regulation of human melanoma cell growth via regulating ITCH, which might be a potential biomarker and therapeutic target of human melanoma.

TNFRSF12A and a new prognostic model identified from methylation combined with expression profiles to predict overall survival in hepatocellular carcinoma.

BACKGROUND: It has been proved that DNA methylation, as an epigenetic regulatory mode, plays a crucial role in the initiation, progression and invasion of hepatocellular carcinoma (HCC). However, there still are some pathways and factors that regulates the carcinogenesis of HCC remains unclear. METHODS: The original datasets comparing DNA methylation, clinical information and transcriptome profiling between HCC and normal controls were downloaded from The Cancer Genome Atlas (TCGA) database. R software was used to screen for methylation-differential genes (MDGs) and methylation-driven genes. Gene-functional enrichment analysis, ConsensusPathDB pathway analysis, protein-protein interaction (PPI) network construction and survival analysis were performed; methylation-specific polymerase chain reaction (MSP) and real-time quantitative polymerase chain reaction (RT-qPCR) were used for validation. RESULTS: One hundred and sixty-seven MDGs and 285 methylation-driven genes were identified. Function and pathway enrichment analysis revealed that they are associated with sequence-specific DNA binding, nuclear nucleosome, regulation of insulin-like growth factor transport, etc. An eight-gene (HIST1H1D, RP11-476B1.1, OR2AK2, TNFRSF12A, CTD-2313N18.8, AC133644.2, RP11-467L13.4 and LINC00989) prognostic model was identified from the MDGs; its methylation degree can strongly predict the overall survival of HCC. Among them, TNFRSF12A being the only one belongs to both MDGs and methylation-driver genes, shows a significant independent correlation with the prognosis of HCC. That was validated in further details. CONCLUSIONS: Our research has identified a registry of novel genes and pathways that's important for regulating the carcinogenesis of HCC. In addition, we identified a strong molecular model for prognostic prediction. These findings will not only provide guidance for clinical individualized treatment, but also to set us targets for further research on the molecular mechanism of HCC.

Vasoactive intestinal peptide induces vascular endothelial growth factor production in human HaCaT keratinocytes via MAPK pathway.

Psoriasis is a chronic skin disease characterized by abnormal keratinocyte proliferation and differentiation, inflammation, and angiogenesis. Although dysfunction of the immune system is known to be an important factor in the pathogenesis of psoriasis, there is also strong evidence that psychological stresses are involved. Neuropeptides are thought to be main mediators of neurogenic inflammation, presumably involved in the pathogenesis of psoriasis. Vasoactive intestinal peptide (VIP) is one of the major neuropeptides in human and rodent skin. In the present study, we examined the effect and mechanism of VIP on vascular endothelial growth factor (VEGF) production by HaCaT cells which is a spontaneous, immortalized, human keratinocyte cell line. Our data indicate the mRNA and protein levels of VEGF by VIP were increased in a concentration-dependent manner. However, this increase was abrogated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor PD98059 or p38MAPK inhibitor SB203580; pretreatment with c-Jun N-terminal kinase (JNK) inhibitor SP600125 did not attenuate the effects of VIP on the expression of VEGF. In addition, VIP treatment induced rapid phosphorylation of ERK1/2 and p38MAPK, and PD98059 and SB203580 were able to inhibit VIP-induced phosphorylation of ERK1/2 and p38MAPK, respectively. These results suggest that VIP increases the expression of VEGF through the ERK1/2 and p38MAPK signaling pathway in human HaCaT cells.

Corticotropin-releasing hormone attenuates vascular endothelial growth factor release from human HaCaT keratinocytes.

OBJECTIVES: Corticotropin-releasing hormone (CRH) is a central component of the local hypothalamic-pituitary-adrenal (HPA) axis, which has a functional equivalent in the skin. To determine whether CRH and its receptor, CRH-R1, modulate the expression of vascular endothelial growth factor (VEGF), which is overexpressed in psoriatic epidermis and plays a causal role in the pathogenesis of psoriasis, we investigated the effect of CRH on the expression of VEGF in a human keratinocyte cell line (HaCaT) and whether this effect is via the mitogen-activated protein kinase (MAPK) signal transduction pathways. METHODS: Real-time RT-PCR, ELISA assay and western blot were used in the present study to investigate the expression of VEGF in CRH-treated HaCaT cells. RESULTS: The mRNA and protein levels of VEGF in CRH-treated HaCaT cells were significantly attenuated. However, this downregulation was abrogated by pretreatment with antalarmin, SB203580 and SP600125; pretreatment with PD98059 did not attenuate the effects of CRH on the expression of VEGF. In addition, CRH treatment induced rapid phosphorylation of p38 MAPK and JNK1/2, and antalarmin, SB203580 and SP600125 inhibited CRH-induced phosphorylation of p38 MAPK and JNK1/2. CONCLUSIONS: CRH might downregulate the expression of VEGF through the CRH-R1 and MAPK (p38 MAPK and JNK1/2) signaling pathways in human HaCaT cells.

Calcitonin gene-related peptide increases proliferation of human HaCaT keratinocytes by activation of MAP kinases.

Psoriasis is a chronic disease characterized by keratinocyte hyperproliferation and inflammation. It has been demonstrated that the expression of calcitonin gene-related peptide (CGRP) is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. CGRP has been previously described to influence proliferation of several cell types, such as Schwann cell, tracheal epithelial cells, and human gingival fibroblasts. In the present study, we determined the effect of CGRP on HaCaT keratinocyte proliferation and the role of mitogen-activated protein kinases (MAPKs) in CGRP induced keratinocyte proliferation. Our data indicate CGRP increased [(3)H]-thymidine incorporation and MTT activity of HaCaT in a concentration-dependent manner. CGRP also enhanced serum-induced HaCaT cell proliferation. HaCaT cells cultured with CGRP had a significant increase in phosphorylated ERK1/2, p38 and JNK, and CGRP induced DNA synthesis was inhibited by PD 98059 or SB 203580, selective inhibitors of MAP kinase kinase (MEK, which is upstream from ERK) and p38, respectively. These findings suggest that HaCaT cell proliferate in response to CGRP, which is mediated by phosphorylation of ERK1/2 and p38 MAPK.

Expression and localization of the activated mitogen-activated protein kinase in lesional psoriatic skin.

Abnormalities in several signaling pathways and in the expression and/or activation of different transcription factors in psoriatic keratinocytes have been hypothesized to play a role in the pathophysiology of psoriasis. The mitogen-activated protein kinase (MAPK) cascades are among the best characterized of intracellular signaling pathways, and they play important roles in cell proliferation, differentiation, gene expression, and inflammation. We investigated the expression, activation and distribution of extracellular signal-regulated kinases (ERKs), p38 mitogen-activated protein kinases (p38 MAPK) and c-Jun N-terminal kinases (JNKs), using immunohistochemistry and Western blot in lesional psoriatic skin and normal control skin, to clarify the possible roles of these kinases involved in the pathogenesis of psoriasis. The immunoblot analysis demonstrated that activation of ERK1/2 and p38 MAPK increased in the lesional psoriatic skin. In addition, a significant increase in p-MEK (the upstream activator of ERK), and p-CREB (a downstream transcription factor of active ERK) was also found in our experiment. The immunohistochemical study showed that the levels of phosphorylated ERK1/2 and p38 MAPK were enhanced in lesional psoriatic skin compared with controls. Phosphorylated ERK1/2 and p38 exhibited clear nuclear localization throughout the epidermal part of lesional psoriatic skin. These findings suggested that ERK1/2 and p38 pathways were involved in the pathophysiology of psoriasis.

[Changes of NO and ET-1 in the blood of patients with chronic pulmonary heart disease].

OBJECTIVE: To study the changes of nitric oxide (NO), and endothelin-1 (ET-1) in patients with chronic pulmonary heart disease (CPHD), and to understand its pathophysiology, observe its severity, assess its prognosis and find clues to some new management methods. METHODS: Forty-five patients with CPHD at the acute exacerbation stage, 20 patients with CPHD at the stable stage and 18 healthy controls were studied. The NO and ET-1 levels were measured by spectrophotometry and radioimmunoassay respectively. RESULTS: 1. The ratio of ET-1/NO in the patients with CPHD at the acute exacerbation stage was higher than that in the patients at the stable stage (P < 0.05), and heathily controls (P < 0.05), the ratio of ET-1/NO in the patients with CPHD at the stable stage was higher than that in the control group (P < 0.05); 2. The NO level in the patients with CPHD at the acute exacerbation stage or stable stage was positively related with PO2(r = 0.85, P < 0.05, r = 0.72, P < 0.05), but negatively related with PCO2(r = -0.54, P < 0.05, r = -0.52, P < 0.05); the ET-1 level in the patients with CPHD at the acute exacerbation stage or stable stage was negatively related with PO2(r = -0.72, P < 0.05, r = -0.53, P < 0.05), but positively related with PCO2(r = 0.55, P < 0.05, r = 0.53, P < 0.05); 3. The serial changes of NO and ET-1 also demonstrated these correlations; 4. The NO level was elevated in those patients with better symptomatic improvement following management (P < 0.05), while the ET-1 level and the ratio of ET-1/NO decreased significantly in the patients with symptomatic improvement (P < 0.05), and there was no significant change in the patients with no symptomatic improvement following management or in the dead patients(P > 0.05). CONCLUSION: The imbalance between ET-1 and NO exists in patients with CPHD, and it is closely associated with hypoxia and may play an important role in the occurrence and development of pulmonary hypertension and CPHD. NO and ET-1 can serve as indicators to observe the disease severity and assess its prognosis.