Expression of STAT1 and STAT2 in malignant melanoma does not correlate with response to interferon-alpha adjuvant therapy

Interferon-alpha (IFN-) is used as an adjuvant therapy in patients with malignant melanoma and who have undergone surgical resection of high-risk lesions. Defective expression or activation of STAT1 or STAT2 has been shown to correlate with IFN- or resistance in vitro; however, recent data from our laboratory suggest that the anti-tumor effects of IFN- are dependent on STAT1 signaling within host immune cells. We measured STAT1 and STAT2 expression in 28 melanoma biopsies (8 cutaneous lesions; 1 lung metastasis; 19 nodal metastases) obtained from patients prior to the initiation of adjuvant IFN- therapy. Disease recurrence following IFN- treatment did not correlate with the staining intensity of either STAT1 (P=0.61) or STAT2 (P=0.52). Tumors with minimal STAT1 or STAT2 expression (<20% positive) were present in four patients with tumor-positive lymph nodes, who exhibited prolonged relapse-free survival (>44 months) following adjuvant therapy. Conversely, high levels of STAT1 were present in a patient who recurred during the course of IFN- therapy. A case study of one patient who experienced recurrent disease during IFN- treatment revealed that STAT1 levels were greater in the recurrent tumor when compared to the original lesion. These studies provide direct evidence to suggest that levels of STAT1 and STAT2 within the tumor do not influence a patients response to adjuvant IFN-.     

The nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate RNA encapsidation

Severe fever with thrombocytopenia syndrome virus (SFTSV), a member of the Phlebovirus genus from the Bunyaviridae family endemic to China, is the causative agent of life-threatening severe fever with thrombocytopenia syndrome (SFTS), which features high fever and hemorrhage. Similar to other negative-sense RNA viruses, SFTSV encodes a nucleocapsid protein (NP) that is essential for viral replication. NP facilitates viral RNA encapsidation and is responsible for the formation of ribonucleoprotein complex. However, recent studies have indicated that NP from Phlebovirus members behaves in inhomogeneous oligomerization states. In the present study, we report the crystal structure of SFTSV NP at 2.8 ? resolution and demonstrate the mechanism by which it processes a ringshaped hexameric form to accomplish RNA encapsidation. Key residues essential for oligomerization are identifi ed through mutational analysis and identifi ed to have a signifi cant impact on RNA binding, which suggests that correct formation of highly ordered oligomers is a critical step in RNA encapsidation. The fi ndings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.     

Deep sequencing and phylogenetic analysis of severe fever with thrombocytopenia syndrome virus from the tick,Haemaphysalis longicornis,in Korea

The Asian longhorned tick, Haemaphysalis longicornis, is widely distributed in China, Japan, and Korea and may transmit infectious diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an important tick‐borne disease caused by the SFTS virus (SFTSV). Deep sequencing to confirm the presence of SFTSV in ticks has not been reported in Korea. To detect SFTSV, RNA was extracted from tick samples and analyzed using high‐throughput deep sequencing. Based on BLASTN results, numerous SFTSV reads were identified. Moreover, a nearly complete genome of SFTSV (JNU‐1 isolate) was obtained using Sanger sequencing. The genome of the JNU‐1 isolate includes three segments of 6,286, 3,299 and 1,642 nucleotides (nt) termed large (L), medium (M), and small (S), respectively. Also, phylogenetic and recombination analyses for each segment of SFTSV were performed using the JNU‐1 isolate. The three segments of JNU‐1 isolate were closely related to the genotype B known human‐derived Korean SFTSV isolate; the JNU‐1 isolate showed no recombination sites with other isolates. This study is the first report of detection of SFTSV from ticks using deep sequencing in Korea and provides information on the genetic diversity of SFTSV in East Asia.     

Poxviruses and paramyxoviruses use a conserved mechanism of STAT1 antagonism to inhibit interferon signaling

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Isolation,characterization, and phylogenic analysis of three new severe fever with thrombocytopenia syndrome bunyavirus strains derived from Hubei Province,China

Hubei Province is a major epidemic area of severe fever with thrombocytopenia syndrome bunyavirus(SFTSV) in China. However, to date, a few SFTSV strains have been isolated from Hubei Province, preventing effective studies of epidemic outbreaks. Here, we report three confirmed patients(2015–2016) with typical symptoms of severe fever with thrombocytopenia syndrome disease(SFTS) who were farmers resident in different regions in Hubei Province. Three new SFTSV strains were isolated from the serum samples of each patient. Characterization of viral growth properties showed that there were no significant differences in virus production. All strains were completely sequenced, and phylogenetic analysis showed that unlike the other strains from Hubei province, which belonged to the SFTSV C3 genotype, one of the three strains belonged to the SFTSV C2 genotype. These results suggested that multiple SFTSV genotypes have been circulating in Hubei Province, providing insights into SFTSV evolution and improving our understanding of SFTSV prevalence in Hubei Province.     

Tilianin inhibits the human ovarian cancer (PA-1) cell proliferation via blocking cell cycle,inducing apoptosis and inhibiting JAK2/STAT3 signaling pathway

Ovarian cancer is one of the deadliest gynecologic malignancies and is the seventh leading cause of mortalities and morbidities globally. Although there are various therapeutic strategies, a major challenge for scientific community is to come up with effective strategy to treat ovarian cancer. Tilianin, a polyphenol flavonoid is well known for its extensive biological actions like cardioprotective, neuroprotective, anti-oxidant, anti-inflammatory, anti-diabetic and anti-tumor properties. The current study is designed to investigate the anti-cancer action of Tilianin in ovarian cancer (PA-1) cells. The findings of this study revealed that Tilianin treatment results in significant and concentration dependent decrease in cell viability. The growth inhibiting action of Tilianin is associated with apoptosis which was confirmed by DAPI and AO/EtBr staining. The Tilianin-triggered apoptosis in PA-1 cells was correlated with elevated generation of ROS, loss of mitochondrial membrane potential, alterations in pro-apoptotic (upregulated mRNA expression of Bax) and anti-apoptotic (downregulated mRNA expression of Bcl2) factors and activation of caspase-8, −9 and −3. Cell cycle analysis revealed that Tilianin treatment prevented G1/S transition through reduced mRNA expression of cyclin D1. Additionally, the findings of this study also showed Tilianin inhibited JAK2/STAT3 signaling (downregulated expression of pJAK2, JAK2, pSTAT3, and STAT3) with no change in mRNA expression level of ERK indicating its non-involvement in the apoptotic and/or growth inhibition of ovarian cancer cells. In conclusion, the findings of this exploration provided clear evidence of anti-cancer effects of Tilianin in PA-1 cells through its anti-proliferative action, ability to induce apoptosis both through extrinsic and intrinsic pathways, cell cycle (G1/S) arrest and JAK2/STAT3 signaling inhibition.     

Targeting EphA2 suppresses hepatocellular carcinoma initiation and progression by dual inhibition of JAK1/STAT3 and AKT signaling

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Synthetic derivatives of the natural product 13-amino 2-desoxy-4-epi-pulchellin inhibit STAT3 signaling and induce G2/M arrest and death of colon cancer cells

2-Desoxy-4-epi-puchellin (PCL) is a sesquiterpene-lactone, which naturally occurs in many traditional Chinese medicinal plants, and has antitumor and anti-inflammatory activities. In this work, a series of 13-amino derivatives of PCL were synthesized through Michael addition reaction. Inhibition of IL-6-induced STAT3 signaling pathway and in vitro cytotoxicity of these compounds were evaluated, and their effect on the cell cycle was also studied. The methyl hydroxyethylamine derivatives showed higher potency than PCL, which could induce significant mitotic arrest via G2/M arrest in HCT116 cancer cells, indicating that these derivatives have the potential to be developed into anti-colon cancer drugs.     

BST2 regulates interferon gamma-dependent decrease in invasion of HTR-8/SVneo cells via STAT1 and AKT signaling pathways and expression of E-cadherin

ABSTRACT

The mechanism by which interferon-gamma (IFN-γ) downregulates trophoblast invasion needs further investigation. Treatment of HTR-8/SVneo cells with IFN-γ led to a decrease in their invasion concomitant with an increased expression of BST2. Silencing of BST2 by siRNA showed a significant increase in their invasion and spreading after treatment with IFN-γ as well as downregulated expression of E-cadherin. Further, STAT1 silencing inhibited the IFN-γ-dependent increase in the expression of BST2 and E-cadherin. Treatment of HTR-8/SVneo cells with IFN-γ led to the activation of AKT, and its inhibition with PI3K inhibitor abrogated IFN-γ-mediated decrease in invasion/spreading and downregulated BST2 and E-cadherin expression. Collectively, IFN-γ decreases the invasion of HTR-8/SVneo cells by STAT1 and AKT activation via increased expression of BST2 and E-cadherin.     

Essential role of Pin1 via STAT3 signalling and mitochondria‐dependent pathways in restenosis in type 2 diabetes

Type 2 diabetes (T2D) is associated with accelerated restenosis rates after angioplasty. We have previously proved that Pin1 played an important role in vascular smooth muscle cell (VSMC) cycle and apoptosis. But neither the role of Pin1 in restenosis by T2D, nor the molecular mechanism of Pin1 in these processes has been elucidated. A mouse model of T2D was generated by the combination of high‐fat diet (HFD) and streptozotocin (STZ) injections. Both Immunohistochemistry and Western blot revealed that Pin1 expression was up‐regulated in the arterial wall in T2D mice and in VSMCs in culture conditions mimicking T2D. Next, increased activity of Pin1 was observed in neointimal hyperplasia after arterial injury in T2D mice. Further analysis confirmed that 10% serum of T2D mice and Pin1‐forced expression stimulated proliferation, inhibited apoptosis, enhanced cell cycle progression and migration of VSMCs, whereas Pin1 knockdown resulted in the converse effects. We demonstrated that STAT3 signalling and mitochondria‐dependent pathways played critical roles in the involvement of Pin1 in cell cycle regulation and apoptosis of VSMCs in T2D. In addition, VEGF expression was stimulated by Pin1, which unveiled part of the mechanism of Pin1 in regulating VSMC migration in T2D. Finally, the administration of juglone via pluronic gel onto injured common femoral artery resulted in a significant inhibition of the neointima/media ratio. Our findings demonstrated the vital effect of Pin1 on the VSMC proliferation, cell cycle progression, apoptosis and migration that underlie neointima formation in T2D and implicated Pin1 as a potential therapeutic target to prevent restenosis in T2D.     

LncRNA-NEAT1对妊娠期高血压大鼠JAK2/STAT3信号通路、炎症反应和妊娠结局的影响

摘要 目的：探讨LncRNA-NEAT1对妊娠期高血压大鼠JAK2/STAT3信号通路、炎症反应和妊娠结局的影响。方法：采用注射L-精氨酸甲酯的方法构建妊娠期高血压大鼠模型。采用Western blot检测JAK2/STAT3信号通路蛋白表达;采用ELISA法检测炎症因子和血管内皮损伤因子。观察并记录大鼠24 h蛋白尿、尾静脉压和死胎率。结果：与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组JAK2、STAT3的蛋白表达水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组JAK2、STAT3的蛋白表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组JAK2、STAT3的蛋白表达水平明显更高（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组ET-1和sICAM-1水平明显更高,而NO水平明显更低（P＜0.05）;与模型组相比,LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组ET-1和sICAM-1水平明显更高（P＜0.05）,而NO水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组ET-1和sICAM-1表达水平明显更高,而NO明显更低（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组TNF-α、IL-1β、IL-18表达水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组TNF-α、IL-1β、IL-18表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组TNF-α、IL-1β、IL-18表达水平明显更高（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组尾静脉压和24h蛋白尿水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组尾静脉压和24 h蛋白尿表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组尾静脉压和24 h蛋白尿表达水平明显更高（P＜0.05）;LncRNA-NEAT1过表达组（21.56%）死胎率显著高于模型组（16.72%）和LncRNA-NEAT1抑制组（5.65%）。结论：妊娠期糖尿病大鼠LncRNA-NEAT1的表达下调可抑制JAK2/STAT3信号通路的表达并下调下游促炎因子的表达,进而缓解血管内皮损伤降低死胎率。     

MicroRNA-216b targets HK2 to potentiate autophagy and apoptosis of breast cancer cells via the mTOR signaling pathway

Patients suffering from breast cancer (BC) still have a poor response to treatments, even though early detection and improved therapy have contributed to a reduced mortality. Recent studies have been inspired on the association between microRNAs (miRs) and therapies of BC. The current study set out to investigate the role of miR-216b in BC, and further analyze the underlining mechanism. Firstly, hexokinase 2 (HK2) and miR-216b were characterized in BC tissues and cells by RT-qPCR and Western blot assay. In addition, the interaction between HK2 and miR-216b was analyzed using dual luciferase reporter assay. BC cells were further transfected with a series of miR-216b mimic or inhibitor, or siRNA targeting HK2, so as to analyze the regulatory mechanism of miR-216b, HK2 and mammalian target of rapamycin (mTOR) signaling pathway, and to further explore their regulation in BC cellular behaviors. The results demonstrated that HK2 was highly expressed and miR-216b was poorly expressed in BC cells and tissues. HK2 was also verified as a target of miR-216b with online databases and dual luciferase reporter assay. Functionally, miR-216b was found to be closely associated with BC progression via inactivating mTOR signaling pathway by targeting HK2. Moreover, cell viability, migration and invasion were reduced as a result of miR-216b upregulation or HK2 silencing, while autophagy, cell cycle arrest and apoptosis were induced. Taken together, our findings indicated that miR-216b down-regulates HK2 to inactivate the mTOR signaling pathway, thus inhibiting the progression of BC. Hence, this study highlighted a novel target for BC treatment.     

Design,synthesis, and in vitro evaluation of BP-1-102 analogs with modified hydrophobic fragments for STAT3 inhibition

Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel compounds was evaluated using several human and two mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were further tested in additional six human and two murine cancer cell lines, in all of which they manifested the cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure–activity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.     

The JAK2/STAT3 Signal Pathway Regulates the Expression of Genes Related to Skeletal Muscle Development and Energy Metabolism in Mice and Mouse Skeletal Muscle Cells

The gene encoding a β-galactosidase from Entevobacter cloacae GAO was cloned and expressed in Escherichia coli. The nucleotide sequence of the insert of a positive clone had an open reading frame of 3084 bp that encoded a polypeptide of 1028 amino acid residues with a calculated molecular mass of 116,677 daltons. The amino acid sequence of β-galactosidase deduced from the nucleotide sequence, especially the sequence around the putative active site and of the fourteen regions, showed significant homology to β-galactosidases of other microorganisms, E. coli, Klebsiella pneumoniae, Lactobacillus bulgaricus, and Clostridium acetobutylicum.