Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is an RNA helicase required for the translation of 5’ structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5’ structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity. Cytosolic sensor melanoma differentiation-associated gene-5 (MDA5) specifically detects long-duplex RNAs in the genome of double-stranded (ds)RNA viruses such as Encephalomyocarditis virus (EMCV) whereas retinoic acid-inducible gene-I (RIG-I) preferentially recognizes vesicular stomatitis virus (VSV) to trigger signaling cascades that lead to production of interferons and cytokines. However, weak RNA binding capacity of MDA5 significantly attenuates the antiviral response. Here, we reveal that DHX29 as a co-sensor of MDA5 ensures the specific and efficient MDA5-RNA interactions leading to a complete MDA5-mediated antiviral signaling. Depletion of DHX29 substantially reduces the activity of MDA5 whereas activity of RIG-I remains intact. These findings provide a mechanism for DHX29 coactivation of MDA5 and a biological context for MDA5-RNA filaments in antiviral response. RIG-I-like receptors, Toll-like receptors (TLRs), Nod-like receptors, and DNA sensors are germline-encoded pattern-recognition receptors (PRRs) that serve as the first line of defense against invading pathogens, including bacteria and viruses , 2. PRRsIn this study, our investigation directed towards the identification of factors required for recognition of MDA5 for viral mRNA revealed that the RNA helicase, DHX29 functions as a dsRNA co-sensor and exclusively interacts with MDA5 for enhanced RNA recognition in response to high molecular weight polyinosinic-polycytidylic acid [HMW-Poly(I:C)] treatment or EMCV infection in immune cells. In this respect, it is worth-mentioning that RNA helicases play critical roles in multiple biological processes, including protein translation initiation and stress granule formation –27. In aIFN-β transcription as well as TBK1 and IRF3 phosphorylation in response to intracellular HMW Poly(I:C), but not to intracellular LMW Poly(I:C) or Poly(dA:dT) treatment . Similar increase in IFIT1 (encodes ISG56), IFIT2 (encodes ISG54), and CCL5 mRNA expression, was observed in response to intracellular HMW Poly(I:C) in DHX29 overexpressed cells  . In addiDHX29 expression or induction, and found that DHX29 expression was not affected by HMW Poly(I:C) or EMCV stimulation (DHX29-specific lentivirus short hairpin RNA (shRNA) constructs could effectively knockdown endogenous DHX29 expression (DHX29 (We first assessed the effect of HMW Poly(I:C) stimulation on mulation . We nextpression . Furthern (DHX29 .DHX29 KD markedly reduced TBK1 and IRF3 phosphorylation in response to intracellular HMW Poly(I:C) treatment, but not in response to LMW Poly(I:C) or Poly(dA:dT), in 293T cells (DHX29 KD greatly reduced ISRE-Luc and IFN-β-Luc activities in 293T cells stimulated with intracellular HMW Poly(I:C), which could be rescued by ectopic expression of HA-DHX29 (DHX29 KD had little or no effect on ISRE-Luc and IFN-β-Luc activities in 293T-TLR3 cells stimulated with LMW Poly(I:C) or 293T cells stimulated with intracellular LMW Poly(I:C) or Poly(dA:dT) , but not intracellular LMW Poly(I:C) or poly(dAdT) (3T cells . ConsistHA-DHX29 . Howevery(dA:dT) . Furtherly(dAdT) , suggestDHX29 KD on the expression of type I IFN-stimulated genes and IFN-β production was determined in human and mouse immune cells. Human THP-1 cells, human peripheral blood mononuclear cells (PBMCs), and mouse RAW cells were transfected with DHX29-specific small interfering RNA (siRNA). High efficiency siRNA-mediated KD of DHX29 was observed in all three types of cells  or HMW Poly(I:C) in lyo/vec form (Vec), but not to Poly(dA:dT)  . Taken tDHX29 knockout (KO) experiments in human peripheral blood mononuclear cells (PBMCs), mouse bone marrow-derived dendritic cells (BMDCs) and peritoneal macrophages using a lentiviral-based Cas9-P2A-puromycin fusion CRISPR . Of note, both human and murine DHX29-sgRNA/Cas9 lentiCRISPR could efficiently knock out DHX29 in puromycin-selected bulk PBMCs, BMDCs, and peritoneal macrophages, as determined by western blot and a surveyor assay (DHX29 KO markedly reduced TBK1 and IRF3 phosphorylation and IFN-β production in response to HMW Poly(I:C) in lyo/vec form (Vec) and EMCV, but did not affect IRF3 phosphorylation and IFN-β production in response to LMW Poly(I:C) Vec, Poly(dA:dT) or VSV in human PBMCs  treatment and EMCV infection.To further substantiate the biological role of DHX29 in type I IFN signaling in both human and mouse primary cells, we performed or assay . DHX29 Kan PBMCs . Similarrophages . Thus, tal cells . It shoual cells –33, 44, DHX29 stably transfected (mDHX29) mouse embryonic fibroblasts (MEFs), when compared with empty vector-transduced MEFs  MEFs, compared with scrambled siRNA transfected (WT and RIG-I-deficient) MEFs after EMCV infection  and deficient MEFs demonstrated the lack of IFN-β secretion in 9 status . In contced MEFs . By contnfection . Overallnfection . Howevernfection , suggest WT MEFs . As expe WT MEFs . Taken tHA-DHX29 and Flag-RIG-I, Flag-MAVS, or Flag-MDA5. Both human and mouse DHX29 interacted with MDA5, but not with RIG-I or MAVS  assays were performed using 293T cells transfected with  or MAVS . After d293T cells were co-transfected with expression vectors encoding proteins RIG-I, MDA5, MAVS and TBK1 together with increasing amounts of expression vector that encodes DHX29 protein along with the ISRE or NF-ĸB luciferase reporter. We found that DHX29 specifically enhanced activation of the luciferase reporters induced by MDA5, but not others . To furtInterestingly, under stringent condition in the presence of our lysis buffer containing 1% Triton X-100, DHX29 did not interact with LGP2 or RIG-I, even after various kinds of stimulations. But in much lesser stringent condition such as in the presence of cell lysis buffer containing 0.1% NP-40 which was used in a recent report , we deteDHX29 KD decreased the MDA5-MAVS interaction in response to HMW Poly(I:C) stimulation - or MDA5-induced ISRE-Luc activity, whereas DHX29c and DHX29d had no effect on the ISRE-luc activity compared with the vector control  were generated and tested for their abilities to interact with MDA5 . DHX29a  control , suggestTo identify the molecular mechanisms by which DHX29 enhances MDA5-mediated type I IFN signaling, we tested whether DHX29 has the ability to bind to HMW Poly(I:C). To test this possibility, we performed a pull-down experiment with NeutrAvidin-beads from cell lysates of 293T cells cotransfected with biotin-labeled HMW Poly(I:C) along with MDA5, DHX29 fragments , or MDA5 in combination with DHX29 fragments. We found that unlike DHX29a, FL-DHX29, DHX29c and MDA5 alone or in combination could be immunoprecipitated with anti-biotin beads , suggestIt has been known that MDA5 has a poor binding affinity for HMW Poly(I:C) and viral dsRNA , 15. TheBecause DHX29 contains two ATP binding motifs  in the DEXD motif-containing domain , we nextDephosphorylation of MDA5 and RIG-I is a key step of their activation , 45, 46.Several recent studies show that MDA5 forms filaments upon binding to dsRNA, which is critical for the activation of the IFN-β signaling pathway , 48. To RIG-I and MDA5 are key RNA sensors that recognize different sets of viruses. MDA5 primarily senses positive-strand viruses from the Picornaviridae family, such as EMCV, whereas RIG-I recognizes SeV, VSV, and influenza virus , 9, 10. DHX29 in various cell types, further substantially demonstrate the specificity and requirement of DHX29 as an RNA co-sensor in the innate immune response to HMW Poly(I:C) treatment and EMCV infection. Moreover, the presence of DXH29 in both human and mouse cells supports for its functional conservation between human and mouse. Hence, our results unequivocally establish that DHX29 is expressed in all types of immune cells tested, including PBMC, monocyte, B cells, T cells, macrophages and BMDC, and is essentially required for cell proliferation and survival; Importantly, our results have clearly demonstrated that DHX29 functions as an MDA5-specific RNA sensor in response to HMW Poly(I:C) and EMCV, but not to LMW Poly(I:C) or VSV, in immune cells tested. Further evidence shows that DHX29 alone cannot trigger type I IFN signaling in immune cells; it must interact with MDA5, but not RIG-I, to activate the type I IFN signaling pathway in various immune cell types, including THP-1, human PBMCs, murine macrophages and DCs. Of note, DHX29 does not interact with MDA5 in uninfected or untreated cells, but rather is induced after HMW Poly(I:C) treatment or EMCV infection. DHX29 does not interact with LGP2 or RIG-I even after viral infection, suggesting that the N-terminus of DHX29 specifically binds to the N-terminal (CARD-containing) domain of MDA5. However, unlike full-length DHX29, the truncated N-terminus of DHX29 (without the C-terminus) inhibits MDA5-mediated type I IFN signaling, suggesting that full-length DHX29 is required for its ability to enhance type I IFN signaling.To demonstrate the importance of DHX29 expression, several studies have shown that DHX29 is essentially required for protein translation, cell proliferation and survival –33, 44. The role of LGP2 in antiviral immunity is controversial. Both positive and negative effects on RIG-I- and MDA5-mediated type I IFN signaling have been reported in response to different viruses , 21–24. DHX29 and MDA5 markedly increases the binding capacity of MDA5 to HMW Poly(I:C), suggesting that DHX29 and MDA5 function as co-sensors for detecting EMCV infection. Further experiments demonstrate that the DEXD  and helicase domains of DHX29 are responsible for HMW Poly(I:C) binding. Thus, it appears that DHX29 specifically interacts with MDA5 through its N-terminus and binds to HMW Poly(I:C) through its C-terminal DEXD and helicase domains, thus bringing MDA5 and HMW Poly(I:C) together. Indeed, our EMSA and AFM results show that the dsRNA-binding activity of DHX29 and MDA5 is much better than MDA5 alone. Overall, further analysis is needed to clarify whether the helicase activity of DHX29 is required for its function as co-sensor for MDA5-dsRNA. The potential role of DHX29 in the modulating of ATPase activity and oligomerization of MDA5 [To understand the molecular mechanisms by which DHX29 enhances MDA5-mediated type I IFN signaling, we show that DHX29 has the ability to bind to HMW Poly(I:C) with a strong binding affinity compared with the low HMW Poly(I:C)-binding affinity of MDA5. This is in agreement with previous studies showing that MDA5 has a poor binding affinity for HMW Poly(I:C) and viral dsRNA , 15. Coe of MDA5 –59 requiBased on the results presented here and previously published data, we propose a working model to illustrate the role of DHX29 in MDA5-mediated innate immune response to HMW Poly(I:C) treatment and EMCV infection . During Buffy coats of blood from healthy donors  were used for isolation of PBMCs  by density-gradient centrifugation with Lymphoprep (Nycomed Pharm). All blood samples were anonymized. The use of PBMCs was in accordance with institutional guidelines on human cell research and the approved protocol (Protocol #IBC00000357) by the Institutional Review Board of Houston Methodist Research Institute. Animal experiments in this study were approved and carried out in accordance with protocol (Protocol #AUP-0115-0005) provided by the Institutional Animal Care and Use Committee (IACUC) at Houston Methodist Research Institute. IACUC uses the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals, which is based on the U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training.5) cells  were seeded into 24-well plates the day before transfection. Cells were cotransfected with the IFN-β or ISRE promoter luciferase reporter and Renilla luciferase internal control (pRL-TK) using Lipofectamine 2000 (Invitrogen) as previously described [Human embryonic kidneys (HEK) 293T  and Vesicular stomatitis virus  were amplified in Vero cells  and 293T cells, respectively, and stored at -80°C until use. After three cycles of freeze-thawing to release intracellular virus, the supernatant was collected by spinning and used in plaque-forming assays.g at 4°C for 2 h) and frozen at -80°C. Murine Bone marrow-derived dendritic cells (BMDCs) were generated by flushing bone marrow cells from femurs and tibiae of mice and were cultured in RPMI media containing 10% FBS supplemented with GM-CSF and IL-4. Peritoneal macrophages (pMAC) were obtained by injecting mice with 4% (v/v) thioglycollate (Beckton Dickson), and peritoneal cavities were flushed after 3 days with RPMI media/2% FBS. Isolated PBMCs, BMDCs, and pMAC were transduced with 4 mixed DHX29-sgRNA-containing lentiCRISPR viruses, respectively. Transduced cells were selected in the presence of puromycin (Invivogen) for 48 h and subjected to ligand stimulation or viral infection.We designed human and murine DHX29 sgRNA as previously described . Four hDThe immunofluorescence assays and confocal microscopy were conducted as described previously . 293T ceFor in-cell pull-down assays, clarified whole cell lysates (WCL) transfected with plasmid constructs expressing HA- or Flag-tagged protein were preincubated with NeutrAvidin beads for 2 h (Pierce). Biotin-labeled Poly (I:C) was added, and WCL were incubated for an additional 4 h at 4°C. For cell-free pull-down assays, WCL transfected with HA- or Flag-tagged protein were incubated with anti-HA or anti-Flag beads (Sigma), and proteins were eluted from the beads using the corresponding peptide. The purified proteins were then added to the NeutrAvidin beads and coincubated with Poly(I:C) for 4 h. After incubation, protein complexes were washed 5 times, boiled in 4X loading buffer, and subjected to SDS-PAGE.24bp HPLC-grade RNA oligonucleotide 5´-GCGUCGUACGCUAG CGUACGACGC-3´ was purchased from Integrated DNA Technologies. Sample preparation was performed as previously described . Samplest-test.Unless otherwise indicated, all data are plotted as means ± S.D. Significant differences between groups were determined by two-tailed Student's S1 Text(DOCX)Click here for additional data file.S1 FigA-C) Real-time PCR analysis of (A) IFIT1, (B) IFIT2, and (C) CCL5 mRNA expression in Flag-DHX29- and empty vector-transfected 293T cells stimulated with IC HMW Poly(I:C). Data are plotted as the mean ± s.d. Results of (A-C) are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to ((TIF)Click here for additional data file.S2 FigA) Immunoblot analysis showing DHX29 expression in THP-1 cells stimulated with intracellular (IC) HMW Poly(I:C)  or EMCV  at the indicated time points. β-actin was used as a loading control. (B) Real-time PCR analysis of IFNB and DHX29 mRNA levels in THP-1 cells stimulated with IC HMW Poly(I:C). (C) Real-time PCR analysis of the KD efficiency of DHX29-specific shRNAs. Scrambled (Scr) siRNA was used as a control. (D) 293T cells were cotransfected with HA-vector or HA-DHX29 and Scr shRNA or DHX29 shRNA. Cell viability was determined at various time points post-transfection using bromophenol blue. (E) ISRE-luciferase (Luc) (left panel) and IFN-β-Luc activities (right panel) in Scr shRNA- and DHX29 shRNA-transfected 293T-TLR3 cells stimulated exogenously with naked Poly(I:C). (F and G) ISRE-Luc (left panel) and IFN-β-Luc (right panel) activities in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with (F) IC LMW Poly(I:C) and (G) IC Poly(dA:dT). ISRE-Luc and IFN-β-Luc activities are expressed as the fold increase relative to the control. (H-J) Real-time PCR analysis of IFIT1, IFIT2, and CCL5 mRNA levels in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with IC HMW Poly(I:C). Data from (A-J) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to ((TIF)Click here for additional data file.S3 FigA) Real-time PCR analysis of the KD efficiency of DHX29 siRNA in human PBMCs, human THP-1 cells, and mouse RAW cells.  Real-time PCR analysis of IFIT2 and CCL5 mRNA levels in scrambled (Scr) siRNA- and DHX29 siRNA-transfected THP-1 cells stimulated with HMW Poly(I:C) or Poly(dA:dT).  Real-time PCR analysis of IFIT2 and CCL5 mRNA levels in Scr siRNA- and DHX29 siRNA-transfected RAW cells stimulated with HMW Poly(I:C) or Poly(dA:dT) lyo/vec. Data from (A-E) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to ((TIF)Click here for additional data file.S4 FigA, B) Western blot of DHX29-sgRNA-Cas9 LentiCRISPR transduced 293T cells (A) or MEFs (B) after puromycin selection and SURVEYOR assays.  SURVEYOR assay of DHX29-sgRNA-Cas9 LentiCRISPR mixture transduced human PBMC (C) or murine cell  (D) after puromycin selection. (E-G) Immunoblot analysis of p-TBK1 in control sgRNA- or DHX29 sgRNA-transduced PBMC (E), BMDC (F), or peritoneal macrophages (pMAC) (G), left untreated or stimulated by LMW Poly(I:C). VSV, Poly(dA:dT), EMCV or HMW Poly(I:C). (H) Morphology of Control-Cas9 or DHX29-sgRNA-Cas9 LentiCRISPR transduced pMAC at Day 9 post transduction. Scale bar = 10μm. Related to ((TIF)Click here for additional data file.S5 FigA) 293T cells were cotransfected with HA-mouse (m)DHX29 and Flag-mRIG-I, Flag-mMDA5, or Flag-mMAVS. Whole cell lysates (WCL) were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA or anti-Flag antibodies. (B) Scrambled or DHX29 siRNA transfected WT MEF and MAVS knockout MEF were stimulated with Flag-TBK1 overnight. The WCL were subjected to immunoblot with indicated antibodies. (C) Luciferase assay of RIG-I knockout 293T cell transfected with increase amount of DHX29, followed by stimulation of intracellular (IC) LMW Poly(I:C), HMW Poly(I:C), MDA5 or MAVS. (D) RIG-I Knockout 293T cells were cotransfected with HA-DHX29 and Flag-MAVS or Flag-MDA5. WCL were immunoprecipitated with anti-HA beads and immunoblotted with anti-HA or anti-Flag antibodies. Anti-RIG-I antibody is used to verify the RIG-I knockout cell in whole cell lysate. (E) HA-DHX29- and Flag-MDA5 (20 ng)-transfected 293T cells were stimulated with intracellular (IC) HMW Poly(I:C) at the indicated time points. WCL were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA, phosphorylated (p)-IRF3, and IRF3 antibodies. (F) WCL obtained from THP-1 cells stimulated with IC HMW Poly(I:C) at the indicated time points were immunoprecipitated with anti-DHX29 antibody and immunoblotted with MDA5, p-IRF3, and IRF3 antibodies. (G) 293T cells transfected with HA-DHX29 and Flag-RIG-I or Flag-MDA5 were infected with indicated kind of stimulation at 8hr. The cell lysate was immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibodies. (H) WCL obtained from 293T cells transfected with HA-DHX29 and Flag-LGP2 after 6hr EMCV treatment were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA and anti-Flag antibodies. (I) IFN-β- Luc activities in 293T cells transfected with indicated plasmids post HMW Poly(I:C) treatment were determined. (J) 293T cells expressing Flag-MAVS and Flag-MDA5 were transfected with DHX29 siRNA or scrambled (Scr) siRNA and then stimulated with IC HMW Poly(I:C). WCL were immunoprecipitated with anti-HA antibody and immunoblotted with anti-HA, anti-Flag, p-IRF3, and IRF3 antibodies. WCL were immunoprecipitated with anti-biotin beads and immunoblotted with anti-Flag and anti-HA antibodies. Data from  are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to ((TIF)Click here for additional data file.S6 FigA) WCL obtained from 293T cells co-transfected with Flag-tagged full-length (FL)-MDA5, N-MDA5, M-MDA5, or C-MDA5 and HA-DHX29a were immunoprecipitated with anti-Flag beads. The immunoprecipitated product was immunoblotted with anti-HA and anti-Flag antibodies. (B) 293T cells were cotransfected with ISRE-luciferase (Luc) and FL-DHX29, empty vector (vec), DHX29a, DHX29b, DHX29c, or DHX29d plasmids and stimulated by overexpressing MDA5. ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold increase relative to stimulated FL-DHX29 control cells. (C) 293T cells transfected with Flag-MAVS, HA-DHX29, Flag-MDA5, or Flag-MDA5 plus HA-DHX29 were incubated with biotin-labeled LMW Poly(I:C) for 4 h. (D) ISRE-luciferase (Luc) activity in 293T cells transfected with DHX29 and increasing concentrations  of WAM-DHX29, WBM-DHX29, DHX29a, or DHX29b and stimulated with intracellular (IC) HMW Poly(I:C). ISRE-Luc activity is expressed as the fold increase relative to the unstimulated control.  20ng Flag-RIG-I (E) or Flag-MDA5 (F) transfected 293T cells were co-transfected with wildtype DHX29, DHX29c, WAM and WBM of DHX29, followed by stimulation for 6 hrs or not. The lysates were immunoprecipitated with anti-Flag beads and immunoblotted with indicated antibodies. Cal A was added 1hr before lysate collection. Data from  are plotted as the mean ± s.d. Results are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. IC Poly(I:C)-stimulated cells (two-tailed Student's t-test). Related to ((TIF)Click here for additional data file.


Yersinia entercolitica (Ye)-induced ReA in TNFRp55-deficient (TNFRp55-/-) mice. In this study, we assessed the contribution of DCs in this overproduction. First, greater levels of IL-12/23p40, IFN-γand IL-17A were confirmed in supernatants of lipopolysaccharide (LPS)-stimulated TNFRp55-/-splenocytes obtained on arthritis onset (day 14 after Ye infection). Later, DCs were identified as a precise source of IL-12/23p40 since increased frequency of splenic IL-12/23p40+DCs was detected in TNFRp55-/- mice. After robust in vivo amplification of DCs by injection of Fms-like tyrosine kinase 3-Ligand (Flt3L)-transfected BL16 melanoma, DCs were purified. These cells recapitulated the higher production of IL-12/23p40 under TNFRp55deficiency. In agreement with these results, TNFRp55-/- DCs promoted Th1 and Th17 programs by co-culture with WT CD4+lymphocytes. A mechanistic study demonstrated that JNK and p38 MAPK pathways are involved in IL-12/23p40 overproduction in purified TNFRp55-/- DCs as well as in the JAWS II cell line. This deregulation was once again attributed to TNFRp55 deficiency since CAY10500, a specific inhibitor of this pathway, compromised TNF-mediated IL-12/23p40 control in LPS-stimulated WT DCs. Simultaneously, this inhibition reduced IL-10 production, suggesting its role mediating IL-12/23p40 regulation by TNFRp55 pathway. These results provide experimental data on the existence of a TNFRp55-mediated anti-inflammatory circuit in DCs. Moreover, these cells may be considered as a novel target in the treatment of ReA.Dendritic cells (DCs) play critical functions in the initiation of immune responses. Understanding their role in reactive arthritis (ReA) will help delineate the pathogenesis of this arthropathy. In early studies, we detected IL-12/23p40 deregulation in  Reactive arthritis (ReA) is a form of seronegative spondyloarthritis (SpA) characterized by predominantly low limb asymmetric oligoarthritis and extra-articular symptoms following gastrointestinal or urogenital infection ,2. ReA mYersinia enterocolitica (Ye) are Gram-negative bacteria causing food born self-limiting severe diarrhea, enteritis and mesenteric lymphadenitis . In the co-culture experiments, we detected two distinct populations of CD4+ T cells Click here for additional data file.S1 Fig+MHC-II+ gate). B. In this sample gating, cells were first analyzed as explained above and then DCs were further analyzed to measure IL-12/23p40 expression. Fluorescence-minus-one (FMO) control was included to define the selected population.A. In this sample gating, cells were first gated for leucocytes (SSC-H vs FSC-H) and then for dendritic cells (DCs) (CD11c(TIF)Click here for additional data file.S2 Fig+ cells were isolated by using anti-mouse CD11c magnetic beads. In this sample gating the isolated cells were gated for CD11c expression (SSC-H vs CD11c), and then for dendritic cells (DCs) (CD11c+MHC-II+ gate). CD80 and CD86 surface expression was then determined, and histogram analyses are shown. Fluorescence-minus-one (FMO) control (grey histogram) was included to define the selected population.Splenic CD11c(TIF)Click here for additional data file.S3 Fig+ cells were isolated by using anti-mouse CD4 magnetic beads. In this sample gating, cells were first gated for leucocytes (SSC-H vs FSC-H) and then for CD4+ T lymphocytes (CD3+ CD4+). Finally, the cells were further analyzed to measure IFN-γ or IL-17A expression. Fluorescence-minus-one (FMO) controls were used to define the selected population for each cytokine expression.Splenic CD4(TIF)Click here for additional data file.S4 FigTNFRp55-/- CD4+ T cells were co-cultivated at a 1:10 ratio with unlabeled WT or TNFRp55-/- DCs (in Ye-infected or uninfected conditions). On day 5, the cells were collected and immediately analyzed using the FACSCalibur cytometer. A and C. Representative overlaid flow cytometry histogram analysis showing CFSE expression on lymphocytes based upon forward and side light scatter profiles. Numbers indicate percentages of proliferating CD4+cells. Unproliferating cells (grey histogram) were used to define the selected population. Percentages of WT CFSE+ CD4+ T cells (B) and TNFRp55-/- CFSE+ CD4+ T cells (D). ns: not significant.CFSE-labeled WT or (TIF)Click here for additional data file.S5 Fig+ T lymphocytes (CD3+ CD4+), and finally two distinct populations were selected (gate 1 and gate 2). The cells of each gate were further analyzed to measure IFN-γ (A) or IL-17A (F) expression. Fluorescence-minus-one (FMO) controls were used to define the selected population for each cytokine expression. Percentages of WT CD4+ IFN-γ+ (B and C) and TNFRp55-/- CD4+ IFN-γ+ (D and E) T cells. Percentages of WT CD4+ IL-17A+ (G and H) and TNFRp55-/- CD4+ IL-17A+ (I and J) T cells. ** P<0.01; *** P<0.001. ns: not significant.A and F. In this sample gating, cells were first gated for leucocytes (SSC-H vs FSC-H) as showed in (TIF)Click here for additional data file.S6 FigIn this sample gating, cells were initially gated on SSC/FSC and then for live cells (7AAD negative). Finally, these cells were further analyzed to determine F4/80, CD11b, CD11c, MHC-II, CD80, CD86, TNFRp55 and TNFRp75 surface expression. Fluorescence-minus-one (FMO) control (grey histogram) was included to define the selected population in each histogram analysis.(TIF)Click here for additional data file.


Helicoverpa armigera, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of Bt transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement Bt to tackle insect-pests menace. In this study, we report host-delivered silencing of HaAce1 gene, encoding the predominant isoform of H. armigera acetylcholinesterase, by an artificial microRNA, HaAce1-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with HaAce1-amiR1/HaAce1-amiR1* sequences. The recombinant HaAce1-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing HaAce1-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of HaAce1 mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of HaAce1 gene for H. armigera management.The polyphagous insect-pest,  Helicoverpa armigera, a notorious polyphagous insect, feeds on a wide variety of agriculturally important crops like chickpea, pigeonpea, sorghum, soybean, and cotton, causing serious damage to crop yield ; b. Agrobacterium infected explants on selection medium [Pre-culture medium with kanamycin (100 mg/l) and cefataxime (500 mg/l)]; c. Initiation of adventitious shoot bud from callus; d. Well developed shoot (5–6 cm in height) transferred into rooting media [MS medium with kanamycin (100 mg/l) and cefataxime (500 mg/l)]; e. Plant with established roots; f. Plant with well developed roots shifted to pot for hardening. (B) PCR screening of putative tobacco transgenic lines with mirAce-F and NOS terminator-R1 primers spanning 150 bp region. Lanes: L, 100 bp DNA ladder; 1–30, putative HaAce1-amiR1 tobacco transgenic lines; C, vector control tobacco; W, untransformed control tobacco; P, positive control. (C) PCR analysis of HaAce1-amiR1 transgenic tobacco lines using CaMV 35S promoter-F (35SP-F) and Nos terminator-R1 (NT-R1) primers spanning 1 kb region. Lanes: L, 1 kb DNA ladder; 1–30, HaAce1-amiR1 tobacco transgenic lines; C, untransformed control tobacco; P, positive control. (D) RT-PCR analysis of HaAce1-amiR1 transgenic tobacco lines using NPTII forward and reverse primers (N- F & R). Lanes: L, 1 kb DNA ladder; 1–30, HaAce1-amiR1 tobacco transgenic lines; C, untransformed control tobacco. (E) Southern analysis of selected HaAce1-amiR1 tobacco transgenic lines. Lanes: P, HindIII linearized pBI::HAR1; 17, 20, 23, 24, 29, 30, selected HaAce1-amiR1 tobacco transgenic lines; Lane: C, Untransformed control tobacco. Number 17 stands for 17R1 carrying single copy transgene, and number 29 stands for 29R1 carrying two copies of the transgene.(A) Different stages of development of (TIF)Click here for additional data file.S3 FigFifteen second instar larvae were released on detached leaves of 17R1, 29R1 and vector control tobacco lines (five larvae per plate in three replications) for three days. (A) Left panel: Larvae fed on vector control leaves; Right panel; Larvae fed on transgenic lines, 17R1 and 29R1. (B) Larvae fed on transgenic leaves showed growth retardation of 35–55% compared to that of control insect group. One way ANOVA test was used to perform statistical analysis of the data. *** denotes extremely significant difference at P<0.001 whereas ** denotes very significant difference at P<0.01. The test was repeated three times.(TIF)Click here for additional data file.S4 Fig(A) Larvae fed on vector control leaves. (B) and (C) Larvae fed on 17R1 and 29R1 transgenic lines, respectively. Thirty synchronous second instar larvae were fed on these two transgenic lines and vector control leaves continuously till their active feeding stage (one larva per plate). Larvae fed on transgenic lines exhibited retarded growth and were smaller than the vector control fed larvae.(TIF)Click here for additional data file.S5 Figa-c. Greenish yellow jelly like appearance at the anal region of larvae; d-i. Less mobility in larvae; j. Sixth instar larva that suddenly stopped feeding and failed to pupate.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


COX-2) or mothers against decapentaplegic homologue 4 (Smad4) 3'-untranslated region (3'-UTR) and suppressed COX-2 and Smad4 expression. In contrast, a miR-144 inhibitor increased COX-2 and Smad4 expression in mouse granulosa cells (mGCs). Meanwhile, Smad4 upregulated COX-2 expression, but this effect was abolished when the mGCs were treated with the transforming growth factor beta signalling pathway inhibitor SB431542. Moreover, luciferase reporter, chromatin immunoprecipitation and electrophoretic mobility shift assay results showed that the transcription factor CP2 upregulated miR-144 expression, which partially contributed to the suppression of COX-2 in mGCs. Both CP2 and miR-144 alter prostaglandin E2 (PGE2) production by regulating COX-2 expression. In addition, miR-144 regulated mGC apoptosis and affected follicular atresia, but these activities did not appear to be through COX-2 and Smad4. Taken together, we revealed an important CP2/miR-144/COX-2/PGE2/ovulation pathway in mGCs.Mammalian folliculogenesis is a complex process in which primordial follicles develop into pre-ovulatory follicles, followed by ovulation to release mature oocytes. In this study, we explored the role of miR-144 in ovulation. miR-144 was one of the differentially expressed microRNAs, which showed 5.59-fold changes, in pre-ovulatory ovarian follicles between Large White and Chinese Taihu sows detected by Solexa deep sequencing. We demonstrated that overexpression of miR-144 significantly decreased the luciferase reporter activity under the control of the cyclooxygenase-2 ( Several endocrine and intra-ovarian factors are related to ovulation.8 Among these, prostaglandin E2 (PGE2) is a critical mediator of ovulation.9In the mammalian ovary, folliculogenesis begins from primordial germ cells, and proceeds through the primary follicle stage to the mature follicle stage and finally produces a pre-ovulatory follicle and releases an oocyte in response to a surge in luteinizing hormone (LH). A few follicles develop to ovulation, and >99% of ovarian follicles will be lost as a result of atresia.COX) is the key enzyme that controls the rate-limiting step of prostaglandin synthesis.10 To date, two well-characterized isoforms of COX have been identified, COX-1 and COX-2. COX-1 is a constitutive enzyme, whereas COX-2 is undetectable in a variety of cell types and can be a significantly induced by various hormones, growth factors and cytokines.10 In the monkey, COX-1 is not detected in the granulosa cells of large antral follicles. However, COX-2 expression substantially increased in granulosa cells of pre-ovulatory ovarian follicles stimulated by human chorionic gonadotropin (hCG).11 A large number of studies have shown that inhibition of COX-2 resulted in ovulation failure.13 Moreover, mice deficient in COX-1 are fertile, whereas COX-2-deficient mice exhibit multiple failures in female reproductive processes, including ovulation, fertilization, implantation and decidualization.14 Furthermore, ovulation is restored in the COX-2-deficient mice by simultaneous treatment with PGE2.15 Overall, these studies strongly suggest that COX-2-derived PGE2 is crucial for ovulation.Cyclooxygenase  are short, non-coding RNAs, 19–22 nucleotides in length, which act by targeting partially complementary sequences within the mRNA 3′-untranslated region (3′-UTR), leading to functional repression of target transcripts. These molecules are involved in multiple biological processes.in vivo and in vitro analyses demonstrated that transcription factor CP2 bound to and activated the miR-144 promoter in mouse granulosa cells (mGCs). Luciferase activity and expression level analyses showed that COX-2 and Smad4 were targets of miR-144. Smad4 regulated COX-2 levels via the transforming growth factor beta (TGF-β) signalling pathway. Our research indicates that miR-144 is involved in ovulation by suppressing PGE2 production in mammals.In this study, we found that the expression of miR-144 was significantly different between pre-ovulatory ovarian follicles of Large White (LW) and Taihu sows. Both P<0.05, |Fold change|>1) . In CT and LW sows, 679 and 713 known miRNAs were detected, respectively. Three hundred ninety miRNAs were differentially expressed between the two breeds, with 124 miRNAs upregulated and 266 miRNAs downregulated in CT sows (ange|>1) . Quantitange|>1) .COX-2 was predicted to be a target of miR-144. In addition, the miR-144-binding seed sequences in the COX-2 3′-UTR were highly conserved in mammals  into Chinese hamster ovarian (CHO-K1) cells, and luciferase activity was significantly suppressed. However, luciferase activity was unchanged when we co-transfected miR-144 mimics and pmirGLO-COX-2-3′-UTR-Mut into mGCs (COX-2 mRNA and protein expression levels were significantly suppressed after miR-144 mimics was transfected into mGCs and pig kidney (PK-15) cells, whereas inhibition of miR-144 increased COX-2 mRNA and protein in mGCs and PK-15 cells  or siRNA-COX-2 (si-COX-2) was transfected into mGCs, respectively  into CHO-K1 cells, and luciferase activity was significantly suppressed by miR-144. Meanwhile, miR-144 had no effect on a Smad4 3′-UTR mutated dual-luciferase construct  into mGCs knocked down Smad4 expression (COX-2 expression (pcDNA3.1-Smad4 (pc-Smad4) was transfected into mGCs and substantially increased Smad4 expression   . These rCOX-2 expression, which prompted us to examine the transcriptional regulation of miR-144 in ovarian follicles. To identify the core promoter of mouse miR-144, a series of deletions of the mouse miR-144 potential promoter were used to drive luciferase gene expression, and luciferase activity was determined. Luciferase activity analysis in both mGCs and CHO-K1 cells revealed that pGL3-miR-144-D6 (−468 bp to −375 bp) was required for miR-144 transcriptional activity  showed a substantial decrease in promoter activity (CP2 mRNA and protein expression levels were significantly increased or suppressed after pcDNA3.1-CP2 (pc-CP2) or siRNA-CP2 (si-CP2) was transfected into mGCs, respectively  analysis was performed to investigate whether CP2 bound to the mouse miR-144 promoter ipitates . These rin vitro. pCMV-C-HA-CP2-CDS was transfected into mGCs, and then, nuclear extracts were isolated from mGCs. A DNA–protein complex was detected when the CP2 probe (−419 bp to −395 bp) was incubated with nuclear extracts. An excess amount of unlabelled oligo DNA, but not the mutated CP2-binding site, could compete with this binding. Moreover, the supershift band appeared when the CP2 probe was incubated with nuclear extracts and anti-HA antibody (in vitro.Electrophoretic mobility shift assays (EMSAs) were used to further detect CP2 binding to the mouse miR-144 promoter antibody . These rpcDNA3.1-CP2 vector or siRNA-CP2 was transfected into mGCs, respectively. CP2 overexpression significantly promoted miR-144 as determined by qRT-PCR analysis, whereas knockdown of CP2 suppressed miR-144 expression  in vivo and in vitro.31 In this study, we used Solexa deep sequencing and bioinformatics analysis to define 390 known miRNAs differentially expressed between pre-ovulatory ovarian follicles of CT and LW sows. The expression patterns of seven known miRNAs  were successfully validated using qRT-PCR analyses. miRNA microarrays show that let-7 family members are differentially expressed during follicular atresia, and further research demonstrate that let-7 g induces porcine granulosa cell apoptosis by targeting mitogen-activated protein kinase kinase kinase 1 (MAP3K1) in the porcine ovary.32 miR-125a-5p is shown to regulate mGC apoptosis by targeting signal transducer and activator of transcription 3 (STAT3), and miR-125a-3p reduces the number of ovulated oocytes by targeting the Fyn proto-oncogene (Fyn).34 However, many studies on miR-144 focus on cancer cell proliferation, invasion and erythropoiesis, and few studies have examined the role of miR-144 in folliculogenesis.38Many previous studies have indicated that miRNAs have a vital role in the folliculogenesis. Deletion of Dicer in mGCs lead to female sterility and multiple reproductive defects.COX-2 gene was a target of miR-144 in both mGCs and PK-15 cells. COX-2 is primarily produced from the secondary follicle stage to the Graafian follicle stage. The production of this enzyme is stopped after the Graafian follicle stage and is resumed with exposure to the LH surge.39COX-2-deficient mice have poor ovulation and low fertilization rates.14 Moreover, ovulation can be restored by treatment of the COX-2-deficient mice with PGE2.15COX-2-derived PGE2 is essential for ovulation because it activates PGE2, which is involved in the MAPK, NF-κB and phosphatidylinositol 3-kinase/Akt pathways.41 PGE2 upregulates EGF-like growth factor biosynthesis, and EGF-like growth factors induce COX-2 expression and PGE2 production by activating the ERK1/2 signalling pathway in human granulosa cells.43 We confirm that miR-144 suppresses PGE2 production by targeting COX-2. Therefore, this study provides a new mechanism by which miR-144 regulates ovulation via targeting COX-2 and then suppressing PGE2 production.Importantly, we revealed that the β signalling pathway regulates ovulation. TGF-β1 induces COX-2 expression and PGE2 production by activating Smad signalling pathways in human granulosa cells.22 TGF-β1 induces mGC proliferation by upregulating miR-224 expression.44 TGF-β1 downregulates steroidogenic acute regulatory protein (StAR) expression and decreases progesterone production by activating the Smad3 and ERK1/2 signalling pathways in human granulosa cells.45 In our study, we demonstrate that Smad4 is a target of miR-144 in mGCs, and Smad4 regulates COX-2 expression via the TGF-β signalling pathway. A previous study suggests that miR-144 regulates COX-2 by targeting c-Fos.46 We found that miR-144 regulated c-Fos expression in mGCs directly through dual-luciferase reporter assays, qRT-PCR and western blot analyses  expression, and SRY can affect sex determination in mammals.55 We further demonstrate that CP2 affects PGE2 production through the CP2/miR-144/COX-2/PGE2 axis. These results suggest that CP2 has an important role in ovulation.Several previous studies have indicated that miRNA expression is also regulated by transcription factors in granulosa cells.25 Paired box gene 4 (PAX4), which regulates human epithelial cancer metastasis, decreases miR-144 and miR-451 expression levels by binding to the promoter region of miR-144/451.25 GATA-binding protein 4 (GATA4) has been shown to activate the promoter of miR-144/451 and protect against simulated ischaemia/reperfusion-induced cardiomyocyte death.24 Here, we find that CP2 regulates the expression of miR-144 and miR-451 in mGCs, which is consistent with previous studies.miR-144 and miR-451 are transcribed in the same pri-miRNA.TNF) and X-linked inhibitor of apoptosis protein (XIAP), are involved in follicular atresia in pigs.57 miR-26b promotes porcine granulosa cell apoptosis and induces follicular atresia by targeting ataxia telangiectasia mutated (ATM) in porcine ovary.58 let-7 g targets MAP3K1 and TGF-β type 1 receptor (TGFBR1) and regulates porcine granulosa cell apoptosis.26 In this study, we demonstrate that miR-144 regulates apoptosis of mGCs but not via Smad4 and COX-2. These data indicate that miR-144 may be involved in follicular atresia.Many factors, such as tumour necrosis factor   and 500 IU hCG (SanSheng) as described previously.60 Only healthy ovarian follicles with a diameter of >5 mm were isolated and snap-frozen in liquid nitrogen. Total RNA was isolated from ovarian follicles with TRIzol reagent . Twenty- to 22-day-old Kunming White female mice used in this study were obtained from the Centre of Laboratory Animals of Hubei Province . All animal treatment procedures were approved by the ethical committee of the Hubei Research Centre of Experimental Animals  2008-0005).Three multiparous CT cyclic sows (≥2 parities) from Xuqin Corporation  and three multiparous LW cyclic sows (≥2 parities) from the Jinpin farm of Huazhong Agricultural University, Wuhan, China that exhibited normal oestrous cycles were treated as reported previously.61 Solexa sequencing was carried out at the Beijing Genomics Institute (BGI). Raw data from the Illumina 1G Genome Analyser were processed using Solexa software (Illumina). Low-quality reads were filtered according to the base quality value.A RNA pool from LW pre-ovulatory ovarian follicles and a RNA pool from Taihu pre-ovulatory ovarian follicles were submitted to Illumina sequencing as described previously.http://www.mirbase.org/), by BLASTn search to identify the conserved miRNAs in pigs. The differentially expressed small non-coding RNAs were identified by comparing their expression between a RNA pool from LW pre-ovulatory ovarian follicles and a RNA pool from Taihu pre-ovulatory ovarian follicles.61After redundancies were removed, sequences ≥18 nt were perfectly mapped to the swine genome Sscrofa10.2 using SOAP. The unique sequences were compared with the miRNA database, miRBase 16.0 (2. The cells were plated and grown until they were 70–80% confluent at the time of transfection. miRNA or siRNA was transfected into the cells using Lipofectamine RNAiMAX (Invitrogen). Plasmids were transfected into the cells using Lipofectamine 3000 (Invitrogen). For luciferase assays, each wild-type or deletion construct was transfected into the cells at 500 ng, together with 10 ng/well of pRL-TK . After transfection for 24 h, the luciferase activities were measured with a PerkinElmer 2030 Multilabel Reader .Granulosa cells from pre-ovulatory ovarian follicles were obtained from murine ovaries after Kunming White female mice were treated for 48 h with 10 IU PMSG (SanSheng). The CHO-K1 cells (GDC018) and PK-15 cells (GDC061) were obtained from the China Centre for Type Culture Collection . Cells were cultured with Dulbecco's minimum essential medium/nutrient F-12 (DMEM/F-12)  or DMEM (Gibco) supplied with 10% foetal bovine serum (Gibco) at 37 °C in a humidified atmosphere of 5% COKpnI and HindIII and cloned into the pGL3-basic vector (Promega). Mouse COX-2 (NM_011198.4), Smad4 (NM_008540.2) and c-Fos (NM_010234.2) CDS were amplified and double-digested with NheI and HindIII and cloned into the pcDNA3.1(+) vector (Invitrogen). The mouse CP2 (NM_001289603.1) CDS was amplified, double-digested with HindIII and XbaI, and then cloned into the pCMV-C-HA vector . The mouse COX-2, Smad4 and c-Fos 3′-UTRs were amplified, double-digested with PmeI and XhoI, and then cloned into the pmirGLO vector (Promega). Binding site mutants were generated using a MutanBEST Kit  and mutagenic primers. The primers are described in Nine deletion fragments of the mouse miR-144 potential promoter region were amplified and double-digested with The synthesized siRNA-CP2, siRNA-COX-2, siRNA-Smad4 siRNA-c-Fos or siRNA NC  was transfected into cells with Lipofectamine RNAiMAX (Invitrogen). After 48 and 72 h, cells were harvested for qRT-PCR assays and western blot assays, respectively. The siRNA sequences are as follows: siRNA-CP2 sense sequence: 5′-CCAUAGCAGUUUCUCUCUUdTdT-3′ antisense sequence: 3′-dTdTGGUAUCGUCAAAGAGAGAA-5′ the siRNA-COX-2 sense sequence: 5′-CACAGGAUUUGACCAGUAUdTdT-3′ antisense sequence: 3′-dTdTGUGUCCUAAACUGGUCAUA-5′ the siRNA-Smad4 sense sequence: 5′-ACAGUGAAGGUGAAUUAAAdTdT-3′ antisense sequence: 3′-dTdTUGUCACUUCCACUUAAUUU-5′ siRNA-c-Fos sense sequence: 5′-GGAGACAGAUCAACUUGAAdTdT-3′ antisense sequence: 3′-dTdTCCUCUGUCUAGUUGAACUU-5′.μl DNase I  to remove DNA contamination. Reverse transcription was performed using a RevertAid First Strand cDNA Synthesis Kit . Random primers, oligo(dT)18 or miRNA specific stem-loop primers were added to initiate cDNA synthesis. qRT-PCR was performed on a Bio-Rad CFX96 system  using the iTaq Universal SYBR Green Supermix (Bio-Rad). All PCR reactions were performed in triplicate. Gene expression levels were normalized to the expression of β-actin, and miRNA expression levels were normalized to the expression of U6 using Gene Expression Macro software (Bio-Rad) by using the 2−ΔΔCt method.61Total RNA (including miRNA) was extracted from tissues or cells with TRIzol reagent (Invitrogen). Cellular RNA was extracted 48 h after transfection. Primers used in the qRT-PCR are shown in β-actin  and CP2  were used for immunoblotting. An Image Quant LAS4000 mini  was used to detect protein expression.Cell protein lysates were generated using RIPA Lysis Buffer (Beyotime). Cellular proteins were extracted 72 h after transfection. Proteins were separated by SDS-PAGE, and a Mini Trans-Blot Cell (Bio-Rad) was used to transfer protein onto polyvinylidene fluoride membranes . Primary antibodies specific for COX-2 , Smad4 , c-Fos , http://www.gene-regulation.com/pub/ programs.html) was used to predict the transcription factor binding sites in the promoters of both mouse miR-144 and pig miR-144. Neural network promoter prediction software (http://www.fruitfly.org/seqtools/promoter.html) was used to analyze the potential promoters, and TargetScan (http://www.targetscan.org/) was used to predict the potential target genes of miRNAs.The BIOBASE software (pCMV-C-HA-CP2-CDS and then transfected this vector into mGCs. ChIP was performed using the EZ-ChIP Kit (Millipore). The AVCX130 system  was used for cell sonication. Anti-HA , anti-OCT1  and normal anti-mouse-IgG (Millipore) were used for the immunoprecipitation reactions. DNA from the immunoprecipitated complex was amplified via PCR. The primer sequences are described in As specifical immunoprecipitate CP2 antibodies are not available, we constructed 62 Briefly, pCMV-C-HA-CP2-CDS was transfected into mGCs, and then, the nuclear extracts were isolated with a Nucleoprotein Extraction Kit (Beyotime). Oligos corresponding to the CP2-binding sites of the miR-144 core promoter were synthesized and annealed into double strands. The DNA-binding activity of CP2 protein was detected by LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific).EMSA was performed as described previously.Mouse GCs were cultured in a six-well plate with 2 ml medium. Mouse GCs were transfected with oligonucleotides or plasmids. After 48 h, cell media were collected to measure PGE2 levels using an ELISA kit . PGE2 levels were normalized to the protein concentrations. Normalized PGE2 values were determined by extrapolating from a standard curve.Mouse GCs were transfected with miR-144 mimics, mimics NC, miR-144 inhibitor or inhibitor NC and harvested 48 h after transfection. Fluorescence-activated cell sorting (FACS) was used to measure apoptosis. The experiments were performed according to the manufacturer's protocol of the Annexin V-FITC Apoptosis Detection Kit (Invitrogen).t-test was used when two groups were compared. Significant differences were evaluated using an independent-samples t-test. P<0.05 was considered to be statistically significant.All results are presented as the mean±S.D. Each treatment had three replicates. Two-tailed


In vivo, intraperitoneal injection of AT7867 inhibited HT-29 xenograft tumor growth in nude mice. AKT activation was also inhibited in AT7867-treated HT-29 tumors. Together, the preclinical results suggest that AT7867 inhibits CRC cells via AKT-dependent and -independent mechanisms.AKT is often hyper-activated in human colorectal cancers (CRC). This current study evaluated the potential anti-CRC activity by AT7867, a novel AKT and p70S6K1 (S6K1) dual inhibitor. We showed that AT7867 inhibited survival and proliferation of established  and primary human CRC cells. Meanwhile, it provoked caspase-dependent apoptosis in the CRC cells. Molecularly, AT7867 blocked AKT-S6K1 activation in CRC cells. Restoring AKT-S6K1 activation, via expression of a constitutively-active AKT1 (“ca-AKT1”), only partially attenuated AT7867-induced HT-29 cell death. Further studies demonstrated that AT7867 inhibited sphingosine kinase 1 (SphK1) activity to promote pro-apoptotic ceramide production in HT-29 cells. Such effects by AT7867 were independent of AKT inhibition. AT7867-indued ceramide production and subsequent HT-29 cell apoptosis were attenuated by co-treatment of sphingosine-1-phosphate (S1P), but were potentiated with the glucosylceramide synthase (GCS) inhibitor PDMP.  Colorectal cancer (CRC) is a leading cause of cancer-related mortality around the world ,2,3. CRCAKT, or protein kinase B (PKB), is a serine/threonine kinase that lies downstream of phosphatidylinositol 3-kinase (PI3K) ,8. Over-et al. has recently developed a dual inhibitor of AKT and p70S6K1 (“S6K1”), named AT7867 -2,5 diphenyltetrazolium bromide (MTT) assay as described previously .4 per treatment) were suspended in agar-containing complete medium or plus AT7867 treatment, which were then added on top of a six-well plate. After 8 days, colonies were stained and manfully counted.As described , cells (Cells with/out the AT7867 treatment were incubated with BrdU (10 μM). Cells were then fixed, and BrdU incorporation was determined by the BrdU ELISA kit (Roche Diagnostics) according to the attached protocol.As described , after aAfter applied treatment, the single strand DNA (“ssDNA”) Cell Apoptosis ELISA Kit was applied to detected denatured DNA in ELISA format to reflect cell apoptosis .The adherent and floating cells were collected and washed. Cells were then incubated in Annexin V solution  for 15 minutes. Immediately prior to reading on a FACS Calibur flow cytometer , 10 μg/mL of propidium iodide (Invitrogen) was added to the mix. Annexin V positive cells were gated as apoptotic cells.The detailed protocols of TUNEL staining assay and caspase activity assay were described in detail in other studies ,19.After treatment, both floating and adherent cells were collected and washed. Cells were then harvested using the RIPA buffer . Aliquots of 30 μg lysates per sample were separated by SDS-PAGE and transferred to PVDF membranes . The blots were blocked and incubated with designated primary and secondary antibodies. Targeted protein bands were visualized with ECL reagents and developed with Hyper-film . Results were quantified via the ImageJ software (NIH).The two lentiviral AKT1 shRNAs (“-a/-b”), with non-overlapping sequences, were designed by Genepharm . The AKT1shRNA (10 μL/mL) or the scramble control shRNA  was added to cultured cells for 24 hours. Puromycin (5.0 μg/mL) was then included to select stable colonies for 4–6 passages. The AKT1 knockdown in the stable cells was verified by Western blot assay.As previously described , cells wAnalyzing sphingosine kinase 1 (SphK1) activity and cellular ceramide content were described in detail in our previous study .s.c.) in the right flanks of female nude mice . Animals were randomized into three groups, and treatment was started with vehicle or AT7867 when established tumors were ~100 mm3 in average volume. Control mice received vehicle only  and treatment mice received 10 or 50 mg/kg AT7867 intraperitoneally (i.p.). Tumor volumes and body weights were monitored every four days. The tumor size was calculated using the formula: V (volume) = 0.5328 × Long × Width × High (mm3). For recording mouse body weight, the estimated tumor weight (tumor volumes × 1g/cm3) was subtracted from total weight of each mouse. Mice were maintained under the following conditions: 12-hour dark/12-hour light cycle, 24±2°C temperatures, and 50±10% humidity. Mice were observed extremely carefully throughout the experimental period. The clinical signs of mice were recorded daily, and if the criteria of humane endpoints were met, animals were sacrificed. Humane endpoints were considered as rapid weight loss (>15%), abnormal changes in behavior and motion , tumor size greater than 2 cm3 or skin problems (wounds or signs of inflammation). If animals reached these endpoints, they were euthanized by exsanguination under 2,2,2-tribromoethanol anesthesia . All injections in this study were performed via the above anesthesia method [HT-29 cells were injected subcutaneous . Statistics was analyzed by one-way ANOVA followed by a Scheffe’s f-test using the SPSS 18.0 software . P< 0.05 is considered statistically significant.HT-29 cells were first treated with gradually-increasing concentrations of AT7867 (0.1–25 μM). Cells were then cultured for 24–96 hours, and then cell viability was determined using an MTT assay. Results in The potential effect of AT7867 on other human CRC cells was also tested. As demonstrated, AT7867 (10 μM) was cytotoxic when added to the two other established CRC cell lines (HCT116 and DLD-1). It also decreased the percentage of viable patient-derived primary human CRC cells  . RemarkaWe next tested the potential effect of AT7867 on CRC cell apoptosis. Under many apoptotic stimuli, cytochrome c released from mitochondria will associate with procaspase-9 and Apaf-1, causing caspase-9 activation ,23. The Next, different caspase inhibitors were applied. z-DEVD-fmk (the caspase-3 inhibitor), z-LEHD-fmk (the caspase-9 inhibitor) and z-VAD-fmk (the pan caspase inhibitor) significantly attenuated AT7867-induced HT-29 cell MTT OD reduction  and cellAT7867 is a newly developed AKT-S6K1 dual inhibitor ,25. We tTo study the link between AKT inactivation and AT7867-induced actions, a constitutively-active AKT1   was intrTo further support our hypothesis, shRNA strategy was applied to knockdown AKT1 in HT-29 cells. As shown in Above results suggest that AKT-independent mechanisms could also contribute to AT7867-induced cytotoxicity in CRC cells. Growing evidences have indicated that SphK1 is an important oncotarget of CRC ,29. OverTo study the role of ceramide production in AT7867-induced HT-29 cell death, pharmacological strategy was applied. Sphingosine 1-phosphate (S1P), an anti-ceramide sphingosine, inhibitin vivo, HT-29 cells were injected s.c. to the nude mice to establish the xenograft tumors. Tumor growth curve results in i.p. injection of AT7867 (10 and 50 mg/kg) significantly inhibited HT-29 tumor growth in nude mice. AT7867 at 50 mg/kg was more efficient than 10 mg/kg in inhibiting HT-29 tumor growth (i.p. injection ofAT7867efficientlyinhibits HT-29 tumor growth in nude mice.In order to examine the effect of AT867 on tumour growth r growth . Estimati.e. EGFR), PIK3CA/PTEN mutations, AKT amplification and/or mutation[In vivo, AT7867 i.p. injection suppressed HT-29 xenograft tumor growth in nude mice.Several mechanisms are responsible for the sustained activation of AKT in CRC and other malignancies, including constitutive activation of upstream receptor tyrosine kinases  mechanisms could be responsible for its superior actions against CRC cells.Intriguingly, AT7867-induced ceramide production appears independent of AKT inhibition. As ca-AKT1 failed to inhibit AT7867-indued ceramide production. Meanwhile, in AKT1-silenced HT-29 cells, a compare ceramide production was still noticed. In summary, the results of this preclinical study suggest that AT7867 inhibits human CRC cells S1 FigRelative expression of the proteins (vs. Tubulin) was quantified.(EPS)Click here for additional data file.


Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with an increased risk of lung cancer. However, the mechanisms underlying Cr(VI)-induced carcinogenesis remain unclear. MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. Studies have shown that miR-21 exerts its oncogenic activity by targeting the tumor suppressor gene programmed cell death 4 (PDCD4). The present study examined the role of miR-21-PDCD4 signaling in Cr(VI)-induced cell transformation and tumorigenesis. Results showed that Cr(VI) induces ROS generation in human bronchial epithelial (BEAS-2B) cells. Chronic exposure to Cr(VI) is able to cause malignant transformation in BEAS-2B cells. Cr(VI) caused a significant increase of miR-21 expression associated with an inhibition of PDCD4 expression. Notably, STAT3 transcriptional activation by IL-6 is crucial for the Cr(VI)-induced miR-21 elevation. Stable knockdown of miR-21 or overexpression of PDCD4 in BEAS-2B cells significantly reduced the Cr(VI)-induced cell transformation. Furthermore, the Cr(VI) induced inhibition of PDCD4 suppressed downstream E-cadherin protein expression, but promoted β-catenin/TCF-dependent transcription of uPAR and c-Myc. We also found an increased miR-21 level and decreased PDCD4 expression in xenograft tumors generated with chronic Cr(VI)-exposed BEAS-2B cells. In addition, stable knockdown of miR-21 and overexpression of PDCD4 reduced the tumorogenicity of chronic Cr(VI)-exposed BEAS-2B cells in nude mice. Taken together, these results demonstrate that the miR-21-PDCD4 signaling axis plays an important role in Cr(VI)-induced carcinogenesis. Hexavalent chromium compounds [Cr(VI) compounds] have been classified as human carcinogens by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) based on epidemiological studies results linking Cr(VI) to lung cancer . Cr(VI) MicroRNAs (miRNAs) are a novel class of endogenous, small, noncoding RNAs that negatively regulate approximately 30% of the gene expression by a site-specific interactions at the 3′-UTR of target-mRNAs, causing translational repression or degradation , 5. AmonPDCD4 is a novel tumor suppressor reported to inhibit neoplastic transformation, tumor promotion and progression , 17. It Persistent activation of signal transducer and activator of transcription-3 (STAT3) is frequently associated with malignant transformation . Recent In the present study, we investigated the interactive role of miR-21-PDCD4 signaling in Cr(VI)-induced malignant cell transformation. We found that chronic Cr(VI) exposure increased miR-21 levels and was associated with inhibition of PDCD4 expression and malignant cell transformation. Importantly, Cr(VI)-induced ROS was essential for the miR-21 elevation and PDCD4 reduction. In addition, STAT3 transcriptional activation by IL-6 was crucial for the Cr(VI)-induced miR-21 elevation. Furthermore, PDCD4 reduction by Cr(VI) suppressed downstream protein E-cadherin expression, and led to the β-catenin/TCF-dependent transcription of uPAR and c-Myc. These results suggest that miR-21 and PDCD4 are critical components of Cr(VI)-induced malignant transformation.p < 0.05) elevation in the miR-21 levels associated with a dose-dependent decrease in PDCD4 expression by RT-PCR and Western blot analysis respectively in human bronchial epithelial BEAS-2B cells treated with Cr(VI)  Figure . Similar) Figure . There w) Figure . These rPrevious studies demonstrated that knock-down of PDCD4 down-regulates E-cadherin and increases β-catenin and TCF4 protein expression . In the 2 − and H2O2 generation in BEAS-2B cells, as indicated by an increase of DHE  and inhibited by MnTMPyP, cell-permeable SOD mimetic (O2 − scavenger)   -induced ROS plays any role in miR-21 –PDCD4 signaling. Cr(VI)-induced ROS production was quantified by flow cytometry using the fluorescent probes DHE and DCFDA. Cr(VI) exposure dramatically stimulated OE Figure  and DCFDE Figure  fluoresc) Figure . Similar) Figure . The flu) Figure . As show) Figure . Taken tNext we sought the role of Cr(VI)-induced ROS generation in miR-21- PDCD4 signaling. As outlined above, we demonstrated that ROS plays an essential role in Cr(VI)-induced miR-21 elevation and PDCD4 suppression. However, the Cr(VI)-induced miR-21 elevation and PDCD4 suppression were markedly reversed by treatment with the ROS inhibitors, NAC or Catalase Figure . These rp < 0.05) increased the IL-6 levels  elevated by Cr(VI) or IL-6, while STAT3 knock down (siSTAT3) inhibited miR-21 promoter activity.Previous studies showed that Cr(VI) stimulates IL-6 mRNA levels and STAT3 phosphorylation in human airway epithelial cells . STAT3 bs Figure , STAT3 as Figure , and STAs Figure  in a doss Figure . A genomp < 0.05) and dose-dependent increase in colony number  exposure induces malignant transformation in BEAS-2B cells . Malignar Figure .p < 0.05) increase in the miR-21 levels  of Cr(VI) and measured the miR-21 levels and PDCD4 expression at two, four and six months. We found a dose-dependent and significant  decreased the chronic Cr(VI)-induced malignant cell transformation -induced malignant transformation and invasion, BEAS-2B cells with stable knocked down of miR-21 were treated with Cr(VI) (0.5 μM) for six months. As shown in Figure n Figure  and invan Figure . These rTo investigate the role of antioxidants on chronic Cr(VI)-induced miR-21 elevation and PDCD4 suppression, we treated BEAS-2B cells overexpressing catalase with Cr(VI) (0.5 μM) for six months. Our study shows that overexpression of catalase noticeably decreased the miR-21 elevation induced by chronic Cr(VI) treatment Figure  and suppin vivo, BALB/c mice were nasally exposed to insoluble Cr(VI) particles (1.2 mg/ml) for up to 3 months. The results show that chronic Cr(VI) exposure at 1.2 mg/ml induced an increase in miR-21 levels  exposure causes miR-21 elevation and PDCD4 suppression s Figure  and decrs Figure  in mouses Figure . Consists Figure  associats Figure  in xenogs Figure , and tums Figure  were smas Figure  and decrs Figure  in the lThe expression levels of miR-21 and its downstream target protein PDCD4 were determined by real time PCR and florescence immunohistochemistry respectively in lung tissues from lung adenocarcinoma patients. As shown in Figure Occupational exposure to Cr(VI) is a well-established cause of lung cancer –41, therMicroRNA's (miRs) are important post-transcriptional regulators that influence more than 30% of human mRNAs. Significant changes in microRNA (miR) expression in response to environmental exposure in humans have recently been reported , 47. NotWe also explored the mechanisms by which Cr(VI) activates miR-21. STAT3, known to be important for cancer initiation and tumor progression, was reported to bind directly to the miR-21 promoter upon IL-6 induction , 30, 52.2O2) can up-regulate miR-21 levels and decrease PDCD4 expression in vascular smooth muscle cells [phox, one of the NOX subunits is the key source Cr(VI)-induced ROS. Moreover, exogenous treatment of cells with catalase or NAC significantly inhibited the Cr(VI)-induced a rise in miR-21 and decreased PDCD4 levels. To substantiate a role for antioxidants in the Cr(VI)-induced malignant cell transformation mediated by increased miR-21 levels and PDCD4 suppression, we chronically treated BEAS-2B cells overexpressing catalase with Cr(VI) for six months. Interestingly, catalase overexpression in BEAS-2B cells significantly inhibited Cr(VI)-induced miR-21 elevation, PDCD4 suppression and malignant cell transformation.Studies suggest that hydrogen peroxide  particles for 3 months. We found an increase in miR-21 levels associated with PDCD4 suppression in the lungs of mice exposed to Cr(VI). Moreover, we found an significantly increased miR-21 levels associated with decreased PDCD4 expression in xenograft tumors generated with chronic Cr(VI) exposed BEAS-2B cells. In addition we confirmed that stable knockdown of miR-21 or overexpression of PDCD4 reduce the tumorigenicity of chronic Cr(VI) exposed BEAS-2B cells in nude mice. Similar results were observed in lung adenocarcinoma tissues from patients compared with normal tissues from same patients.To explore whether Cr(VI) exposure causes miR-21 elevation and PDCD4 suppression In summary, we found that chronic Cr(VI) exposure increases miR-21 levels with an associated inhibition of PDCD4 expression, and cause malignant transformation in BEAS-2B cells. Cr(VI) triggers the miR-21 increase via the IL-6/STAT3 pathway. Cr(VI)-induced PDCD4 suppression down-regulates the E-cadherin protein expression along with an upregulation of active β-catenin (nuclear translocated form) and TCF4 in BEAS-2B cells. In addition, Cr(VI) exposure increases uPAR and c-Myc expressions, and also activates β-catenin/TCF4 complex binding to the uPAR and c-Myc promoters. Stable knockdown of miR-21 or overexpression of PDCD4 reduces the tumorogenicity of chronic Cr(VI) exposed BEAS-2B cells in nude mice Figure . In shor2Cr2O7), apocynin (APO), 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), were purchased from Sigma-Aldrich . Both Dichlorodihydrofluoresceine acetate (DCFDA) and dihydroethidium (DHE) were purchased from Molecular Probes . Manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP) was purchased from Cayman Chemical . Human miR-21 hairpin inhibitor was purchased from Thermo Fisher Scientific Dharmacon . Antibodies against PDCD4 (CST#9535), E-cadherin (CST#3195), β-catenin (CST#8480), c-Myc (CST#5605), uPAR (CST#9692), pSTAT3 Tyr705 (CST#9145) and STAT3 (CST#9139) were purchased from Cell Signaling Technology . Anti–active-β-catenin (clone 8E7#05-665) antibody was purchased from EMD Millipore . Antibodies against TCF4 (sc-13027) and GAPDH (sc-25778) were purchased from Santa Cruz Biotechnology, Inc. .Potassium dichromate (K2 in serum-free LHC-9 medium. BEAS-2B and CrT cells were cultured in Dulbecco's modified Eagle's medium (DMEM), while H2030, H460, H23, and A549 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 5% penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO2 in air.Human bronchial epithelial cell lines BEAS-2B, NL20 and other lung cancer cell lines H2030, H460, H23, A549 cells were obtained from the American Type Culture Collection . Chromium transformed cells (CrT) were generated as described previously . NL20 ce2Cr2O7 was used for Cr(VI) treatments. For short-term exposure of Cr(VI), cells were grown to 80–90% confluent, and then the medium was replaced with DMEM medium containing 0.1% FBS for overnight before Cr(VI) treatment at the dose and duration indicated. In some experiments, cells were first pretreated with various inhibitors for 2 h and then exposed to Cr(VI). For chronic exposure of Cr(VI), the cells were continuously cultured in growth medium with the indicated concentration Cr(VI).KPlasmid DNA encoding human catalase and STAT3 shRNA were purchased from Origene . PDCD4 3′-UTR reporter plasmid was kindly provided by Dr. Yong Li . The STAT3-miR-21 reporter was constructed as described previously . PcDNA3.The overexpression of catalase or PDCD4 and knockdown of STAT3 in BEAS-2B cells were performed using Lipofectamine™2000  according to the manufacturer's protocol. Briefly, BEAS-2B cells were seeded at approximately 50% confluency in 6-well culture plates and transfected with 4 μg plasmid. Cell clones resistant to G418 were isolated; overexpression of CAT and PDCD4 protein production were confirmed by immunoblotting.The pLenti-III-miR-Off-has-miR-21-puro-GFP expression vector and the negative control vector pLenti-III-miR-Off- puro-GFP were purchased from Applied Biological Materials, Inc. . Human embryonic kidney 293T cells  were transfected with lentiviral packaging vectors  and lentiviral vectors expressing miR-Off-has-miR-21-puro-GFP or miR-Off- puro-GFP by Lipofectamine™2000  according to the manufacturer's protocol .For generating stable miR-21 knockdown cell lines, two days after transfection, supernatants containing viral particles were harvested and used to infect BEAS-2B cells at approximately 70% confluence in DMEM supplemented with 8 μg/ml of polybrene using lentifectin reagent (ABM technologies) following the manufacture's protocol. 72 h after transfection, the medium was changed to the selection DMEM with 10% FBS and supplemented with puromycin 1 μg/mL (sigma) to screen stable cell lines for further assay. Three weeks later, the cell clones were screened and further cultured in DMEM medium containing 1 μg/mL puromycin and selected through FACS (Fluorescent Assisted Cell Sorting system).2O2 and O2.- generations were examined using the fluorescent dye DCFDA and DHE, respectively, as described previously [5 cells/well), treated with 5 μM of Cr(VI) for 12 h and incubated with DCFDA or DHE (10 μM) for 40 min at 37°C. Trypsinized cells were washed twice with cold PBS, and analyzed by fluorescence-activated cell sorting . The fluorescence intensity of DCFDA was measured at an excitation wavelength of 492 nm and an emission wave length of 517 nm. The fluorescence intensity of DHE was measured at an excitation wavelength of 535 nm and an emission wavelength of 610 nm.Heviously . The cel3CrO8) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) as reference standards. Reactants were mixed in test tubes to a total final volume of 0.5 mL. The reaction mixture was then transferred to a flat cell for ESR measurement.ESR measurement was conducted using a Bruker EMX spectrometer  and a flat cell assembly. The intensity of ESR signal was used to measure the amount of hydroxyl radical generation. DMPO was used as a spin or radical trap. DMPO was charcoal purified and distilled to remove all ESR detectable impurities before use. Hyperfine couplings were measured (to 0.1 G) directly from magnetic field separation using potassium tetraperoxochromate (K5 cells) were seeded into each well of a 6-well plate and allowed to attach overnight. After the indicated exposure, cells were collected by trypsinization, three hundred cells were then reseeded into each of three dishes (60 mm diameter), and grown for 10 days. The cells were fixed with 2% formalin for 10 min and stained with 0.5% crystal violet stain and counted.BEAS-2B cells . Homogenates were centrifuged at 800 × g at 4°C for 10 min to remove the unbroken cells and debris, and aliquots were used immediately. To start the assay, 100-μl of homogenate supernatants were added to 900 μl of 50 mM phosphate buffer, pH 7.0, containing 1 mM EGTA, 150 mM sucrose, 5 μM lucigenin, and 100 μM NADPH. Photon emission in terms of relative light units was measured in a Glomax luminometer (Promega) every 30 s for 5 min. There was no measurable activity in the absence of NADPH. Superoxide anion production was expressed as relative chemiluminescence (light) units (RLU)/mg protein.NOX activity was measured by the lucigenin enhanced chemiluminescence method as described previously . Briefly4/well) and subjected to various treatments when cultures reached 80–90% confluence. Cellular lysates were subjected to a luciferase reporter assay  using Glomax luminometer (Promega) as described previously [BEAS-2B cells transfected with the luciferase reporter constructs were seeded into 24-well plates (5 × 10eviously . The resBEAS-2B cells were cultured in DMEM supplemented with 10% FBS containing Cr(VI)  for 24 h. Subsequently, the culture media was collected and used to estimate IL-6 level using commercially available IL-6 ELISA kit  according to the manufacturers' recommendation.AM ELISA kit  was used following the manufacturer's protocol. For this assay, the nuclear extracts of cell samples from various treatment groups were prepared using the Nuclear Extraction kit (Active Motif) according to the manufacturer's direction. Absorbance was recorded at 450 nm with reference taken at 650 nm. The assay was performed in duplicate and the results are expressed as the percentage absorbance of control group.For quantitative analysis of STAT3, Trans4 cells were suspended in 3 mL culture medium containing 0.35% agar and seeded into 6-well plates with 0.5% agar base layer, and maintained in an incubator for 4 weeks. The colonies greater than 0.1 mm in diameter were scored by microscopic examination.Soft agar colony formation assay was performed as described previously . BEAS-2BThe Cr(VI)-transformed cells from anchorage-independent colonies were selected and grown in DMEM. Passage-matched cells without Cr(VI) treatment were used as control.5 cells/Transwell) chronically exposed to Cr(VI) were seeded to the top chamber of invasion chamber and incubated 24 h. Cells in top chamber (non-invaded) were removed, and cells on bottom of filter insert (invaded) were fixed with 3.7% paraformaldehyde and stained with 0.5% crystal violet in 2% ethanol. Membranes were washed and the dye was eluted with 10% acetic acid. Absorbance was measured at 595 nm using a microtiter plate reader (Beckman coulter). The number of invaded cells was presented relative to untreated controls. Experiments was performed in duplicate and repeated 3 times.Collagen matrix invasion assay was performed as described previously . Briefly® thermal cycler .ChIP analysis was performed using a PierceTM Agarose ChIP Kit . Sheared chromatin was diluted and immunoprecipitated with 2 μg of anti-STAT3, anti-TCF4 or control IgG antibody. DNA protein complexes were eluted from the protein A/G agarose beads using a spin column and were reverse cross-linked by incubating with NaCl at 65°C. The relative TCF4 binding to the c-MYC and uPAR promoters or STAT3 binding to miR-21 promoter was analyzed by MyiQ™ Single-Color Real-Time PCR Detection System  with SYBR Green PCR master mix using following primer sequences, TCF4 binding to c-Myc promoter: (F) GCGCCCATTAATACCCTTCT and (R) TCTCCCTTTCTCTGCTGCTC; TCF4 binding to uPAR promoter: (F) GGAAGCAAAGCAAGGGTTAAG and (R) GCCCTGACTCATGGAGTTGT; STAT3 binding to miR-21 promoter/enhancer (F) CCTCTGAGAAG AGGGGACAA and (R) ACCGCTTCCAGCA AAAGAGT. General PCR amplification also performed in a Mastercycler® microRNA reverse transcriptase kit  according to the manufacturer recommendations. Expression of miR-21-microRNA was determined by the TaqMan miRNA-assay , and normalized using the 2−ΔΔCT -method relative to U6-snRNA. All TaqMan-PCR reactions were performed in triplicates using a Bio-Rad's MyiQTM single-color real-time PCR detection system.Total RNA was extracted using Trizol (Invitrogen), and cDNA was synthesized by using TaqManHuman lung adenocarcinoma tissue (stage IA or IIA) was provided from the Biospecimen and Tissue Procurement Shared Resource Facility of the University of Kentucky Markey Cancer Center. Lung tissues were freshly frozen and fixed with 10% formalin for real-time PCR and immunofluorescence analysis, respectively.Cells lysates were prepared in ice-cold RIPA buffer (Sigma-Aldrich) containing freshly added protease inhibitor cocktail. The lysate was then centrifuged at 12000 g for 10 min at 4°C and the supernatant  was collected, aliquoted and stored at −80°C. The protein concentration was determined using Coomassie Protein Assay Reagent . About 40 μg cellular proteins were separated using 6%–12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane . Membranes were blocked with 5% fat-free dry milk in 1X Tris-buffered saline (TBS) and incubated with antibodies. Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies  and visualized with enhanced chemiluminescence reagent . Tumor tissues were homogenized with MagNA Lyser Green Beads using MagNA Lyser Instrument  and Western blot analysis performed.6–8 weeks old, female BALB/c mice were purchased from Jackson Laboratory . Endotoxin-free basic zinc chromate particle was crushed using mortar and pestle and then washed with distill water and acetone to generate a particle of 4.7 μm and a purity of 99–100%. Cr(VI) particle was suspended in sterile 0.9% sodium chloride solution at a concentration of 1.2 mg/ml and prepared as previously described . Animals6 cells per mouse) from different treatments were resuspended in serum-free medium with matrigel basement membrane matrix (BD Biosciences) at a 1:1 ratio  and subcutaneously injected into the flanks of nude mice. Mice were checked daily for tumor appearance, and tumor volume was measured every 3 days for 30 days. Tumor volume was determined by Vernier caliper, following the formula of A × B2 × 0.52, where A is the longest diameter of tumor and B is the shortest diameter. At the end of the experiment, mice were sacrificed, the tumors excised and snap frozen.Athymic nude mice  were housed in a pathogen-free room in the animal facilities at the Chandler Medical Center, University of Kentucky. Cells , and non-specific binding sites were blocked with 10% horse serum in PBS. Staining was performed using Vectastain ABC Kit according to the manufacturer's protocol . Briefly, the sections were incubated with rabbit anti-PDCD4 (1:100) antibody for 2 h at room temperature, washed and then incubated with biotinylated secondary antibody for 45 min followed by incubation with ABC reagent. After washing in PBS, color was developed with DAB solution until the desired staining intensity was achieved. Finally, the sections were counterstained with hematoxylin.BEAS-2B cells or BEAS-2B cells chronically exposed to Cr(VI) were grown on coverslips in 6-well plates and treated with Cr(VI). The cells were fixed in 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100, blocked with 10% horse serum in PBS solution, and incubation with antibodies PDCD4 (1:500), E-cadherin (1:100), β-Catenin (1:100), TCF4 (1:100) in buffer A  for 1 h at 37°C. The cells were then incubated with Alexa Fluor 594 goat anti-rabbit or Alexa Fluor 488 goat anti-rabbit secondary antibody and mounted using DAPI. The cells were visualized using digital confocal microscopy  or Olympus BX53 fluorescence microscope.For the formalin-fixed lung tissue fluorescence immunohistochemical analysis, air-dried tissue sections were rinsed, blocked, and incubated with primary antibody overnight. Second antibody was added for 1 h. 300 μl of the diluted DAPI solution was added and incubated 2–5 min at room temperature. DAPI binds to DNA and is a convenient nuclear counterstain. The sections were mounted with an anti-fade mounting media. Olympus BX53 fluorescence microscope was used for visualization.p < 0.05 was considered significantly different.Presented values are means ± SD. One-way analysis of variance (ANOVA) was used for statistical analysis, with


Syntrophomonas wolfei subsp. methylbutyratica” strain JCM 14075T (=4J5T), isobutyrate-degrading Syntrophothermus lipocalidus strain TGB-C1T, and non-BCFA-metabolizing S. wolfei subsp. wolfei strain GöttingenT. We demonstrated that 4J5 and TGB-C1 both encode multiple genes/gene clusters involved in β-oxidation, as observed in the Göttingen genome, which has multiple copies of genes associated with butyrate degradation. The 4J5 genome possesses phylogenetically distinct β-oxidation genes, which may be involved in 2-methylbutyrate degradation. In addition, these Syntrophomonadaceae strains harbor various hydrogen/formate generation systems  and energy-conserving electron transport systems, including electron transfer flavoprotein (ETF)-linked acyl-CoA dehydrogenase, ETF-linked iron-sulfur binding reductase, ETF dehydrogenase (FixABCX), and flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr). Unexpectedly, the TGB-C1 genome encodes a nitrogenase complex, which may function as an alternative H2 generation mechanism. These results suggest that the BCFA-degrading syntrophic strains 4J5 and TGB-C1 possess specific β-oxidation-related enzymes for BCFA oxidation as well as appropriate energy conservation systems to perform thermodynamically unfavorable syntrophic metabolism.The syntrophic degradation of branched-chain fatty acids (BCFAs) such as 2-methylbutyrate and isobutyrate is an essential step in the production of methane from proteins/amino acids in anaerobic ecosystems. While a few syntrophic BCFA-degrading bacteria have been isolated, their metabolic pathways in BCFA and short-chain fatty acid (SCFA) degradation as well as energy conservation systems remain unclear. In an attempt to identify these pathways, we herein performed comparative genomics of three syntrophic bacteria: 2-methylbutyrate-degrading “ Furtherproaches . HoweverSyntrophomonadaceae  provideS. wolfei subsp. methylbutyratica” strain JCM 14075T (=4J5T) draft genome (DDBJ/GenBank/EMBL accession: BBQT01000001–BBQT01000092)  (S. wolfei strain Göttingen complete genome (CP000448)  , S. lipoP000448) . As reponome CP0048 . Ba. BaS. woostridia  and BLASostridia . Transpoostridia .Syntrophomonadaceae strains 4J5  and pilus and flagellum formation. Syntrophic BCFA degradation is considered to proceed through β-oxidation , affinities to specific concentration ranges, or adaptation to other environmental conditions  reduction to hydrogen (H2) (Rhodobacter nitrogen fixation (Rnf) complex, these Syntrophomonadaceae syntrophs may not perform reverse electron transport from NADH to oxidized ferredoxin (Fdox) through the Rnf found in several syntrophs  , this system may support the capacities of these organisms to perform crotonate reduction (Syntrophomonadaceae syntrophs encode a syntroph-associated flavin oxidoreductase-heterodisulfide reductase (Flox-Hdr) system  along with FixABCX  , 31. Sinobacter) , 39. Insobacter) , 22, 30 obacter) . These robacter) , 27. Alteduction , 18. In ) system  expresse FixABCX , suggestThere may be multiple hydrogenase and formate dehydrogenase genes in the 4J5 and TGB-C1 genomes . Both st2-producing organism Clostridium butyricum strain CWBI1009 has been proposed to generate H2 and enhance tolerance to acidification through the consumption of protons in the reaction, which produces ammonia as a base (Thermacetogenium phaeum strain PB and Syntrophobacter fumaroxidans strain MPOB) (A nitrogenase gene cluster (NifBDEHIK) was detected in the genome of strain TGB-C1 (Slip_2124–2130) along with a molybdate transporter  and ammonia transporter  . A previin MPOB) . Among sSyntrophomonadaceae strains revealed multiple genes for the β-oxidation of BCFA/SCFA and energy conservation systems employing various types of electron carriers for electron disposal. In theory, the syntrophic degradation of 2-methylbutyrate and isobutyrate have Gibbs free energy values identical to butyrate under standard conditions :Syntrophomonadaceae strains degrading the above fatty acids have unique substrate-specific genes , they share similar energy conservation systems reflecting the thermodynamic similarity shown above. TGB-C1 has several distinct energy conservation pathways from Göttingen and 4J5 potentially due to differences in their temperature preferences. These genome-based results further provide insights into how syntrophic BCFA-degraders interact with H2/formate-utilizing methanogen in an anaerobic ecosystem.Thus, while