Fourier-transform infrared spectroscopy confirms the embedding of undoped and doped MnFe2O4 nanoparticles in the SiO2 matrix at all annealing temperatures. In all cases, the X-ray diffraction (XRD) confirms the formation of MnFe2O4. In the case of undoped, di-, and trivalent metal-ion-doped gels annealed at 1200 °C, three crystalline phases  belonging to the SiO2 matrix are observed. Doping with mono- and trivalent ions enhances the nanocomposite’s structure by forming single-phase MnFe2O4 at low annealing temperatures (500 and 800 °C), while doping with divalent ions and high annealing temperature (1200 °C) results in additional crystalline phases. Atomic force microscopy (AFM) reveals spherical ferrite particles coated by an amorphous layer. The AFM images showed spherical particles formed due to the thermal treatment. The structural parameters calculated by XRD  and AFM , as well as the magnetic parameters , are contingent on the doping ion and annealing temperature. By doping, the saturation magnetization and magnetocrystalline anisotropy decrease for gels annealed at 800 °C, but increase for gels annealed at 1200 °C, while the remanent magnetization and coercivity decrease by doping at both annealing temperatures (800 and 1200 °C).This work presents the effect of monovalent (Ag The percentage of Fe3+ ions occupying the A sites dictates the inversion degree. Thus, in normal spinel structure the A sites are occupied by Fe3+ ions, while in inverse spinel structure the A sites are occupied by Mn2+ ions [2O4 nanoparticles. MnFe2O4 has attracted significant interest due to its controllable grain size, superparamagnetic nature, low coercivity (CH), high magnetic permeability, moderate saturation magnetization (SM), good chemical stability, high catalytic performance, capacity to be guided by an external magnetic field, surface tailoring possibility, good biocompatibility, and high crystal symmetry [2O4 is also a non-toxic, non-corrosive, environmentally friendly, high thermal, and shock-resistant material often used for application in medicine, electronics, as well as in the paint and coating industry [Nanoparticles display enhanced properties relative to microparticles and bulk materials, allowing their use in various applications . Spinel rocesses ,4,5,6. On2+ ions . The invsymmetry ,5,7. MnFindustry .2+-doped MnFe2O4, Zn has a strong tendency to occupy A sites enhancing the magnetic properties [2O4, allowing the degradation of refractory organic pollutants [2Fe2O4 composites also display superparamagnetic and remarkably antibacterial activity [2O4 nanoparticles coated with citrate obtained by the sol-gel method revealed that high Ca content improves their capacity to be used as a hyperthermia agent without compromising their cytocompatibility or cellular internalization [2O4 by doping with rare earth ions has drawn attention as a novel technique to enhance its physical characteristics. Of these, the non-magnetic La3+ ion stands out due to its larger ionic radius compared to that of Fe3+ and Mn2+ ions that change the structural and magnetic properties of MnFe2O4 by the higher electron–hole pair recombination in the ferrites, supporting the shift of the electronic states [3+ doping promotes a higher production of oxygen vacancies and photocatalytic degradation [Doping with various cations enhances the ferrites’ magnetic, optical, and electrical properties. The doped spinel ferrites have various benefits, i.e., they are less expensive, easy to produce, have good stability, and have different magnetic properties compared to undoped ferrites . Cation operties ,12. Prevoperties ,13. The llutants . Ag–Mn2Factivity . A previlization . Recentlc states ,16. Moreradation ,16.2O4 ferrites, the sol–gel route offers a flexible approach due to its low cost, low reaction temperature, simplicity, and good control of chemical composition, structural, physical–chemical, and magnetic properties [Given the diversity of experimental techniques  used to obtain MnFeoperties ,4,5. Theoperties . Solvo/hoperties . Microwaoperties . Baublytoperties  showed a2O4 in mesoporous SiO2 plays an important role in enhancing the stability in water, improving biocompatibility, and diminishing the degradation of MnFe2O4 nanoparticles. The SiO2 coating also prevents agglomeration by controlling the dipolar attraction between the magnetic nanoparticles. Moreover, the silanol groups from the surface of mesoporous SiO2 promote the binding of biomolecules, directing targeted ligands and drug loading on the nanocarrier surface [2O4 embedded in the SiO2 matrix sol–gel method consist in the mixing of reactants with tetraethylorthosilicate (TEOS) and the formation of strong networks with moderate reactivity that permit the incorporation of various inorganic and organic molecules [2O4/SiO2 nanocomposites recommend their use in various technological and scientific systems, such as magnetic devices, catalysis, and sensors [2O4/SiO2 nanocomposites display remarkable electrical and magnetic properties, high chemical and thermal stability, improved microwave absorption performance owing to the strong eddy current loss, excellent attenuation characteristic, better impedance matching, and multiple Debye relaxation processes [2O4/SiO2 nanocomposites are widely studied due to their potential applications in different areas such as electronic, microwave, and communication devices, information storage systems, ferrofluid technology, gas sensors, magnetocaloric refrigeration, and for photocatalytic activity [2O4 and SiO2 integrated into a single entity (nanocomposites particle) is of particular interest in magnetic fluid hyperthermia due to MnFe2O4 superior magnetization and biocompatibility of SiO2 [2O4 nanoparticles  [The embedding of MnFe surface ,4,5. Ourolecules ,4,5. Theolecules ,4,5,6. T sensors ,5,21,22.rocesses . The magactivity ,25,26,27 of SiO2  and drugelivery) . 2O4 has become important for various biomedical applications. Additionally, tailoring MnFe2O4 by doping with various ions could enhance its magnetic, optical, and electrical properties. Thus, producing homogenous doped MnFe2O4 nanoparticles with tailored magnetic properties and crystalline structures is challenging, but it is important to discover novel approaches to increase their potential for existing and new conceivable applications. In this regard, this study was conducted to assess the changes in structure, morphology, surface, and magnetic properties of MnFe2O4 doped with monovalent , divalent , and trivalent  metal ions embedded in a SiO2 matrix synthesized through a modified sol–gel method, followed by annealing at 500, 800, and 1200 °C. The architecture adopted by ferrites depends on the metal ion(s) size, charge and concentration, crystal field effects and electrostatic contribution to the lattice energy, while the particle size increase and the volume-to-surface ratio decreases with annealing temperature. Moreover, due to its high degree of magnetization compared to other nanoferrites, MnFe3)2∙4H2O, Merck, Darmstadt, Germany), ferric nitrate nonahydrate (Fe(NO3)3∙9H2O, 98%, Merck, Darmstadt, Germany), silver nitrate , sodium nitrate , calcium nitrate tetrahydrate (Ca(NO3)2∙4H2O, 99%, Carlo Erba, Milan, Italy), cadmium nitrate tetrahydrate (Cd(NO3)2∙4H2O, 99%, Carlo Erba, Milan, Italy), lanthanum nitrate hexahydrate (La(NO3)3∙6H2O, 98%, Carlo Erba, Milan, Italy) 1,3 propanediol , TEOS , and ethanol . All chemicals were used as received without further purification and purchased from different commercial sources as follows: manganese nitrate tetrahydrate  molar ratios, namely 0/1/2 (MnFe2O4), 1/9.5/20 , 1/9/20 , and 1/10/19 (La0.1CoFe1.9O4). The key advantages of the sol-gel method are versatility, simplicity, effectiveness, achievement of high purity products, narrow particle size distribution, and uniform nanostructure at low temperatures. The main disadvantages refer to the presence of amorphous phases at low annealing temperatures and secondary crystalline phases at high annealing temperatures, respectively [2 network, followed by the thermal-assisted formation of carboxylate precursors and their decomposition to a multicomponent system (mixed oxide). Generally, to obtain spinel ferrites via the sol–gel method, nitrate salts are preferred as precursors, as they are a convenient source of aqueous metal ions and act as low-temperature oxidizing agents for the synthesis [3–/1,3–PD/TEOS molar ratio of 1/1/1. The resulting sols were vigorously stirred over a 1 h and kept at ambient air until gelation occurred. The obtained gels consisting of a homogenous mixture of TEOS, 1,3–PD and metal nitrates were ground thoroughly using an agate mortar pestle and heated at 40 °C for 5 h and 200 °C for 5 h, respectively, in an UFE 400 universal oven . Finally, the powder samples were annealed at different temperatures  for 5 h at 10 °C/min using a LT9  muffle furnace, at ambient temperature.MnFeectively . Brieflyynthesis ,30. Acco−1 were recorded in transmittance mode with a resolution of 2 cm−1 and 8 scans on KBr pellets containing 1% sample using a Perkin–Elmer Spectrum BX II  spectrometer equipped with DTGS detectors. For atomic force microscopy (AFM), the thermally treated powders were dispersed in ultrapure water by stirring to enable the finest particles to release. Bigger particles sediment on the bottom of the vials, while the finest ferrite particles remained dispersed due to Brownian motion. They were transferred onto a glass slide via vertical adsorption, and the formed thin films were dried at room temperature and investigated using a JSPM 4210  microscope. The AFM was operated in alternative current mode with NSC 15  cantilevers with a nominal force constant of 40 N/m frequency of 325 kHz. At least three macroscopic areas of 1 µm2 of the thin films were scanned to obtain the topographic images. The thin ferrite films’ height, surface roughness (Rq), particle diameter, and surface area were obtained by analyzing the images with WinSPM System Data Processing Version 2.0 by JEOL. The magnetic measurements were carried out at room temperature using a cryogen free vibrating-sample magnetometer (VSM), CFSM—12 T . The hysteresis loops were recorded with a maximum field of 2 T, while the magnetization was measured in a high magnetic field up to 7 T. The powder samples were embedded into epoxy resin to avoid the motion of particles.X-ray diffraction patterns were recorded on a D8 Advance  diffractometer equipped with an X-ray tube  and 1–dimensional LynxEye detector; data collection was carried out in the 2θ range of 15–80°, with a step size of 0.015° and counting time of 1s/step. The Fourier-transform infrared (FT–IR) spectra in the range of 400–4000 cm2O4 thermally treated at 40, 200, 500, 800, and 1200 °C are presented in The functional groups, molecular geometry and inter-molecular interactions remarked in the FT–IR spectra of undoped and doped MnFe2 matrix, namely: O–H bond vibration in the Si–OH group (3435–3458 cm−1), H–O–H bond bending vibration (1633–1651 cm−1), Si–O–Si bonds stretching vibration (1083–1098 cm−1), Si–O chains vibration in SiO4 tetrahedron (791–800 cm−1), Si–O bond vibration (450–462 cm−1) and Si–O–Si cyclic structures vibration (542–575 cm−1) [2CH3)4 groups of TEOS in gels dried at 40 °C shown by the shoulder at 939–945 cm−1 disappears at higher temperatures. The water is present on the surface and in the volume of SiO2 particles in physically and chemically (bounded to the surface and molecularly dispersed) bound forms [In all cases, the FT–IR spectra show the representative bands of the SiOnd forms ,32. The −1 is attributed to –O–H bond vibrations in the diol, physically and chemically bound water molecules [−1 and is attributed to the C=O bond vibration in the carboxylate precursors and the chemically absorbed water molecules [For gels dried at 40 °C, the intense band at 1633 cmolecules ,16,32, wolecules . Increas−1 is attributed to the overlapping of C–O and H–O deformation vibrations [−1 in the case of gels thermally treated at 200 °C and is attributed to the C=O bond vibration in the carboxylate precursors; by further increasing the thermal treatment temperature, it progressively decreases till it disappears at 1200 °C [−1 and 3195–3205 cm−1 are attributed to O–H stretching in precursors and to intermolecular hydrogen bonds [−1 present only in gels dried at 40 °C is characteristic of N–O bond’s asymmetric vibration in nitrates. The absence of this band at high temperatures suggests that the reaction between nitrates and 1,3–PD with the formation of metal–carboxylate precursors has already occurred [−1 and 2885–2897 cm−1 present only in gels at 40 °C, are characteristic of C–H bond vibration in the methylene groups of 1,3–PD and carboxylates precursors and disappear at higher temperatures when the precursors decompose [In the case of gels dried at 40 °C, the intense band at 1633 cmbrations ,3,4,5,16en bonds . The intecompose .−1 and in B sites by the band at 450–462 cm−1 [–1 (1200 °C) and 694–706 cm–1 (200 °C) are attributed to O–Fe–O and Fe–OH bond vibration [3+ ion [The vibration of M–O bonds in A sites is indicated by the band at 542–575 cm462 cm−1 . The banibration ,34. The 2O4 gels match the reflection planes of (111), (220), (311), (222), (400), (422), (511), (440), (531), (620), (533), (622) and (444) confirming the presence of pure, low–crystallized MnFe2O4 (JCPDS #00–010–0319) phase with a cubic spinel structure (space group Fd3m) [2O4 is remarked, while at 800 and 1200 °C, the better crystallized MnFe2O4 is accompanied by α–Fe2O3 (JCPDS #00–033–0664) and cristobalite (JCPDS #00–074–9378) [2O4. Contrary to undoped MnFe2O4, for MnFe2O4 doped with monovalent (Ag+ and Na+) ions annealed at 800 °C, the presence of single-phase crystalline MnFe2O4 is remarked. The XRD patterns of gels annealed at 500, 800 and 1200 °C are presented in up Fd3m) . At 500 74–9378) . XRD pat0.1Mn0.95Fe2O4 annealed at 1200 °C, besides the MnFe2O4 crystalline phase, cristobalite and metallic Ag (JCPDS #00–033–0664) are also formed, indicating the presence of unreacted Ag in the SiO2 matrix. The diffraction patterns of MnFe2O4 doped with divalent (Ca2+ and Cd2+) ions annealed at 800 °C display various secondary phases. For Cd0.1Mn0.9Fe2O4, the MnFe2O4 crystalline phase is accompanied by Fe2SiO4 (JCPDS #00–071–1400), while for Ca0.1Mn0.9Fe2O4, the barely crystalline MnFe2O4 is attended by Fe2SiO4, α–Fe2O3, and cristobalite. At 1200 °C, the MnFe2O4 is accompanied by the crystalline phases belonging to SiO2 matrix ) and α–Fe2O3 for Ca0.1Mn0.9Fe2O4, and cristobalite, quartz (JCPDS #00–079–1910), and α–Fe2O3  for Cd0.1Mn0.9Fe2O4. For MnFe2O4 doped with trivalent metal ions (La0.1MnFe1.9O4), single-crystalline-phase MnFe2O4 at 800 °C, and additional secondary phases ) are observed at 1200 °C. Generally, the micron-sized SiO2 is crystalline, while the amorphous SiO2 refers to particle sizes up to 100 nm. Thus, in the case of MnFe2O4 doped with divalent  and trivalent (La3+) ions annealed at 1200 °C, three polymorph crystalline phases of the SiO2 matrix  are formed, while in the case of doping with divalent (Ca2+ and Cd2+) ions annealed at 800 °C, the presence of crystalline Fe2SiO4 is remarked. For AgXRD), degree of crystallinity (DC), lattice constant (a), unit cell volume (V), distance between magnetic ions—hopping length in A (dA) and B (dB) sites, physical density (dp), X-ray density (dXRD), and porosity (P) of gels annealed at 500, 800, and 1200 °C determined by XRD are displayed in The structural parameters, i.e., crystallite size , 16.7–14.7 nm (800 °C), and 66.3–40.1 nm (1200 °C) compared to undoped MnFe2O4. By increasing the temperature, the diffraction peaks become narrower and sharper indicating that the crystallite size increases and the surface area decreases. The DXRD increases with the annealing temperature due to the crystallite agglomeration without recrystallization, leading to the formation of a single crystal instead a polycrystalline structure at high temperatures (1200 °C) [2O4 gels increases with the annealing temperature, but the replacement of Mn2+ ion by metal ions leads to a decrease in lattice constant and the formation of a compositionally homogeneous solid solution. Moreover, the variation in the lattice constant causes internal stress and defeats further grain growth during the annealing process [3+ ion (1.6061 Å) replaces the small-sized Fe3+ ion (0.645 Å) initiates higher asymmetry in the lattice structure [2O4 than doped MnFe2O4  and 800 °C (20 nm) and 1200 °C (45 nm) decreases compared to that of undoped MnFe2O4. The decrease in the ferrite particle size by doping with monovalent  ions and annealing at 1200 °C is most probably due to the shrinkage of the crystal lattice and dependence of particle size on annealing temperature [At low annealing temperatures, Agnd 13 nm d at 500 nd 13 nm e. Howevend 13 nm f. Simila+ doping g–i does perature ,38. 2+ doping leads to the formation of spherical nanoparticles with a diameter depending on the annealing temperature. Accordingly, low temperature annealing generates fine nanoparticles (12 nm), in good agreement with the crystallite sizes of about 10 nm  MnFeperature . The graperature ,43,44. Tperature .M(µ0H) and the first derivatives dM/d(μ0H) of gels annealed at 800 °C (dM/d(μ0H) vs. μ0H curves close to the coercive field indicates the occurrence of a single magnetic phase, specific for crystalline samples [HC) ascribed to particle coalescence during annealing and to relatively low saturation magnetization (SM) values [The magnetic hysteresis loops, t 800 °C  and 1200t 800 °C  indicate samples . The mag) values ,16,33,35SM of gels annealed at 800 °C decreases from 21.5 to 6.4 emu/g for doped MnFe2O4 compared to the undoped MnFe2O4. The small-size particles have a large surface-to-volume ratio, indicating that the high surface defect density is responsible for the SM ‘depreciation [SM value of the nanoparticles [The eciation ,4,5. Morarticles ,33,35. ISM value increases with doping from 23.3 to 32.2 emu/g  decreases for the doped MnFe2O4 gels compared to undoped MnFe2O4, from 5.9 to 1.0 emu/g (at 800 °C) and from 6.5 to 5.4 emu/g (at 1200 °C). Generally, the behavior of SM and RM can be assessed using Neel’s theory via cation distribution between the A and B sites. The Ag+, Na+, Ca2+, Cd2+ and La3+ ions tend to occupy the A sites, Mn2+ ions occupy both A and B sites, while Fe3+ ions can be found mainly in B sites [The remnant magnetization . The lower HC values also suggest a spin distortion due to the surface effects, which affect the magnetocrystalline anisotropy [Compared to undoped MnFeisotropy ,44.SM, the magnetocrystalline anisotropy constant (K) for the doped MnFe2O4 gels annealed at 800 °C decreases (from 1.57 × 10−3 to 0.40 × 10−3 erg/cm3), while for those annealed at 1200 °C increases (from 2.34 × 10−3 to 3.68 × 10−3 erg/cm3) compared to undoped MnFe2O4. A possible explanation could be the decrease in particle size which results in increased surface spin disorder and related surface effects. K is affected by the shape and surface anisotropy of nanoparticles [K.Similar to articles . Since t2O4 nanoparticles can be further enhanced by adjusting the dopant ions content, annealing temperature, or the SiO2 to ferrite ratio, this study introduces valuable information on the properties of doped MnFe2O4/SiO2 nanocomposites. Metal ion doping and annealing temperature can have remarkable effects on the structure, morphology, and magnetic properties of the MnFe2O4/SiO2 nanocomposites, which allow the control of the physical properties of these nanocomposites to make them potential candidates for various applications such as microwave and communication devices, information storage systems, ferrofluid technology, gas sensors, and medical applications for magnetic hyperthermia, magnetic resonance imaging, and photocatalytic activity.While the properties of the obtained doped MnFe+, Na+), di– , and trivalent (La3+) ions and annealing temperature on the structural, morphological, and magnetic properties of MnFe2O4 were studied. Low-crystalline MnFe2O4 at low annealing temperatures and well-crystallized MnFe2O4 at high annealing temperatures were remarked. For the gels annealed at 1200 °C, in MnFe2O4 doped with divalent and trivalent metals, three crystalline phases associated with the SiO2 matrix  were also observed. The crystallite size, degree of crystallinity, lattice constant, unit cell volume, hopping length, and density increased, while the porosity decreased with the annealing temperature. The crystallite size estimated by XRD was in good agreement with the particle size measured by AFM, suggesting that the observed nanoparticles contain a single ferrite crystallite. Doping slightly reduced the ferrite particle diameter depending on the doping ion radius. Uniformly self-assembling ferrite nanoparticles in thin films by adsorption from an aqueous solution may be a straightforward approach for doped ferrite nanocrystalline coatings. The saturation magnetization (SM) of doped MnFe2O4 gels annealed at 800 °C decreased compared to the undoped MnFe2O4 (from 21.5 to 6.4 emu/g), while MS of gels annealed at 1200 °C increased for the doped MnFe2O4 gels (from 23.3 to 32.2 emu/g). Similar behavior was found for the magnetocrystalline anisotropy constant (K). The coercive field (cH) decreased by doping for the gels annealed at 800 °C (from 116 to 100 Oe) and 1200 °C (from 160 to 120 Oe). The magnetocrystalline anisotropy constant (K) of the doped MnFe2O4 gels was lower at 800 °C (from 1.57 × 10−3 to 0.40 × 10−3 erg/cm3) and higher at 1200 °C (from 2.34 × 10−3 to 3.68 × 10−3 erg/cm3) compared to the undoped MnFe2O4. The results obtained confirm that doping and annealing temperature play an important role in tailoring the structural, morphological, and magnetic properties of doped MnFe2O4/SiO2 nanocomposites making them important for catalysts and biomedical applications, such as magnetic resonance imaging, biomolecule detection, and magnetic hyperthermia. Although their use in biomedical applications is still in the beginning stage, some challenges, such as tuning size, shape, and magnetic properties of nanoparticles, exploring additional dopants, and optimizing annealing conditions, require further study. Additionally, adjusting MnFe2O4′ s surface as key performance for biomedical applications should be further explored as one of the most important challenges to obtaining ferrite nanoparticles. The effect of doping with mono- (Ag


Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) is an RNA binding protein with multiple roles in regulation of gene expression at the post-transcriptional level and is implicated in tumorigenesis and progression of numerous cancers including gastric cancer (GC). Circular RNAs (circRNAs) are a diverse endogenous noncoding RNA population that have important regulatory roles in cancer. However, circRNAs that regulate the expression of IGF2BP3 in GC is largely unknown.CircRNAs that bound to IGF2BP3 were screened in GC cells using RNA immunoprecipitation and sequencing (RIP-seq). The identification and localization of circular nuclear factor of activated T cells 3 (circNFATC3) were identified using Sanger sequencing, RNase R assays, qRT-PCR, nuclear-cytoplasmic fractionation and RNA-FISH assays. CircNFATC3 expression in human GC tissues and adjacent normal tissues were measured by qRT-PCR and ISH. The biological role of circNFATC3 in GC was confirmed by in vivo and in vitro experiments. Furthermore, RIP, RNA-FISH/IF, IP and rescue experiments were performed to uncover interactions between circNFATC3, IGF2BP3 and cyclin D1 (CCND1).We identified a GC-associated circRNA, circNFATC3, that interacted with IGF2BP3. CircNFATC3 was significantly overexpressed in GC tissues and was positively associated with tumor volume. Functionally, the proliferation of GC cells decreased significantly after circNFATC3 knockdown in vivo and in vitro. Mechanistically, circNFATC3 bound to IGF2BP3 in the cytoplasm, which enhanced the stability of IGF2BP3 by preventing ubiquitin E3 ligase TRIM25-mediated ubiquitination, thereby enhancing the regulatory axis of IGF2BP3-CCND1 and promoting CCND1 mRNA stability.Our findings demonstrate that circNFATC3 promotes GC proliferation by stabilizing IGF2BP3 protein to enhance CCND1 mRNA stability. Therefore, circNFATC3 is a potential novel target for the treatment of GC.The online version contains supplementary material available at 10.1186/s12967-023-04235-y. Gastric cancer (GC) is a common malignancy that affects over 1 million people annually with a mortality of 7.7% worldwide , 3. ThusRNA binding proteins (RBPs) are key regulators of RNA metabolism and have been implicated in both cancer initiation and progression . The insCircular RNAs (circRNAs) are a class of noncoding RNAs with covalently closed single-stranded loop produced by back-splicing of precursor mRNAs that lack 5' G-caps and 3' A tails , 20. CirCyclin D1 (CCND1) is an effector gene that promotes tumor progression in a variety of cancers –28. In cHere, we identified an IGF2BP3-binding circRNA, circNFATC3, that was upregulated in GC and its expression promoted proliferation of GC cells in vivo and in vitro. Mechanistically, circNFATC3 directly bound to IGF2BP3 and enhanced the stability of IGF2BP3 by preventing tripartite motif-containing 25 (TRIM25)-mediated ubiquitination, thereby promoting Cyclin D1 (CCND1) mRNA stability.GC tissues and corresponding adjacent non-tumour (NC) tissues (16 paired samples) were obtained from Southwest Hospital of the Army Medical University. All patients did not receive any neoadjuvant therapy before operation and were diagnosed as by postoperative pathology. The surgically excised specimens were collected in EP tubes filled with RNA Later  and stored at − 80 °C. Clinical characteristics are listed in Additional file 2 (standard incubation conditions).The human GC cell lines SGC7901, BGC823 and HGC-27 cells were obtained from the Army Medical University . SGC7901 and BGC823 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% foetal bovine serum (FBS) . HGC-27 cells were cultured in RPMI 1640 medium  supplemented with 10% FBS. All cells were cultured at 37 °C in a humidified incubator with 5% COTotal RNA (5 μg) was incubated with or without 6 U RNase R  at 37 °C for 10 min, followed by incubation at 85 ℃ for 5 s. After treatment, expression levels of NFATC3 and circNFATC3 were determined using qRT-PCR.CircNFATC3 stability assays utilized GC cells seeded into 12-well plates that were treated with medium containing actinomycin D (5 μg/ml)  or DMSO for 0, 3, 6 and 9 h. The cells were then lysed in RNAiso Plus  for RNA extraction to assess NFATC3 mRNA and circNFATC3 stability using qRT-PCR. For mRNA stability assays, GC cells separately containing IGF2BP3 knockdown and overexpression and circNFATC3 knockdown systems were cultured in 12-well plates and treated with actinomycin D (5 μg/ml) or DMSO for the indicated times. The cells were lysed in RNAiso Plus for RNA extraction and qRT-PCR measurements.RNA from nuclear and cytoplasm fractions of GC cells were separated using a commercial Paris kit  according to the manufacturer’s instructions. CircNFATC3, NFATC3 and GAPDH cDNA were obtained using a PrimeScript RT Master Mix (Takara) and snoU6 cDNA was synthesized by stem-loop methods . The expression levels of circNFATC3, NFATC3, GAPDH and snoU6 in nuclear and cytoplasm were determined by qRT-PCR.RNA fluorescence in situ hybridization (FISH) assays were conducted using GC cells seeded in μ-Slide 8 well chamber slide  that were incubated for 24 h. And then FISH assays were performed using a commercial kit  according to the manufacturer’s instructions. Biotin-labeled probes  at a 1:400 dilution at 4 °C overnight. The cells were washed for 3 × with PBS and incubated with fluorescent secondary antibody  at 1:400 dilution for 1 h at room temperature and washed 3 × with PBS. Images were acquired with a confocal microscope .−ΔΔCt method was used to analyze the relative expression levels of indicated genes and GAPDH was used as the normalizing control. The primers are listed in Additional file Total RNA was extracted using RNAiso Plus (Takara) and then converted into cDNA with a PrimeScript RT Master Mix (Takara). Real-time qPCR was performed using SYBR Green Master Mix  with CFX connect Real-Time PCR System . The 22O2 and distilled water (1:10) at room temperature for 5 min to inactivate endogenous enzymes, and then digested for 2 min at 37 ℃ with a freshly diluted pepsin drops of 3% citric acid. Next, the specimens were washed 3 × with in situ hybridization PBS and again with distilled water. Then the specimens were fixed with fixing solution for 10 min and washed 3 × with distilled water. The digoxin-labelled probe hybridization solution was added to the specimens and pre-hybridized for 2 h at 50 °C, and then hybridized at 50 °C overnight. The specimens were washed with sodium citrate buffer for 40 min at 50 ℃ and then closed with blocking solution for 30 min at 37 ℃. The specimens were incubated with biotin-conjugated anti-digoxin antibody for 1 h at 37 ℃ and then washed 4 × with in situ hybridization PBS. The specimens were reacted with SABC for 20 min at 37 ℃ and washed 3 × with in situ hybridization PBS. The specimens were incubated with biotinylated peroxidase for 20 min at 37 ℃ and washed 4 × with in situ hybridization PBS. And then the specimens stained with NBT/BCIP (Roche) and photographed. CircNFATC3 expression was quantified using staining scores utilized the staining intensity that was ranked as follows: 0, no expression; 1, mildly positive; 2, moderately positive; 3, markedly positive and the scores were then multiplied by numbers of positive stained cells as follows: 0, < 5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, > 75%.TMAs were produced from 180 paraffin-embedded samples by Outdo Biotech  and clinical characteristics for the sample sources are listed in Additional file Small interfering RNAs (siRNA) were designed and synthesized by GenePharma . The protein samples were separated on SDS-PAGE gels and electrotransferred to polyvinylidene fluoride membranes (PVDF). The membranes were blocked with 5% BSA, and then incubated with primary antibodies overnight at 4 ℃. The membranes were washed with PBST and incubated with HRP-conjugated secondary antibodies (Beyotime) at room temperature for 1 h. The membranes were incubated with ECL chemiluminescent reagent (BioScience) for visualization. The antibodies used in these experiments were as follows: β-actin (Beyotime), IGF2BP3 and TRIM25 , CCND1 , GFP  and HA .RIP experiments were performed using a Magna RIP kit . Briefly, cell lysates from GC cells possessing IGF2BP3 or circNFATC3 knockdowns or IGF2BP3 truncations and overexpression plasmids were incubated with RIP buffer containing antibody against IGF2BP3 (Abcam) or GFP (Roche) or control IgG conjugated magnetic beads overnight at 4℃. Subsequently, immunoprecipitated RNA was extracted and purified from the samples and quantitative analysis of the enriched RNA was performed using qRT-PCR.RNA pull-down assays were performed with RNA pull-down kit . Briefly, cell lysates were incubated with streptavidin magnetic beads that bound the biotin-labeled circNFATC3 pull-down probe or negative control probe  to measure cell viability.The CCK-8 kit  was employed with 3 × 103 cells seeded in 12-well plates containing 1 ml complete culture media that were cultured for 1–2 weeks. The cell spheres were fixed with 4% paraformaldehyde for 10 min and then stained with 1% crystal violet for 10 min. The plates were washed 3 × with water and air-dried at ambient temperature. Images were scanned using an Epson scanner .Plate colony formation assays utilized 1 × 104 cells seeded in 48-well plates containing 250 μl complete culture media and cultured for 24 h. A commercial EdU kit (Ribobio) was used to measure cell proliferation ability according to the manufacturer’s instructions. Cells were photographed with a fluorescent inverted microscope .EdU (5-ethynyl-2′-deoxyuridine) assays utilized 3 × 106 HGC-27 cells were suspended in 100 μl PBS and injected subcutaneously on the backs of BALB/c nude mice. One week later, the size of the xenograft tumor was measured with a caliper and for every 2 days thereafter. Tumor volumes were calculated using the formula: length × width2/2. After the xenograft tumor grew to an appropriate volume, the nude mice were randomly divided into groups. To study the proliferative function of circNFATC3 in vivo, cholesterol modified siRNA targeting circNFATC3 or negative control  was dissolved in 25 μl sterile physiological saline and mixed with 5 μl in vivo transfection reagent  and then injected into the tumor each 2 days for 2–3 weeks. In vivo rescue assays utilized tumor- bearing nude mice treated with a cholesterol-modified siRNA targeting circNFATC3 or negative control combined with lentivirus containing CCND1 or pCDH  using siRNA levels as per above. The lentivirus containing CCND1 was also injected into the tumor mass once a week for 2 weeks. The animals were sacrificed when the tumor size of the nude mice was appropriate. All animal studies were approved by the Ethics Review Committee of Chongqing Medical University.Male BALB/c nude mice  aged 3–4 weeks were employed for our in vivo study. To construct subcutaneous xenografts, 5 × 10IHC was performed as previously described . The priCHX is an inhibitor of protein synthesis in eukaryotes and is used to detect protein stability. GC cells were transfected with siRNA targeting circNFATC3 using Lipofectamine 2000 (Invitrogen) for 40 h and then treated with CHX (20 µg/ml)  for 0, 6 and 12 h. MG132 can effectively reduce the activity of 26S proteasome complex towards ubiquitin binding proteins. GC cells were transfected with si-circNFATC3 for 30 h, and then treated with MG132 (10 µM)  for 12 h. IGF2BP3 protein expression was determined using Western blotting.IP analysis was performed using a commercial Kit . Briefly, cells were lysed with IP lysis/wash buffer containing PMSF and phosphatase inhibitor (Beyotime). Lysates were incubated with magnetic beads cross-linked with IGF2BP3 (Abcam), HA (Abmart), GFP (Roche) or IgG antibodies overnight at 4 ℃. Proteins enriched by magnetic beads were eluted with elution buffer and identified using Western blotting.siRNAs targeting IGF2BP3 were transfected into SGC7901 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The transfected SGC7901 cells were lysed with Trizol (Takara) and subjected to RNA sequencing by Sinotech Genomics .P values < 0.05 was considered as significant.Statistical analysis was performed using Prism 8 . Student’s t tests were used to evaluate differences of continuous variables between the two groups. One-way ANOVA was used to compare the differences of continuous variable among multiple groups. The Mann–Whitney U test was used for ISH and IHC score analyses. We initially employed RIP-seq assays to identify circRNAs that bound to IGF2BP3 in GC cells. We found > 50 circRNAs that interacted with the IGF2BP3 protein and circELK4, circARID1A, circFNDC3B and circNFATC3 displayed the greatest levels of binding  was composed of exons 2 and 3 of NFATC3 gene  and evaluated the expression of circNFATC3 and linear NFATC3 after circNFATC3 knockdowns in GC cells. We found that the both siRNAs targeting circNFATC3 inhibited expression of circNFATC3 in both SGC7901 and BGC823 cells while no detectable change of NFATC3 expression was observed  circRNA Mini Vector and Pcd-ciR plasmid, commercial vector used for circRNA overexpression. No matter how we changed the transfection conditions, circNFATC3 was not successfully overexpressed in GC cell lines, while the linear sequence of circNFATC3 was elevated, which demonstrated that the cyclization of circNFATC3 was inhibited in GC cells. A previous study has shown that the biogenesis of exon-derived circRNAs required coordination between intronic repeats and exons and the sequence or length of exons is also a major factor affecting exon circularization. In addition, short intronic repeats are required for some exon circularization . To testCircRNA-protein interactions are one of the ways that circRNAs exert their biological functions. Here, we found that circNFATC3 was primarily localized in the GC cell cytoplasm and could interact with both the N- and C-terminal regions of IGF2BP3. Interestingly, IGF2BP3 protein but not mRNA expression was significantly downregulated following circNFATC3 knockdown. Moreover, circNFATC3 knockdown in GC cells induced IGF2BP3 degradation through the ubiquitin–proteasome pathway. Similar to our results, previous studies have indicated that other circRNAs could also regulate protein expression. For example, circPFKFB4 inhibits DDB2 degradation in breast cancer  and circUbiquitination is a post-translational modification that mediates protein degradation through the proteasome, thereby influencing tumor progression , 50. E3 IGF2BP3 is an RBP and can affect tumor progression by targeting mRNA for MMP1, CD44, CDK164, ABCG2, IGF2 and other genes . In our Nucleic acid therapy can effectively regulate coding and non-coding target genes with high specificity and is attracting special attention. At present, there are increasing numbers of nucleic acid drugs that are approved or are in clinical trials including siRNA drugs and antisense oligonucleotides , 55. In Here, we identified an IGF2BP3-binding circRNA, circNFATC3, that was significantly overexpressed in GC tissues and was positively associated with tumor volume. Functionally, circNFATC3 promoted the proliferation of GC cells in vivo and in vitro. Mechanistically, circNFATC3 bound to IGF2BP3 in the cytoplasm and enhanced the stability of IGF2BP3 by preventing ubiquitin E3 ligase TRIM25-mediated ubiquitination, thereby enhancing the regulatory axis of IGF2BP3-CCND1 and promoting CCND1 mRNA stability. Our results indicated that circNFATC3 may be a novel therapeutic target for GC Fig. .Fig. 8A Additional file 1: Table S1. Characteristics of GC patients for circNFATC3 measurement. Table S2. Probes for RNA-FISH. Table S3. Primers for amplification of indicated circRNAs and genes. Table S4. Characteristics of GC patients for TMA. Table S5. Probes targeting circNFATC3 for TMA. Table S6. SiRNAs sequences. Table S7. Probes for RNA pull-down. Table S8. CircRNAs interacting with IGF2BP3 in SGC7901 cells (Top 50). Table S9. Upregulated genes in STAD of the TCGA database (Log2(Fold Change)＞2). Table S10. mRNAs interacting with IGF2BP3 in SGC7901 cells evaluated by RIP-seq. Table S11. Downregulated genes evaluated by RNA-seq in SGC7901 cells following IGF2BP3 knockdown.Additional file 2: Fig. S1. The expression of circELK4, circARID1A, circFNDC3B, and circNFATC3 in GC tissues and corresponding adjacent tissues (n=16). The P-values were calculated using the Mann-Whitney U test. ns, not significant, **P < 0.01. Fig. S2. CircNFATC3 sequencing results of GC tissues and the corresponding adjacent tissues, as well as results from SGC7901 and BGC823 cell lines. Fig. S3. Effect of circNFATC3 knockdown on the expression levels of circNFATC3 or NFATC3 mRNA in GC cells. A The interference efficiency of si-circNFATC3-1 and si-circNFATC3-2 in SGC7901 and BGC823 cells. B The expression of NFATC3 mRNA in SGC7901 and BGC823 cells treated with circNFATC3 knockdown by siRNAs. The P-values were calculated using Student’s t test. ns, not significant, *P < 0.05, **P < 0.01. Fig. S4. Knockdown efficiency of IGF2BP3 and circNFATC3 in SGC7901 and BGC823 cells. A The expression of IGF2BP3 in SGC7901 and BGC823 cells after IGF2BP3 knockdown by siRNA. B The expression of circNFATC3 in SGC7901 and BGC823 cells after circNFATC3 knockdown by siRNA. The P-values were calculated using Student’s t test. **P < 0.01, ***P < 0.001. Fig. S5. Effect of circNFATC3 knockdown by si-circNFATC3-1 on proliferation of GC cells. A The viability of SGC7901 and BGC823 cells after transfected with si-circNFATC3-1. B Plate colony formation of SGC7901 and BGC823 cells after transfected with si-circNFATC3-1. C The EdU assay of SGC7901 and BGC823 cells after transfected with si-circNFATC3-1. The P-values were calculated using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S6. Effect of circNFATC3 stable knockdown on the proliferation of SGC7901 cells. (A) Efficiency of lentivirus sh-circNFATC3 transduction in SGC7901 cells. (B) Cell viability of SGC7901 cell following circNFATC3 stable knockdown. (C) Plate colony formation assay of SGC7901 cells with circNFATC3 stable knockdown. (D) The EdU assay of SGC7901 cells with circNFATC3 stable knockdown. The P-values were calculated using Student’s t test. **P < 0.01, ***P < 0.001. Fig. S7. Overexpression of circNFATC3 in GC cells by using different vectors. A-B Plasmid profile of pLC5-ciR (A) and overexpression efficiency of circNFATC3 in SGC7901 and BGC823 cells, as well as linear product of circNFATC3 in SGC7901 cells (B). C-D Plasmid profile of pcDNA3.1(+) CircRNA Mini Vector (C) and overexpression efficiency of circNFATC3 in SGC7901 and BGC823 cells, as well as linear product of circNFATC3 in BGC823 cells (D). E-F Plasmid profile of pCD-ciR (E) and overexpression efficiency of circNFATC3 or linear product in SGC7901 cells (F). G The overexpression of pLC5-ciR-circPDHK1 in SGC7901 and BGC823 cells. H The overexpression of pCD-ciR-circTNPO3 in SGC7901 and BGC823 cells. The P-values were calculated using Student’s t test. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S8. Regulation of circNFATC3 and IGF2BP3 to each other in GC cells. A-B The expression of IGF2BP3 at RNA (A) and protein (B) level in SGC7901 and BGC823 cells transfected with si-IGF2BP3. C The expression of circNFATC3 in SGC7901 and BGC823 cells after IGF2BP3 knockdown. D-E The expression of IGF2BP3 at RNA (D) and protein (E) level in SGC7901 and BGC823 cells transfected with pCDH-IGF2BP3. F The expression of circNFATC3 in SGC7901 and BGC823 cells after IGF2BP3 overexpression. G The expression of IGF2BP3 at RNA level in SGC7901 and BGC823 cells after circNFATC3 knockdown. The P-values were calculated using Student’s t test. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S9. Quantitative analysis of IGF2BP3 protein stability after circNFATC3 knockdown in SGC7901 and BGC823. Fig. S10. Expression of LC3 in SGC7901 and BGC823 cells after circNFATC3 knockdown. Fig. S11. Overexpression efficiency of TRIM25 (HA-tagged) and IGF2BP3 truncations (GFP-tagged) in SGC7901 and BGC823 cells. Fig. S12. Expression of RCC2, ARL6IP1, CCND1, and MUC13 mRNA in SGC7901 and BGC823 cells treated with si-IGF2BP3, pCDH-IGF2BP3, or si-circNFATC3. A Expression of RCC2, ARL6IP1, CCND1, and MUC13 mRNA in SGC7901 and BGC823 after IGF2BP3 knockdown. B Expression of RCC2, ARL6IP1, CCND1, and MUC13 mRNA in SGC7901 and BGC823 treated with IGF2BP3 overexpression. C Expression of RCC2, ARL6IP1, CCND1, and MUC13 mRNA in SGC7901 and BGC823 after circNFATC3 knockdown. The P-values were calculated using Student’s t test. ns, not significant, *P < 0.05, **P < 0.01，***P < 0.001. Fig. S13. The proliferation of SGC7901 and BGC823 cells after CCND1 knockdown. A The viability of SGC7901 and BGC823 cells transfected with si-CCND1. B Plate colony formation of SGC7901 and BGC823 cells transfected with si-CCND1. C The EdU assay of SGC7901 and BGC823 cells transfected with si-CCND1. The P-values were calculated using Student’s t test. *P < 0.05, **P < 0.01，***P < 0.001. Fig. S14. Expression of CCND1 protein in SGC7901 and BGC823 cells transfected with blank vector or pCDH-CCND1 and co-transfected with NC or circNFATC3 siRNA. Fig. S15. Expression of circNFATC3 and CCND1 in xenograft tumor tissues. A Expression of circNFATC3 in xenograft tumor tissues treated with lentivirus containing CCND1 or cholesterol modified si-circNFATC3. B Expression of CCND1 in xenograft tumor tissues treated with lentivirus containing CCND1 or cholesterol modified si-circNFATC3. The P-values were calculated using Student’s t test. ns, not significant, *P < 0.05, ***P < 0.001. Fig. S16. Representative images of IGF2BP3 expression evaluated by IHC in xenograft tumor tissues. Three different visual fields were randomly selected for each slice. The P-values were calculated using Mann-Whitney U test. ***P < 0.001.


One potential practical solution to eliminate CO2 is to convert CO2 into formate using hydrogen (H2) (CO2 hydrogenation), which can be accomplished with inexpensive hydrogen from sustainable sources. While industrial flue gas could provide an adequate source of hydrogen, a suitable catalyst is needed that can tolerate other gas components, such as carbon monoxide (CO) and oxygen (O2), potential inhibitors. Our proposed CO2 hydrogenation system uses the hydrogenase derived from Ralstonia eutropha H16 (ReSH) and formate dehydrogenase derived from Methylobacterium extorquens AM1 (MeFDH1). Both enzymes are tolerant to CO and O2, which are typical inhibitors of metalloenzymes found in flue gas. We have successfully demonstrated that combining ReSH- and MeFDH1-immobilized resins can convert H2 and CO2 in real flue gas to formate via a nicotinamide adenine dinucleotide-dependent cascade reaction. We anticipated that this enzyme system would enable the utilization of diverse H2 and CO2 sources, including waste gases, biomass, and gasified plastics.It is challenging to capture carbon dioxide (CO To address this issue, we propose a CO2 hydrogenation system that utilizes multiple enzymes, taking advantage of their high reactivity and substrate specificity under ambient temperature and pressure conditions.Industrial flue gas can address this issue as a cheap Hindustry . The steindustry . In partarly 60%   for H2 oxidation , etc., in addition to the substrate H2 and CO2. The H2ase and FDH used in the previous study as well as most other H2ases and FDHs were derived from anaerobic microorganisms, and so their metal-active sites are often attacked and inactivated even in the presence of trace amounts of O2  consisting of heterodimeric [NiFe] hydrogenase (HoxHY) subunits and diaphorase (HoxFU) subunits, stably oxidized H2 and reduced NAD+ to NADH under oxic conditions  . Although metal-independent FDH exhibits O2-independent activity, the strategies like mutation and immobilization have been reported to enhance its activity and stability due to its inherently low activity , consisting of alpha and beta subunits, as FDH to produce formate via enzymatic NAD+ regeneration stably . Ni-NTA agarose and a gravity-flow polypropylene column  were purchased from Qiagen . Strep-Tactin XT 4 Flow high-capacity resin was obtained from IBA Life Sciences . Disposable PD-10 desalting columns were purchased from Cytiva . Unless otherwise stated, all other chemical reagents were purchased from Sigma-Aldrich .Strains and expression conditions of ReSH and MeFDH1 were described in previous studies . To puriThe purification of MeFDH1 was started with the cell lysis by sonication in an anaerobic chamber. The wet cell pellet was suspended into the buffer A (50 mM (3-(N-morpholino) propanesulfonic acid (MOPS), 200 mM NaCl, 20 mM imidazole, 2 mM dithioerythritol (DTE) and 2 µM resazurin, pH 7.0) with 1 g wet cell/10 mL buffer concentration and sonicated . The lysate was centrifuged at 11,000 rpm at 4 °C for 20 min. The supernatant and Ni-NTA agarose bead were mixed together and incubated for 15 min to bind protein. The protein-bound Ni-NTA agarose bead was separated from the mixture using a gravity flow column. The collected protein-bound Ni-NTA agarose bead was washed with buffer A. The MeFDH1-bound Ni-NTA agarose bead was stored at 4°C until use. For the activity and kinetic property assay, the purified MeFDH1 was eluted by buffer B .−1⋅cm−1, respectively, based on their amino acid sequences.Protein purity was verified by SDS-PAGE . The concentrations of purified ReSH was determined by measuring their absorbance at 280 nm using a microplate reader , as previously reported for other proteins . The con+-dependent oxidation of H2 to H+ in the presence or absence of O2 and CO. The sealing cuvette was filled with 900 μL of 200 mM Kpi buffer (pH 6.5) containing NAD+ and sealed; then, 100% H2 and mix gas containing 1) 100% N2, 2) 96% N2 and 4% O2 3) 60% N2 and 40% CO, and 4) 56% N2, 4% O2 and 40% CO were injected simultaneously for 30 min at 10 mL/min. ReSH  was purged with 10 mL/min N2 gas bubbling in a 25 mL sealing vial for 30 min to remove O2 from the air. The reaction was initiated by mixing 100 μL of 80 nM ReSH with a gas-saturated solution in a sealed cuvette. The final concentration of NAD+ was varied from 0 to 4 mM.The enzyme reaction kinetic parameters of ReSH were measured for the NAD2 to formate in the presence or absence of O2 and CO. The 200 mM Kpi buffer (pH 6.5) was purged with gases containing 1) 100% N2, 2) 100% O2 and 3) 100% CO for 1 h in closed serum bottles. Each gas-purged buffer was mixed to make 1) 100% N2, 2) 98% N2 and 2% O2, 3) 80% N2 and 20% CO and 4) 78% N2, 20% CO and 2% O2 dissolved buffers. The powder of sodium bicarbonate and NADH was dissolved into each buffers (100 mM of sodium bicarbonate and 6.25—100 µM of NADH). The 980 μL of buffers are finally aliquoted into the cuvettes and sealed. By injecting 10 ug of MeFDH1 in 20 uL of buffer B, the reaction was started.The enzyme reaction kinetic parameters of MeFDH1 were measured for the NADH-dependent reduction of COversus the substrate concentration according to the Hanes-Woolf plot to calculate the kinetic parameters.All measurements were performed in triplicate based on the change in the absorbance at 340 nm in the cuvette. The absorbane changes for ReSH and MeFDHS1 were measured using the T60 UV-Vis spectrophotometer and the UV-1650 PC , respectively. The inverse of initial reaction rates was plotted +, and 1% (v/v) antifoam A. All components were resuspended or dissolved in 200 mM Kpi buffer (pH 6.5). The reaction was initiated by a 30 mL/min substrate gas injection. Formate production was sampled every 30 min during incubation for 1.5 h, and 20 μL of 6 N H2SO4 was added to the 200 μL sample to inactivate the enzymes immediately. Additionally, 180 μL of distilled water was mixed with the sample, and the aggregated enzymes were removed by syringe filter. Formate production was quantified by HPLC  equipped with a diode-array detector and an Aminex HPX-87H column  with a mobile phase of 5 μM H2SO4 at a flow rate of 0.6 mL/min.For the cascade reaction, the gas content was controlled in a gas flow reactor . The reaR. eutropha and M. extorquens, respectively. The ReSH and MeFDH1 were immobilized on the strep tactin XT 4flow resin and Ni-NTA agarose affinity resins, respectively, as described in the Materials and Methods section. A fraction of these was eluted separately to obtain purified proteins. The SDS-PAGE analysis was performed to verify the existence of subunits and purity of each. Five bands of purified ReSH subunits were observed, which matched their expected molecular weights   . Two ban,567 Da) . The pur+-dependent H2 oxidation of ReSH and NADH-dependent CO2 reduction of MeFDH1 to determine the buffer conditions in the reactor for the cascade reaction. The specific activity of ReSH tended to increase with increasing pH from 6 to 8 (We investigated the pH-dependent enzyme activities of NADm 6 to 8 . In contm 6 to 8 . Since t2 and CO) present in the flue gas at pH 6.5. The final concentrations of O2 and CO were 2% and 20%, respectively, which is higher than those inside the COG, to evaluate enzyme activity inhibition. The NAD+-dependent H2 oxidation reaction rate by ReSH was measured, and the Hanes-Woolf plot was fitted to calculate the kinetic parameters using Origin 2022 program  and measured the formate produced when supplied with gas substrate H2 and CO2 for 1 h  . On the other hand, metal-dependent FDHs display high catalytic activity (with a range of kcat/Km 100∼103 s-1 mM-1), but most of them face difficulties in utilizing real CO2 sources due to susceptibility to inactivation by O2 (2 and CO.We investigated formate production by feeding real flue gases to the enzymatic reactor. We provided the COG, BFG, and a 1:1 mix of COG and BFG to the enzymatic reactor and monitored the formate production . Contrar2 hydrogenation using hydrogen in the real flue gas from the steel industry via an NAD+-dependent cascade reaction in an enzymatic reactor that combines a specific H2ase and FDH. We confirmed that ReSH and MeFDH1, previously reported as O2-tolerant, are also resistant to CO, a potential inhibitor of metalloenzymes. We used both H2 and CO2 from the flue gas in this study. However, the enzymatic reactor system developed in this study is expected to enable the utilization of CO2 and H2 obtained from other sources, such as industrial waste gases, biomass and gasified plastics. We plan to improve the stability of the enzymes for the long-term operation of the reactor and improve the cofactor co-immobilization system (We have successfully demonstrated COn system  for impl


Several 2, 4-dinitrophenyl and 2,4-dinitrophenylamine tethered 5-FU (5-fluorouracil) compared to their components have shown minimal or no cytotoxicity to HT-29 cell line under aerobic conditions but high cytotoxicity and radiosensitizing effects under hypoxic conditions. In the present study the cytotoxicity and radiation potentiation of three novel analogues of these compounds by replacing 2,4-dinitrophenyl moiety with 2-methyl-5-nitroimidazole, a known radiosensitizer and cytotoxic agent was investigated.7–9 were prepared by the reaction of 1-(t-butoxycarbonyl)-5-fluorouracil 6 with metronidazole esters 2–4 followed by removal of the t-butoxycarbonyl protecting group. Cytotoxicity of compounds in HT-29 cells with or without radiation were determined by 3--2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI)-digitonin and clonogenic assays.Tethered compounds 7–9 induced time-and concentration–dependent cytotoxicity under hypoxia but had no significant effect under aerobic conditions. These compounds also showed selective and concentration- dependent radiosensitization effects under hypoxic conditions.Tethered compounds 7–9 compared to 5-FU 5 showed minimal cytotoxicities under aerobic and selective radiosensitizing activities under hypoxic conditions. Also effects of these compounds were higher than those of metronidazole 1 which is a known cytotoxin and radiosensitizer under hypoxic conditions.Tethered compounds  Oxygen as a radiosensitizer increases the effects of radiation by the formation of free radicals that damage DNA . ElectroNitroimidazoles ,2 specifin vitro aerobic cytotoxicity of 2, 4-dinitrophenylamine tethered 5-FU through three carbon atoms in HT-29 cell line with and without radiation showed that the compound unlike its components has no cytotoxicity but is radiosensitizer [Previously, results of our study on nsitizer . Since insitizer  and 2,4-nsitizer  are due 50 values of compounds which showed selective cytotoxicity were not different significantly it was concluded that they have similar mechanism of action and might produce the same reactive species [Also results of our investigations on 2,4-dinitrobenzyl, 2,4-dinitrobenzoyl, 3,5-dinitrobenzoyl and 2,4-dinitrophenacetyl derivatives of 5-FU showed that with exception of 3,5-dinitrobenzoyl derivatives other compounds have selective- cytotoxicity and radiosensitization effects under hypoxic conditions . Since t species .in vivo hydrolysis to generate active metabolites which might have synergistic actions.In continuation of our investigations on this type of bifunctional compounds -11 in th1, 5-FU 5, di-t-butoxydicarbonate (Boc)2O, triethylamine, sodium hydride and other chemicals and solvents not listed here were from Merck Chemical Co. . Thin layer chromatography was carried out using Kieselge l60, 230–400 mesh,  to monitor the progress of reactions. Melting points were determined on a MEL-270 Sibata melting point apparatus and are uncorrected. 1HNMR spectra were recorded on 500 MHz a Bruker (Germany), using DMSO-d6 or CDCl3 as solvent. Chemical shifts (δ) are reported in ppm relative to TMS as internal standard. Mass spectra were obtained on a Finningan TSQ-70 (USA) instrument. Infrared spectra were recorded on a Nicolet Magna IR-550 (USA) spectrometer. All compounds were characterized by the above techniques. Analytical and spectroscopic data for the known compounds were consistent with the reported literature values and data only for new compounds are presented.Metronidazole 1  in 100 ml of anhydrous dichloromethane was added 1.9 ml (24 mmol) triethylamine at temperature below 5°C and after stirring for 30 min the solution was treated with 3.2 ml (40 mmol) of chloroacetyl chloride dropwise within 5 min and then the resulting solution was stirred at room temperature overnight. The mixture was filtered and the residue after evaporation of the solvent was subjected to flash chromatography using Pet-ether: EtOAC as eluting solvent (80:20) to give 7.9 g (64%) of the compound 3. mp 80-82°C; 1H NMR (DMSO-d6): δ 8.01 , 4.61 , 4.48 , 4.35 , 2.18 ; FT-IR (KBr): 1720 (C = O), 1546, 1350 (NO2) cm-1.To a solution of metronidazole 1  with 3.85 ml (40 mmol) of 3-chloropropionyl chloride under above conditions for the preparation of the ester 3 but purification of the product by crystallization from Pet-ether: EtOAC instead of flash chromatography gave 4.1 g (71%) of the ester 4. mp 89-93°C;1H NMR (DMSO-d6): δ 8.01 , 4.58 , 4.40 , 3.7 , 2.5 , 2.18 ; FT-IR (KBr): 1729 (C = O), 1540, 1345 (NO2) cm-1.The reaction of metronidazole t-butoxycarbonyl)-5-FU 6[2-4 in 10 ml of anhydrous DMF and the mixture stirred at room temperature for 18 h. The mixture was then diluted with the same volume of water, extracted with ethyl acetate (3×20 ml), and the organic layer was dehydrated using anhydrous sodium sulfate. Evaporation of ethyl acetate gave an oily residue which was dissolved in 10 ml of methanol, treated with 0.42 g potassium carbonate and the obtained mixture was stirred at room temperature for 1 h. After evaporation of the solvent under reduced pressure, the residue was dissolved in water-ethyl acetate mixture (1:1), the organic layer was separated and dehydrated using anhydrous sodium sulfate and then evaporated under reduced pressure. For the preparation of compound 7, the residue after evaporation of the solvent was purified by flash chromatography using Pet-ether: EtOAC as eluting solvent (70:30) to give 100 mg (35%) of the product. For the preparation of compounds 8 and 9, residues were crystallized from Hexan-EtOAC to give 310 mg (92%) and 330 mg (93%) of the pure products respectively.To a stirred solution of 225 mg (1 mmol) of 1-(l)-5-FU 6 in 10 ml1HNMR (CDCl3): δ 7.9 , 7.8 , 4.0 , 3.9 , 2.4 ; FT-IR (KBr): 3108 (NH), 1722, 1671 (C = O), 1550, 1350 (NO2) cm-1; MS: m/z 81(100%), 130 (78%), 238 (39%), 155(24%).mp 185-189°C; 1H NMR (DMSO-d6): δ 7.99 , 7.93 , 4.65 , 4.42 , 3.98 , 2.42 ; FT-IR (KBr): 3131 (NH), 1725, 1656 (C = O), 1542, 1349 (NO2) cm-1; MS: m/z 341 (7%), 296 (23%), 212 (80%), 171(74%), 130 (100%), 126 (42%).mp 231–234°C; 1H NMR (DMSO-d6): δ 8.01 , 7.98 , 4.57 , 4.39 , 3.78 , 2.70 , 2.44 ; FT-IR (KBr): 3124 (NH), 1721, 1652 (C = O), 1547, 1350 (NO2) cm-1; MS: m/z 355 (5%), 309 (21%), 226 (86%), 185(75%), 171 (42%), 130 (100%), 126 (59%).mp 247-250°C; HT-29 cells, originally derived from human colorectal carcinoma were obtained from Pasteur Institute . Fetal Bovine Serum (FBS), Penicillin/Streptomycin 100X, Trypsin-EDTA 10X and Trypan blue were from Biosera (England). RPMI 1640 from Gibco (England), MTT and PI from Sigma (USA) and Digitonin from Merck (Germany) were used in this study.2 and 95% air at 37°C [HT-29 cells were grown as an attached monolayer in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (50 unit/ml), and streptomycin (50 μg/ml). Cells were routinely grown in tissue culture flasks and kept in a humidified atmosphere of 5% CO at 37°C .5, metronidazole 1, and tethered compounds 7–9 against HT-29 cells under both aerobic and hypoxic conditions were determined by measurement of the percentage of cell survivals using MTT [4 cells/well in 96-well culture dishes and incubated for 4 h. Stock solutions of the tested compounds in DMSO at concentrations of 50 mmol which were prepared prior to the experiments diluted with culture media and at concentrations of 6.25-400 μmol/ml added to each well of plates. Cells in the control wells were treated with the same volume of medium which contained DMSO at concentration equal to those which were used in incubations treated with the tested compounds. Cell incubations were kept at 37°C under either air by purging with 95% nitrogen and 5% CO2 or hypoxia in sealed GasPack jar by using Anoximat (Netherland) and an anaerobic chamber GasPak Jar  and GasPak pluss  [Cytotoxicity of 5-FUsing MTT  and PI-dsing MTT ,17 and csing MTT . Cells wGermany)  for 3 or25 μl of the reagent (5 mg/ml in PBS) was added to each well, cell cultures were incubated for 4 h at 37°C and then the precipitated formazan was dissolved in 100 μl of DMSO and its absorbance was read in Elisa reader at 550 nm and a background wavelength of 690 nm. Survival was scored by the ratio of the absorbance of the treated cells to untreated cells with the tested compounds (control) and is expressed as percentage of the cell survival .20 μl of a 1.53 mmol solution of PI in PBS for a final concentration of 30 μmol was added to each well, cell cultures were incubated for 60 min at 37°C and then the initial fluorescence intensity from the dead cells was measured in a multi-well plate reader,  with 530-nm excitation and 645-nm emission filters. Then digitonin (600 nmol) was added to each well to permeabilize all cells and label all nuclei with PI and after 30 min incubation, the fluorescence intensity was measured again to obtain a value corresponding to the total number of cells. The percentage of dead cells was calculated as the proportion of fluorescence intensity of the dead cells to that of total cells multiplied by 100 and percentage of the cell viability was calculated by subtracting the percentage of dead cells from 100% .50 values for both MTT and PI-digitonine assays were determined from cell survival curves in which the percentages of cells that survived were plotted as a function of the concentration of the tested compounds.The IC3 cells per well and maintained at 37°C for 14 days. The cells were then fixed in methanol, colonies which were formed stained with crystal violet and those containing more than 50 cells were scored. The number of colonies of treated and untreated dishes with the tested compounds were compared and expressed as the percentage of survival by clonogenic assay [The medium of the cell cultures after treatment with the tested compounds and/or irradiation under aerobic or hypoxic conditions were first separated to remove the tested compounds, and washed with PBS. Cells were seeded in 6-well plates at concentration of 5 × 10ic assay . Clonoge2 by a 60Co source (Theratron 780 MDS – Nordion).HT-29 cells at 37°C under hypoxic or aerobic conditions were incubated with the tested compounds at concentrations of 25, 50, and 100 μmol for 3 h and then at room temperature were exposed to 4 different doses of radiation  at the dose rate of 1.67 Gy/min and Source Surface Distance (SSD) = 80 cmRadiosensitivity of the tested compounds were determined through measurement of the cell growth inhibition using clonogenic assay . ResultsFor the cell survival and cell death assays each experiment was repeated three times and results are mean ± SD of three independent experiments. Statistical analysis was performed using Sigmaplot 12 (Systat Software Inc.) for window and descriptive statistics are shown as arithmetic mean ± standard deviation. Independent samples’ t-test was used to investigate the mean differences between irradiated and un-irradiated cells treated with the tested compounds and for comparison of more than two groups, Holm-Sidak multiple comparison test followed by one way analysis of variance (One way ANOVA) were performed and p value smaller than 0.05 was considered statistically significant.7–9 were prepared by the reaction of 1-(t-butoxycarbonyl)-5-FU 6[1 -5-FU 6 with p-te ester 2 through 5, metronidazole 1, and tethered compounds 7–9 for different incubation periods on viability and proliferation of HT-29 cells were determined by MTT (15), PI-digitonin [7  from each other and results of these tests which are seldom unanimous [Effects of different concentrations of 5FU igitonin ,17, and nanimous  were in 7–9 under hypoxic conditions exhibited remarkable concentration–dependent cytotoxicity  under hypoxic (232.1±21-240±14) compared to aerobic (304±18-330±16) conditions suggesting that compounds have hypoxia-selective cytotoxicity.While tethered compounds y Figure B, these y Figure B. Consis7–9 at various concentrations under aerobic conditions were not different significantly (p > 0.05)  Figure A and at ) Figure B. Howeve) Figure  radiosen) Figure  which uned value  for an i5 against HT-29 cell line under aerobic and hypoxic conditions were not different significantly and this compound at all concentrations showed no radiosensitizing activity, tethered compounds 7–9 showed higher cytotoxicities and selective radiosensitizing activities under hypoxic conditions. Also effects of these compounds relative to metronidazole 1 which is a known cytotoxin and radiosensitizer were comparable under aerobic but higher under hypoxic conditions. Further exploration of these compounds through modification of the linker chain might result in compounds which their uses in conjunction with tumor radiotherapy have greater toxicity to tumor than normal tissues.While the cytotoxicity of 5-FU 5 FU: 5- fluorouracil; MTT: 3--2,5-diphenyltetrazolium bromide; PI: Propidium Iodide; SER: Sensitization enhancement ratio; PBS: Phosphate buffer saline; FBS: Fetal bovine serum.The authors declare that they have no competing interests.KA: Designer of the project, corresponding author of the manuscript. AK: Determination of cytotoxicity and radiosensitizing activity of the tested compounds. OS-N: Supervisor for the determination of the cytotoxocity and radiosensitizing activity of the tested compounds. LN: Synthesis of the tethered compounds. KMR: supervising statistical analyses. All authors read and approved the final manuscript.


Streptomyces thermoviolaceus subsp. thermoviolaceus isolate 76T-2 that can degrade poly(ε-caprolactone) (PCL) was isolated from soil in Taiwan. Isolate 76T-2 grew well in urea fructose oatmeal medium and exhibited clear zones on agar plates containing PCL, indicating the presence of extracellular PCL depolymerases. The PCL powder present in culture medium was completely degraded within 6 h of culture at 45°C. Two PCL-degrading enzymes were purified to homogeneity from the culture supernatant. The molecular weights of these two enzymes were estimated to be 25 kDa and 55 kDa, respectively. A portion of the N-terminal region of the 25-kDa protein was determined, and the sequence Ala-Asn-Phe-Val-Val-Ser-Glu-Ala thus obtained was identical to that of A64-A71 of the Chi25 chitinase of Streptomyces thermoviolaceus OPC-520. The 25-kDa protein was shown to also degrade chitin, suggesting that isolate 76T-2 has the ability to degrade both PCL and chitin.A thermophilic  The powdery colonies with branched hyphae on the plates were then picked and streaked on International Streptomyces Project (ISP) 3 agar plates  and incubated at 45°C for 3–7 days for identification. The PCL-degradation ability of the isolates was detected by the presence of a clear zone around the colonies on agar plates containing emulsified PCL. PCL agar plates were made by dissolving one gram of PCL powder in 50 ml of methylene chloride and then emulsified into 50 ml basal medium containing (per liter) yeast extract, 0.1 g; FeSO4·7H2O, 10 mg; MgSO4·7H2O, 0.2 g; (NH4)2SO4, 1 g; CaCl2·2H2O, 20 mg; NaCl, 0.1 g; Na2MoO4·2H2O, 0.5 mg; NaWO4·2H2O, 0.5 mg; MnSO4·H2O, 0.6 mg; and dishwashing liquid, 1 ml . Methylene chloride was then evaporated in a hume hood.Thermophilic actinomycetes were isolated from soil samples as described previously  plates at 45°C for 7 days, a representative colony was isolated, fixed in 4% osmium tetroxide, dehydrated through a graded ethanol series, dried to critical point, coated with gold, and then examined with a scanning electron microscope , and Urea Fructose Oatmeal (UFO) media. The optimal growth temperature was assessed using UF medium. For examination of PCL-degrading enzymes, 76T-2 cells were grown at 45°C in UF medium to stationary phase and then transferred to basal medium containing 0.1% (w/v) PCL for various lengths of time. To assay extracellular PCL-degrading enzymes, aliquots of culture supernatant were assessed for the decrease in turbidity of the emulsified PCL. To quantify PCL-degrading activity, 1 ml of an overnight liquid culture in UF medium was mixed with 20 ml of basal medium containing 0.3% PCL and incubated at 30-50°C for 14 h. The PCL-degrading activity was measured spectrophotometrically at 650 nm.The growth characteristic was determined by growing 76T-2 cells in Luria Bertani (LB), UF  and dialyzed against four volumes of the buffer. Protein concentration was determined using the Lowry method with bovine serum albumin as a standard. Isolated enzymes were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). PCL-degrading activity was detected by zymography by separating the proteins in a native polyacrylamide gel. After electrophoresis, the gel was placed on another polyacrylamide gel containing 0.1% PCL or chitin and incubated at 45°C for 30 min. Active protein bands were detected as clear zones. The gel was then stained with Coomassie brilliant blue R-250 to determine the sizes of the proteins with PCL-degrading activity.One gram of each soil sample was suspended in 9 mL of sterilized distilled water. A serially diluted 0.1-mL aliquot of each sample was plated on a modified HV agar plate and incubated at 45-50°C for 7–10 days. Many powdery and irregular colonies with branched hyphae were seen in most cultures. Some of these isolates were found to degrade PCL when they were subcultured on oatmeal agar plates containing PCL, and an isolate designated 76T-2 was found to have the highest PCL-degrading activity .Streptomyces  has been deposited in the Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan.Isolate 76T-2 produced round, pale white cottony colonies on oatmeal agar plates. Analyses of its peptidoglycan cell wall revealed that the major constituent was LL-diaminopimelic acid (LL-DAP). No sugars were detected. The cell wall was thus determined to be chemotype I  after 12 h of incubation in UF medium at 40°C or 45°C. A slower growth rate was seen after 12 h (OD650 = 1.6) and 24 h (OD650 = 1.7) of incubation at 50°C. 76T-2 cells did not grow well at 30°C. These results  and dried cell mass of the cultures were measured every two hours for 14 hours  of culture and was maximal (OD650 = 0.05) at 1 h of incubation when 76T-2 cells were grown at 40°C. No further increase in PCL degradation was observed afterward.The effect of temperature on PCL degradation by 76T-2 was then investigated. 76T-2 cells were grown in UF medium for 12 h at 45°C and then in basal medium containing 0.3% PCL at a temperature between 30°C and 50°C. The PCL hydrolytic activity was assayed every 2 h for 14 h by measuring the turbidity of the culture. Changes in PCL turbidity as indicated by OD650 = 0.95) was observed after 1 h of culture, but a significant degradation (OD650 = 0.15) of PCL was seen at the 2 h time point. The PCL degradation reached the maximum (OD650 = 0.05) after 4 h of incubation. A similar trend of PCL degradation was observed at 50°C with an OD650 = 1.1 at 1 h and OD650 = 0.05 at 2 h and no further increses in PCL degradation afterward. However, when the temperature was changed to 30°C, a slow (OD650 = 1.1 after 1 h incubation) and gradual increase in PCL degradation rate with an OD650 = 0.1 at 14 h of incubation was observed. These results suggest that the optimal temperature for PCL degradation was 40°C.At 45°C, a lower degree of PCL hydrolysis  concentration, maximum protein precipitation was achieved. When the precipitate was analyzed by electrophoresis in an 8% native polyacrylamide gel, multiple protein bands were found Figure A. Two baTo investigate chitin degradation activity of the PCL depolymerases, the 60% ammonium sulfate precipitate was analyzed in an 8% native polyacrylamide gel and then overlaid on another gel containing chitin. A clear zone due to chitin hydrolysis was seen at the place corresponding to protein II (25-kDa protein) but not protein I (55-kDa protein) Figure B, lane 2Alcaligenes, Clostridium, Aspergillus, Penicillium, Fusarium, and Streptomyces have been isolated , an important polymer due to its strong mechanical properties, biodegradability, and miscibility with a number of other polymers   have been identified in Streptomyces species have been extensively studied with respect to their structure, function, and regulation of gene expression  (Tsujibo et al. S. thermoviolaceus.Although chitinases from many The authors declare that they have no competing interests.


H. pylori, whose infection increases tumor invasiveness and metastasis, is generally labelled as the strongest risk factor for the development of gastric cancer. It appears not to be a coincidence that there is also an overexpression of CXCR4 and an obvious involvement in gastric cancer metastasis. The aim of this study attempts to investigate and further to establish a link between them. With H. pylori being a potent inducer of TNF-α, whether TNF-α, a tumor promoter, is involved in the induction of CXCR4 expression by H. pylori was also under research in this study.Expression of CXCR4, TNF-α, IL-6 and IL-1β mRNA was determined by real-time PCR. CXCR4 protein expression was detected by Western blotting. Concentrations of TNF-α, IL-6 and IL-1β in cell culture supernatants were measured using the Quantikine Elisa kit. To abrogate TNF-α expression in HGC27 cells, TNF-α RNAi plasmid was used to transfect them.+ ) or Tx30a (cag PAI- ); The induction of CXCR4 expression by H. pylori was inhibited significantly by a neutralizing TNF-α antibody, infliximab; CXCR4 expression was upregulated in MKN45 cells after treatment with exogenous TNF-α or co-culture with macrophage, and was downregulated in HGC27 cells after transfection with TNF-α RNAi plasmid. There was a significant increase in the migration of MKN45 cells treated with H. pylori 26695, and a strong inhibition when AMD 3100, a CXCR4 antagonist, or infliximab, was added.Levels of CXCR4 and TNF-α mRNA were significantly higher in H. pylori-positive gastric cancers (n = 19) compared to H. pylori-negative ones (n = 15). A subsequently Spearman's rank correlation test showed there was a positive correlation between the level of CXCR4 mRNA and that of TNF-α in 34 primary gastric cancers. Other results followed: Expression of CXCR4 and TNF-α was upregulated in gastric cancer cell MKN45 and HGC27 after infection with H. pylori 26695 (cag PAIOur findings demonstrated that H. pylori upregulates CXCR4 expression in gastric cancer through TNF-α. It is well accepted that Helicobacter pylori (H. pylori) is a strong risk factor for the development of various gastric diseases, namely chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer, and it is acknowledged that the interaction between H. pylori and epithelial cells contributes to such development. In fact, H. pylori infection induces inflammation in microenvironment of the stomach associated with induction of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which mH. pylori infection also increases tumor invasiveness and metastasis -6, thougOne of the key chemical mediators implicated in inflammation-associated cancers is TNF-α, and there is now substantial evidence in its involvement in promotion and progression of experimental and human cancers ,11. True2. 34 primary gastric cancer specimens were acquired from patients under operation with all their informed consent at Shengjing hospital, Chinese Medical University, and were frozen in liquid nitrogen immediately after surgical removal. Haematoxylin- and eosin-staining sections were prepared for assessment of the percentage of tumor cells, and only specimens with > 70% tumor cells were selected for analysis. This study was carried out with the approval of the ethical committee of China Medical University. All experiments were done at least three times.The human gastric cancer cell MKN45 and HGC27 were obtained from Keygen Biotech. Co. , and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, at 37°C in a humid incubator with 5% CO2.The macrophage cell RAW 264.7 was provided by the American Type Culture Collection , and was maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37°C in a humid incubator with 5% CO+ ) and Tx30a  were offered by ATCC . They were grown on sheep blood agar plates at 37°C under microaerophilic conditions. Bacteria were transferred after 48 h into Brucella broth containing 5% fetal bovine serum for 24 h. A multiplicity of infection of 100 was used in all studies.H. pylori strain 26695 , 1 μl; RNase inhibitor, 0.25 μl; RNA 1 μl; dH2O, 3.75 μl. Conditions for RT were: 30°C for 10 minutes, 42°C for 25 minutes, 99°C for 5 minutes, and 5°C for 5 minutes. Real-time PCR was performed using the LightCycler system together with the LightCycler DNA Master SYBR Green I Kit (Roche Diagnostics). The total volume is 20 μl, containing 25 mM MgCl2, 3 μl; 10 × Buffer, 5 μl; 10 μM forward Primer, 1 μl; 10 μM reverse Primer, 1 μl; LightCycler DNA Master SYBR Green I, 2 μl; cDNA, 2 μl; dH2O, 6 μl. Conditions for PCR were: 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 5 seconds at 95°C and 20 seconds at 60°C. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Gene expression was quantified by the comparative CT method, normalizing CT values to GAPDH and calculating relative expression values. Primer sequences were provided by Takala  as follows: CXCR4 forward, 5'-GAGGAAATGGGCTCAGGG-3', reverse, 5'-AGTCAGCAGGAGGGCAGGGA-3'; TNF-α forward, 5'-AGTGACAAGCCTGTAGCCC-3', reverse, 5'-GCAATGATCCCAAAGTAGACC-3'; IL-1β forward, 5'-CCACCACTACAGCAAGGG-3', reverse, 5'-GAACTGGGCAGACTCAAA-3'; IL-6 forward, 5'-CCTTCGGTCCAGTTGCCTTCT-3', reverse, 5'-GCATTTGTGGTTGGGTCA-3'; GAPDH forward, 5'-GGGAAACTGTGGCGTGAT-3', reverse, 5'-AAAGGTGGAGGAGTGGGT-3'.Total RNA was isolated from tissues and cell lines by Trizol  according to the protocol supplied by the manufacturers. cDNA was synthesized using Takara RNA PCR 3.0 Kit  in a total volume of 10 μl, containing AMV reverse transcriptase, 0.5 μl; random 9 primer, 0.5 μl; 25 mM MgCLβ-actin  was used as an internal control. Blots were visualized with FX pro plus system (Bio-Rad) and quantified using Scion Image 4.03 software.Cell lysates were prepared with sample buffer  and were subjected to a 12% sodium dodecyl sulfate (SDS)/acrylamide gel. The proteins on acrylamide gel were transferred to a nylon membrane, which was blocked overnight (4°C in PBS with 0.1% Tween and 10% milk powder). Polyclonal antibodies for CXCR4 , and the corresponding secondary antibodies  were applied before immunoblotting. The human gene 5 . On the following day, cells were transfected with TNF-α siRNA or Control siRNA using Lipofectamine 2000  according to the manufacturer's instructions.TNF-α RNAi plasmid and nonsilencing control RNAi plasmid were purchased from Takala . Cells were seeded into a 24-well plate at a density of 2 × 10Concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants were measured using the Quantikine Elisa kit  according to the manufacturer's instructions. The sensitivity of the assay was 2 pg/ml for TNF-α, 4 pg/ml for IL-1β and 4 pg/ml for IL-6.5 ) were added to the upper chamber and medium supplemented with CXCL12  was added to the lower chamber. Migration assays were incubated for 18 hours at 37°C and 5% CO2. Migrated cells on the lower surface were stained using 1% toluidine blue after fixation with 100% methanol. For each transwell, the number of migrated cells in 10 medium power fields (× 20) was counted.The migration of cultured cells was assayed using Matrigel invasion chamber . Cells . Difference was considered significant when P-value was < 0.05.Mann-Whitney GAPDH expression. The results revealed its level significantly higher in H. pylori-positive gastric cancers (n = 19) compared to H. pylori-negative ones (n = 15)  and more secretion of TNF-α protein into the culture supernatant  in MKN45 and HGC27 cells , was then used to treat MKN45 and HGC27 cells after an infection by 26695, and the induction of CXCR4 was inhibited significantly  for 6 hours in attempt to further explore the role of TNF-α in the upregulation of CXCR4 expression, and real time-PCR and Western blotting detection revealed it was upregulated significantly in a dose-dependent manner  and IL-6 (50 ng/ml) respectively, to rule out the possibility that IL-1β and IL-6 may upregulate CXCR4 expression like TNF-α. Neither IL-1β nor IL-6 was found to upregulate CXCR4 expression significantly Figure .P < 0.001, Figure P < 0.01, respectively, Figure Migration analysis was performed using the Matrigel invasion chamber in an attempt to investigate whether the upregulated CXCR4 was functional. In the initial phase of the experiment with 100 ng/ml of CXCL12 in the lower chamber, there was a significant increase (up to 4.5 folds) in the migration of MKN45 cells with 26695 treatment in the upper chamber, compared with control cells without 26695 treatment  was found to increase MKN45 cell migration significantly (H. pylori is blamed to infect about 50% of the world's population as a definitive gastric carcinogen for humans . Pathogevs 3.1 folds in MKN45 cells), which suggested H. pylori products in cag PAI may be also involved in the induction. In fact, it had been reported that cagA of H. pylori could induce TNF-α in gastric cancer biopsy specimens [Though the mechanism of induction of TNF-α by H. pylori remains relatively unclear, a protein family has been disclosed in the last decade, including Helicobacter pylori-membrane protein-1 (HP-MP1) and TNF-α inducing protein (Tipα) -24. Tipαpecimens . In addipecimens .TNF-α, a key cytokine in many chronic inflammatory diseases, was originally labelled as a serum factor for the induction of hemorrhagic necrosis of transplanted solid tumors in mice. However, presently it is commonly identified as a tumor promoter in local tumor microenvironment, and therefore the deletion or inhibition of it is supposed to reduce the incidence of experimental cancers. TNF-α/TNF-R1 knock down mice are resistant to chemical-induced carcinogenesis ,28. It iTNF-α was found not only involved in cell transformation and proliferation, but also in tumor metastasis. Such a finding was initially based on an animal model with colon cancer, in which injection of LPS enhanced the development of lung metastasis dependent on TNF-α production by host cells . The subOverexpression of CXCR4, whose involvement in various human tumors is well known, was frequently observed in gastric cancer tissues to increase gastric cancer metastasis. Some human gastric carcinoma cells also express CXCR4 mRNA and protein at high levels ,8. Our sMacrophages' involvement in the carcinogenesis and tumor invasion and metastasis ,32 generStudies have ultimately attributed the abnormal activation of NF-κB in cancer cells to the excessive secretion of TNF-α, whose role in CXCR4 upregulation is subsequently assumed to be related to pathways mediated by NF-κB. Others findings have revealed TNF-α antagonists can inhibit the upregulation of CXCR4 expression by H. pylori, and both it and CXCR4 antagonists can suppress the increased migration of gastric cancer cells in vitro. These results suggest that these antagonists alone, or in combination with other therapies, may serve as effective therapies for gastric cancer patients.TNF-α is involved in the upregulation of CXCR4 expression in gastric cancer induced by H. pylori.The authors declare that they have no competing interests.ZC designed the study, carried out PCR analysis, analyzed and interpreted the data, and drafted the manuscript. LX performed Western analysis. BM performed cell transfection. ZN was engaged in drafting the manuscript and in statistical analysis. WW performed Elisa analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/419/prepub