Endothelial-specific intron-derived miR-126 is down-regulated in human breast cancer and targets both VEGFA and PIK3R2

Endothelial cells are the key components of vascular intima and play pivotal roles in vasculogenesis, angiogenesis, and tumor growth. Using Northern blot and real-time PCR, we confirmed that miR-126 and its host gene EGF-like domain 7 (EGFL7) were widely expressed in rat tissues but strictly expressed in endothelial cells. In mammals, miR-126 gene is embedded in intron7 of EGFL7. To explore the biogenesis of miR-126, plasmid EGFL7(126)-pEGFPc1 containing segment of exon7-intron7-exon8 of EGFL7 was constructed and expressed in 293T. Expression of spliced exon7-8 and excised mature miR-126 was detected by PCR and Northern blot. Knocking-down of endothelial endogenous miR-126 did not affect EGFL7 expression at mRNA or protein level. To investigate the possible roles of miR-126, PicTar, miRBase, miRanda, Bibiserv, and Targetscan were used to screen the targets. VEGFA and PIK3R2 were confirmed as the targets of miR-126 by luciferase reporter assay and Western blot. Interestingly, Northern blot and western blot showed that miR-126 was down-regulated in breast tumors where the VEGF/PI3K/AKT signaling pathway was activated. Introduction of miR-126 mimics into MCF-7 could effectively decrease VEGF/PI3K/AKT signaling activity. In summary, miR-126 was strictly expressed in endothelial cells and excised from EGFL7 pre-mRNA without affecting splicing and expression of its host gene. In addition, miR-126 could target both VEGFA and PIK3R2, and its expression was decreased in human breast cancer, implying that miR-126 may play a role in tumor genesis and growth by regulating the VEGF/PI3K/AKT signaling pathway.     

MicroRNA-294 promotes cellular proliferation and motility through the PI3K/AKT and JAK/STAT pathways by upregulation of NRAS in bladder cancer

In our study we examined the role of microRNA-294 (miR-294) in bladder cancer and related mechanisms. Realtime polymerase chain reaction (RT-PCR) was performed to determine the expression level of miR-294. Western blot was used to determine the expression of NRAS, mainly factors in the PI3K/AKT and JAK/STAT pathways. Cell counting kit8 assay, clonogenic assay, wound-healing assay, transwell and flow cytometry were used to explore, respectively, cell proliferation, survival, migration, invasion, and apoptosis of bladder cancer cell line T24. The expressions of miR-294 in bladder cancer cells including J82, HT1376, T24, and SW780 were significantly increased compared to those in human bladder epithelium cells (both HCV29 and SV-HUC-1). The proliferation rate, surviving fraction, migration, and invasion of T24 cells in miR-294 mimetic transfected group were significantly increased, while they were significantly decreased by miR294 inhibitor transfection. Moreover, miR-294 suppression could increase the apoptotic rate of T24 cells. In addition, drug resistance of T24 cells to cisplatin was increased in miR-294 mimetic-treated group, while it was decreased by miR-294 inhibitor compared to empty control. Overexpression of miR-294 could upregulate NRAS expression in T24 cells and activate PI3K/AKT and JAK/STAT pathways. We found that miR-294 expression was positively related with proliferation and motility of T24 cells. Moreover, miR-294 suppression could promote the sensitivity of T24 cells to cisplatin. We also found miR-294 could upregulate NRAS and activate the PI3K/AKT and JAK/STAT pathways in T24 cells.     

Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway

Oncostatin M (OSM), a member of the IL-6 superfamily of cytokines, is elevated in patients with rheumatoid arthritis and, in synergy with IL-1, promotes cartilage degeneration by matrix metalloproteinases (MMPs). We have previously shown that OSM induces MMP and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene expression in chondrocytes by protein tyrosine kinase-dependent mechanisms. In the present study, we investigated signaling pathways regulating the induction of MMP and TIMP-3 genes by OSM. We demonstrate that OSM rapidly stimulated phosphorylation of Janus kinase (JAK) 1, JAK2, JAK3, and STAT1 as well as extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun N-terminal kinase 1/2 mitogen-activated protein kinases in primary bovine and human chondrocytes. A JAK3-specific inhibitor blocked OSM-stimulated STAT1 tyrosine phosphorylation, DNA-binding activity of STAT1 as well as collagenase-1 (MMP-1), stromelysin-1 (MMP-3), collagenase-3 (MMP-13), and TIMP-3 RNA expression. In contrast, a JAK2-specific inhibitor, AG490, had no impact on these events. OSM-induced ERK1/2 activation was also not affected by these inhibitors. Similarly, curcumin (diferuloylmethane), an anti-inflammatory agent, suppressed OSM-stimulated STAT1 phosphorylation, DNA-binding activity of STAT1, and c-Jun N-terminal kinase activation without affecting JAK1, JAK2, JAK3, ERK1/2, and p38 phosphorylation. Curcumin also inhibited OSM-induced MMP-1, MMP-3, MMP-13, and TIMP-3 gene expression. Thus, OSM induces MMP and TIMP-3 genes in chondrocytes by activating JAK/STAT and mitogen-activated protein kinase signaling cascades, and interference with these pathways may be a useful approach to block the catabolic actions of OSM.     

Curcumin induces apoptosis in JAK2‐mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti‐proliferative and pro‐apoptotic effects of curcumin in JAK2 V617F‐mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F‐mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.     

Inhibition of miR-21-5p suppresses high glucose-induced proliferation and angiogenesis of human retinal microvascular endothelial cells by the regulation of AKT and ERK pathways via maspin

The aim of the present study is to investigate the role of miR-21-5p in angiogenesis of human retinal microvascular endothelial cells (HRMECs). HRMECs were incubated with 5 mM glucose, 30 mM glucose or 30 mM mannitol for 24 h, 48 h or 72 h. Then, HRMECs exposed to 30 mM glucose were transfected with miR-21-5p inhibitor. We found that high glucose increased the expression of miR-21-5p, VEGF, VEGFR2 and cell proliferation activity. Inhibition of miR-21-5p reduced high glucose-induced proliferation, migration, tube formation of HRMECs, and reversed the decreased expression of maspin as well as the abnormal activation of PI3K/AKT and ERK pathways. Down-regulation of maspin by siRNA significantly increased the activities of PI3K/AKT and ERK pathways. In conclusion, inhibition of miR-21-5p could suppress high glucose-induced proliferation and angiogenesis of HRMECs, and these effects may partly dependent on the regulation of PI3K/AKT and ERK pathways via its target protein maspin.     

Curcumin blocks small cell lung cancer cells migration, invasion, angiogenesis, cell cycle and neoplasia through Janus kinase-STAT3 signalling pathway

Curcumin, the active component of turmeric, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about its anti-tumor mechanism in small cell lung cancer (SCLC). In this study, we found that curcumin can inhibit SCLC cell proliferation, cell cycle, migration, invasion and angiogenesis through suppression of the STAT3. SCLC cells were treated with curcumin (15 μmol/L) and the results showed that curcumin was effective in inhibiting STAT3 phosphorylation to downregulate of an array of STAT3 downstream targets ,which contributed to suppression of cell proliferation, loss of colony formation, depression of cell migration and invasion. Curcumin also suppressed the expression of proliferative proteins (Survivin, Bcl-X(L) and Cyclin B1), and invasive proteins (VEGF, MMP-2, MMP-7 and ICAM-1). Knockdown of STAT3 expression by siRNA was able to induce anti-invasive effects in vitro. In contrast, activation of STAT3 upstream of interleukin 6 (IL-6) leads to the increased cell proliferation ,cell survival, angiogenesis, invasion, migration and tumor growth. Our findings illustrate the biologic significance of IL-6/JAK/STAT3 signaling in SCLC progression and provide novel evidence that the pathway may be a new potential target for therapy of SCLC. It was concluded that curcumin is a potent agent in the inhibition of STAT3 with favorable pharmacological activity,and curcumin may have translational potential as an effective cancer therapeutic or preventive agent for SCLC.     

ETS-1 protein regulates vascular endothelial growth factor-induced matrix metalloproteinase-9 and matrix metalloproteinase-13 expression in human ovarian carcinoma cell line SKOV-3

Matrix metalloproteinase-mediated degradation of extracellular matrix is a crucial event for invasion and metastasis of malignant cells. The expressions of matrix metalloproteinases (MMPs) are regulated by different cytokines and growth factors. VEGF, a potent angiogenic cytokine, induces invasion of ovarian cancer cells through activation of MMPs. Here, we demonstrate that invasion and scattering in SKOV-3 cells were induced by VEGF through the activation of p38 MAPK and PI3K/AKT pathways. VEGF induced the expression of MMP-2, MMP-9, and MMP-13 and hence regulated the metastasis of SKOV-3 ovarian cancer cells, and the activities of these MMPs were reduced after inhibition of PI3K/AKT and p38 MAPK pathways. Interestingly, VEGF induced expression of ETS-1 factor, an important trans-regulator of different MMP genes. ETS-1 bound to both MMP-9 and MMP-13 promoters. Furthermore, VEGF acted through its receptor to perform the said functions. In addition, VEGF-induced MMP-9 and MMP-13 expression and in vitro cell invasion were significantly reduced after knockdown of ETS-1 gene. Again, VEGF-induced MMP-9 and MMP-13 promoter activities were down-regulated in ETS-1 siRNA-transfected cells. VEGF enriched ETS-1 in the nuclear fraction in a dose-dependent manner. VEGF-induced expression of ETS-1 and its nuclear localization were blocked by specific inhibitors of the PI3K and p38 MAPK pathways. Therefore, based on these observations, it is hypothesized that the activation of PI3K/AKT and p38 MAPK by VEGF results in ETS-1 gene expression, which activates MMP-9 and MMP-13, leading to the invasion and scattering of SKOV-3 cells. The study provides a mechanistic insight into the prometastatic functions of VEGF-induced expression of relevant MMPs.     

Leptin介导的JAK/STAT信号通路对脂类代谢调节的研究进展

Leptin介导的JAK/STAT信号通路主要参与脂类代谢的调节。JAK/STAT信号通路激活后,CPT-1的表达水平升高,通过促进脂肪酸分解而参与脂类代谢的调节。本文主要介绍了近年来关于leptin介导的JAK/STAT信号通路的组成、作用机制、活性调节和leptin与受体结合激活细胞内多个信号通路如JAK/STAT、PI3K/Akt、MAPK等,以及这些信号通路对脂类代谢调节的最新研究进展。     

Vascular endothelial growth factor signaling in VE-cadherin expression and tube-like formation by rheumatoid arthritic synovial fibroblast-like cells

An increase in the vasculature is one of most representative changes in the synovial tissue of joints in rheumatoid arthritis (RA) and is closely associated with disease progression. Although the vasculatures are believed to be a result of VE-cadherin-dependent angiogenesis and a possible therapeutic target of the disease, synovial fibroblastic cells express VE-cadherin and form tube-like structures, suggesting that vasculatures in RA synovium may not simply result from angiogenesis. This paper analyzes a mechanism of VE-cadherin expression by rheumatoid arthritic synovial fibroblast-like cells (RSFLs) and their involvement in the tube-like formation. A representative angiogenic factor, vascular endothelial growth factor (VEGF), and its binding to a predominant receptor (VEGFR2) activated VE-cadherin expression and the signaling pathways of ERK/MAPK and PI3K/AKT/mTOR. Treatment of RSFLs with signaling pathway inhibitors, VEGFR2 siRNA and a VEGF-antagonizing mimicking peptide inhibited VE-cadherin expression dose-dependently. VEGF-stimulated tube-like formation by RSFLs on Matrigel was hindered by the mimicking peptide and inhibitor treatment. This data demonstrates that RSFLs activated by VEGF binding of VEGFR2 express VE-cadherin and formed tube-like structure under the control of ERK/MAPK and PI3K/AKT/mTOR pathways suggesting that the inhibition suppresses vascular development in RA synovium.     

The TRAF3 adaptor protein drives proliferation of anaplastic large cell lymphoma cells by regulating multiple signaling pathways

T cells devoid of tumor necrosis factor receptor associated factor-3 (Traf3) exhibit decreased proliferation, sensitivity to apoptosis, and an improper response to antigen challenge. We therefore hypothesized that TRAF3 is critical to the growth of malignant T cells. By suppressing TRAF3 protein in different cancerous T cells, we found that anaplastic large cell lymphoma (ALCL) cells require TRAF3 for proliferation. Since reducing TRAF3 results in aberrant activation of the noncanonical nuclear factor-κB (NF-κB) pathway, we prevented noncanonical NF-κB signaling by suppressing RelB together with TRAF3. This revealed that TRAF3 regulates proliferation independent of the noncanonical NF-κB pathway. However, suppression of NF-κB-inducing kinase (NIK) along with TRAF3 showed that high levels of NIK have a partial role in blocking cell cycle progression. Further investigation into the mechanism by which TRAF3 regulates cell division demonstrated that TRAF3 is essential for continued PI3K/AKT and JAK/STAT signaling. In addition, we found that while NIK is dispensable for controlling JAK/STAT activity, NIK is critical to regulating the PI3K/AKT pathway. Analysis of the phosphatase and tensin homolog (PTEN) showed that NIK modulates PI3K/AKT signaling by altering the localization of PTEN. Together our findings implicate TRAF3 as a positive regulator of the PI3K/AKT and JAK/STAT pathways and reveal a novel function for NIK in controlling PI3K/AKT activity. These results provide further insight into the role of TRAF3 and NIK in T cell malignancies and indicate that TRAF3 differentially governs the growth of B and T cell cancers.