H]-pyrimidinone (UPy) functionalised graphene oxide (GO) and poly (PVA) were readily prepared by self-assembly using a vacuum filtration method. The isocyanate (UPy) functionalisation of the PVA was confirmed from a combination of Fourier transform infrared spectroscopy (FTIR) and changes in d-spacing from X-ray diffraction (XRD) measurements and, of the GO by solid-state NMR measurements reported by the authors previously. This is the first example of nacre mimetic structures where both the nanoplatelet (GO) and polymer (PVA) components are functionalised with complimentary groups. The resulting films displayed substantial increases in Young's modulus (E) of 392% (GO1/PVA1), ultimate tensile strength  of 535% (GO1/PVA1), elongation at break (εmax) of 598% (GO10/PVA5) and tensile toughness (UT) of 1789% (GO1/PVA10) compared to the un-functionalised GO analogues. The binding of UPy to both the GO and the PVA provides multiple routes by which these freestanding nacre mimetic films can dissipate applied loads.Nacre mimetic films based on 2-ureido-4[1 H]-pyrimidinone (UPy) functionalised graphene oxide (GO) and poly (PVA) were readily prepared by self-assembly using a vacuum filtration method.Nacre mimetic films based on 2-ureido-4[1 The high nanoplatelet content results in a ‘brick and mortar’ structure whereby the aragonite platelets are arranged in a layered morphology and the biopolymer penetrates between these layers and behaves as a form of adhesive. Because of the highly structured morphology, cooperative energy dissipation mechanisms are present that efficiently relax localised stress over a large area, thus enabling absorption of large amounts of energy prior to failure.Nacre, otherwise known as ‘mother-of-pearl’ is a remarkable material that displays mechanical properties that far exceed the sum of its component parts.2–4 solvent casting,5–7 and spraying,8–10 amongst others and there are examples where thermo-chromic transitions,11 enhanced thermal12–14 and electrical15,16 conductivity, and self-healing properties17 have been reported. It is widely recognised that cooperative effects between the hard (nanoplatelet) and soft (polymer) phase are key to maximising mechanical properties by introducing competing energy dissipation mechanisms.18,19 Thus, it is important to investigate techniques to increase the interfacial bonding between the two phases.Interest in nacre-mimetic materials has grown substantially in the past decades, in part due to the mechanical properties observed, but also the simplicity of manufacture and potential for production of multifunctional materials. It is widely understood that nacre-mimetic materials can be produced by vacuum-assisted filtration,2,5,20 and non-covalent21–24 bonding between the nanoplatelet and polymer components. Typically, graphene oxide (GO) is used as the nanoplatelet phase due to its reactive surface chemistry that allows for simple functionalisation via multiple methods.25–27 Notably, there are few examples of the introduction of non-covalent bonding to increase interactions. Park et al. reported the use of Mg2+ and Ca2+ ions to introduce ionic interactions between adjacent GO sheets however, the authors did not examine any effect on polymer-containing films.21 Critically, only minimal increases to Young's modulus (E) and tensile strength (σ) were achieved. More recently, Wang et al. functionalised GO with dopamine that was subsequently polymerised. Further treatment with 2-ureido-4[1H]-pyrimidinone (UPy) enabled grafting of the UPy onto the poly(dopamine) (PDA) and facilitated strong hydrogen bonding interactions between the PDA-capped GO sheets.22 Ultimately, this resulted in a 3.6 times increase in σ and a 10 times increase in tensile toughness (UT) when compare to a neat GO control.Previous literature has reported the introduction of both covalent23 This facilitated increased hydrogen bonding between GO sheets and achieved increases in E (↑323%), σ (↑470%), ε (↑214%) and UT (↑1117%). Critically, there was no polymer component in these materials and so the mechanical properties were not optimal. It was hypothesised that a polymer component could be functionalised using the UPy group to generate strong hydrogen bonding dimers with the GO component  that would greatly increase the interfacial interactions and ultimately achieve substantial increases in E, σ, εmax and UT.In our previous work, we reported the successful functionalisation of GO with UPy using a simple reaction between the isocyanate of UPy and the multiple oxygenated surface groups present on GO (GOx).via a simple process. The UPy-functionalised PVA (PVAy) was then dissolved into DMSO before being mixed with a GOx dispersion and filtered under vacuum to produce nacre-mimetic films. To the best of our knowledge, this work represents the first example of nacre mimetic structures where both the nanoplatelet (GO) and polymer (PVA) components are functionalised with complimentary groups. It is also the first example of a nacre mimetic film of GOx containing a polymer component. The reaction of UPy and PVA is confirmed by Fourier-transform infrared (FTIR) spectroscopy. The morphology, chemical and mechanical properties of the GOx/PVAy nacre mimetic films were analysed by scanning-electron microscopy (SEM), X-ray diffraction (XRD) and quasi-static tensile testing, respectively.In this work, we report the functionalisation of poly (PVA) with UPy 2.2.1n-pentane and poly (PVA), with a molecular weight of 89–98k and hydrolysed content of >99%, were purchased from Merck. All chemicals were used as received. Hydrophilic poly(tetrafluoroethylene) PTFE membranes were purchased from Merck. Molecular sieves (3 Å) were purchased from Fischer and activated by heating at 350 °C for at least 4 hours prior to use.GO was purchased from Abalonyx, Norway in powder form and used with no further purification. 2-Amino-4-hydroxy-6-methylpyrimidine and DMF were purchased from Fisher Scientific and, hexamethylene diisocyanate (HDI) was purchased from VWR International. Dichloromethane (DCM), DMSO, isopropyl alcohol (IPA), 2.22.2.123 Simply, 2-amino-4-hydroxy-6-methylpyrimidine  was combined with an excess of HDI , and heated at 100 °C for 24 hours. A white precipitate formed and was filtered under vacuum. The resulting solid was washed multiple times with n-pentane achieving a white powder in excellent yield .UPy was synthesised as outlined in our previous work.2.2.223 previously reported by Stankovich et al.28 Typically GO (≈1.5 g) was combined in solid form with UPy at a weight ratio of GO : UPy = 1 : 1, 1 : 0.1 and 1 : 0.01 and will be referred to as GO50, GO10 and GO1, respectively (unmodified GO will be referred to as GO0 in this context). Anhydrous DMF (75 cm3) was added and the mixture degassed with N2 for 1 hour, then stirred for 24 hours. The resultant dispersion was coagulated in DCM (≈200 cm3) filtered under vacuum and washed with DCM. The brown powder obtained was dried overnight at 40 °C under vacuum.Functionalisation of GO with UPy was achieved using the method outlined in our previous work,2.2.33) by heating at 60 °C until full dissolution was observed. The resulting mixture was then stored over activated molecular sieves (3 Å) for 24 hours to ensure anhydrous conditions. The dried PVA solution (10 cm3) was combined with UPy at a weight ratio of PVA : UPy = 1 : 0.1, 1 : 0.05 and 1 : 0.01 and will be referred to as PVA10, PVA5 and PVA1 from hereon (unmodified PVA will be referred to as PVA0 in this context). The mixture was degassed under a flow of Ar for 15 minutes. The mixture was then heated at 80 °C (whereby the UPy would dissolve) for 16 hours. Viscous products were achieved so DMSO (10 cm3) was added and the mixture agitated to aid dissolution of the polymer product. Precipitation was achieved in IPA (200 cm3) yielding a ‘cotton-like’ product. UPy loadings of 20 wt% and above could not be re-solubilised following the reaction so could not be isolated.PVA (9.96 g) was dissolved in DMSO (100 cm2.2.43) at 80 °C until complete dissolution was observed. GOx powders (≈500 mg) were suspended in DMSO (50 cm3) and stirred for 2 hours. The PVAy solution (≈2.5 g) was then added dropwise under stirring. The mixture was stirred for 1 hour before being filtered through a hydrophilic PTFE membrane. The GOx/PVAy film and PTFE membrane were removed from the filtration setup and dried in a fan-assisted oven at 40 °C for 24 hours. The film could then be peeled from the PTFE membrane.PVAy solutions of 1 wt% were achieved by dissolving PVAy (150 mg) in DMSO  crystal. Spectra were processed using OPUS analysis software between 500 and 4000 cmScanning Electron Microscopy (SEM) micrographs were obtained using a Zeiss Sigma instrument using an InLens detector at 10 kV. The samples imaged were sputter coated using an Au/Pd target prior to imaging.rd generation Malvern Panalytical Empyrean instrument equipped with multicore (iCore/dCore) optics and a Pixcel3D detector operating in 1D scanning mode. A Cu tube was utilised giving Cu Kα1/2 radiation (1.5419 Å) and a beam knife to reduce air scatter at the low angles. Scans were recorded in the range 4° to 30° 2θ with a step size of 0.0263° and a counting time of ∼130 s per step. Using the measured diffraction angle, 2θ, the interlayer spacing (d) was calculated using Bragg's law, nλ = 2d sin θ (where λ = 1.541 Å).XRD measurements were performed on a 3−1. Test specimens were cut into bars of width 10 mm and length 40 mm using a razor blade . The room temperature (RT) tensile mechanical properties were determined from an average of at least five (5) specimens. The Young's modulus was calculated from the gradient of the linear region of the stress–strain curve and tensile toughness calculated from the area under the stress–strain curves.Quasi-static tensile testing was completed using an Instron 5800R machine equipped with a 500N load cell and an extension rate controlled at 1 mm min3.23 Of key importance from that work was that solid-state nuclear magnetic resonance (SSNMR) confirmed UPy reacted readily with the hydroxyl groups on the GO surface. PVA has abundant hydroxyl groups in the polymer chain and so grafting of UPy can be achieved easily  has been described in our previous work.d easily .i.e. >10 wt%), the mixture gelled due to the high degree of hydrogen bonding dimerization of the UPy pendant groups. As a result, these products could not be isolated.Isocyanates are reactive towards water, producing an amine and carbon dioxide. As a result, water could not be used in the reaction medium and anhydrous conditions were required. Dimethyl sulfoxide (DMSO) was found to effectively disperse GOx in our previous work and it was observed that PVA and UPy were soluble in DMSO when heated to 60 °C. Functionalisation of PVA was achieved by stirring an anhydrous mixture of PVA in DMSO with the UPy powder at room temperature for 24 hours. At high UPy content  scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretches of the cyclic ketone and amide carbonyl of UPy, respectively, are detected in PVA1, PVA5 and PVA10 with increasing intensity at higher UPy content. Other peaks show an increase in intensity (e.g. 1253 cm−1 or 1013 cm−1) however, are much less prominent and/or overlap with solvent signals and so cannot be used to confirm a successful reaction. It is important to note there is no isocyanate peak at 2288 cm−1 in the PVAy samples. This confirms the terminal isocyanate of the UPy group has reacted with the PVA chain. The presence of the peaks at 1700 cm−1 and 1662 cm−1, and the absence of an isocyanate peak at 2288 cm−1, confirm the successful functionalisation of PVA with the UPy group.FTIR spectroscopy was utilised to confirm successful functionalisation of PVA . Evolutivia stirring. A DMSO–PVAy solution was added before the mixture was stirred further. The resulting dispersion was filtered under vacuum and dried overnight. This allowed the nacre-mimetic film to be peeled from the substrate yielding freestanding films, see Nacre-mimetic films were produced using a simple vacuum-assisted filtration approach. All GOx/PVAy films were produced at a 95 : 5 by weight ratio of GOx : PVAy and are named according to the UPy content on each component. For example, GO10/PVA5 contains 95 wt% of GO10 (10 wt% UPy content on GO) and 5 wt% PVA5 (5 wt% UPy content on PVA). Initially, GOx was dispersed in DMSO SEM was used to observe the cross-sectional layered structure of the nacre-mimetic films . Distincθ, the interlayer spacing (d) of a crystalline material can be calculated using Bragg's law, nλ = 2d sin θ (where λ = 1.541 Å). The XRD patterns and calculated d-spacings are shown in d-spacing of the GO0/PVAy, GO1/PVAy and GO10/PVAy films increases when the PVA component is added. This is to be expected as the PVA penetrates between the GO layers and forces them to separate but, the slight increase is much less than would be expected given the size of the PVA chains. Perhaps the PVA aids the assembly of a more homogeneous layered structure through hydrogen bonding to the GO, possible tethering to the OH groups on the edge of the GO platelets. However, it is not until the ratio of GO to UPy is 1 : 1, i.e. (GO50), 100 times more UPy than for GO1, that there is a significant increase in d-spacing up to ∼1.419 for PVA10GO50. The XRD diffractogram shows the evolution of several new peaks, including a major intense peak at lower 2θ, ∼6° due to increased spacing (exfoliation) between the GO platelets and resulting in different layered morphology, see Using the measured diffraction angle, 2σ = 62.85 ± 7.27 MPa, whilst GO10/PVAy films had the highest εmax of up to 2.39 ± 0.43%. This highlights the complexity of the competing energy dissipation mechanisms occurring within the films.Quasi-static tensile testing was completed to determine the mechanical properties of the films and representative stress–strain curves are shown in E from 2.75 ± 0.44 GPa for GO0 to 6.25 ± 3.28 GPa for GO0/PVA0 is obtained however, the error is relatively large. E increases for GO0/PVA1 (3.91 ± 0.76 GPa) compared to GO0 as the polymer enables a more efficient shear stress transfer across a layered GO-polymer structure,29 and suggests effective penetration of the PVA0 between the GO0 layers. For GO0/PVA5 and GO0/PVA10, E = 5.17 ± 1.15 GPa and 5.04 ± 1.78 GPa, respectively, confirming an increase in E compared to GO0/PVA1. This trend is supported by the XRD data which confirmed that increasing the y content of PVAy (i.e. from PVA1 to PVA5 to PVA10) results in a reduction in d-spacing. Reduced d-spacing suggests improved packing and thus stiffening by PVAy dominates and E increases.The mechanical properties calculated from the stress–strain curves are displayed as bar charts in E for GO1 (3.12 ± 0.53 GPa) upon addition of PVA0 to 7.14 ± 0.29 GPa for GO1/PVA0, an increase of 229%. For GO1/PVA1 there was a further increase in E to 12.24 ± 1.60 GPa, representing an increase of 392% and 171% compared to GO1 and GO1/PVA0, respectively. The UPy group (present on the GO1 and PVA1) forms strong interactions between the GO1 and PVA1 components, creating a 3D network of hydrogen bonding that increases the modulus by the PVAy. E is reduced for GO1/PVA5 and GO1/PVA10, down to 7.70 ± 0.24 GPa and 7.80 ± 0.26 GPa, respectively. The increasing y content on the PVAy results in a larger steric volume (UPy is much larger than hydroxyl pendant groups) of the PVAy component. This forces the GO1 nanoplatelets to separate and greater PVAy motion occurs. Separation of GO1/PVA5 and GO1/PVA10 is observed through the increased interlayer distance from XRD . However, the decrease in d is small, so other processes, such as tethering of the –OH groups on the GO platelet edges by PVA may also play a role in this behaviour.There was a significant increase in E of GO10 films ranges from 5.81 ± 0.39 GPa (GO10/PVA1) to 7.35 ± 1.88 GPa (GO10). The weight ratio of GOx : PVAy in these films = 95 : 5. As a result, increasing UPy content (x) in GOx (i.e. from GO1 to GO10) has a greater impact on the quantity of UPy groups in the structure compared to an increase to y in the PVAy (i.e. PVA1 to PVA10). It is therefore possible that in the GO10 films, the impact of changing the y content on the PVAy is not large enough to significantly affect E. The same is observed in the GO50 films where for all the GO50/PVAy films E is between 4.91 ± 0.67 GPa (GO50/PVA10) and 5.84 ± 0.45 GPa (GO50/PVA5). Interestingly, for GO50, E = 8.87 ± 1.95 GPa, significantly higher than the GO50/PVAy films. It is likely the PVAy disrupts the strong hydrogen bonding between the GO50 nanoplatelets and thus reduces collaborative stiffening of the film. The schematic diagram shown in The σ increases from 6.45 ± 0.97 MPa for GO0 to 12.29 ± 2.80 MPa for GO0/PVA0, an increase of 191%. σ = 14.78 ± 1.59 MPa for GO0/PVA1, 19.50 ± 2.52 MPa for GO0/PVA5 and 16.45 ± 5.00 MPa for GO0/PVA10. Critically, a significant increase in σ of 302% is observed between GO0 and GO0/PVA5. The GO0 nanoplatelets assemble into a porous structure.30 The pores localise stress and disrupt the dissipation of energy through the film. Most probably coalescence of pores into a major defect also causes a decrease in UTS. The PVAy occupies the pores within the film and bridges the GOx nanoplatelets. As a result, energy is dissipated over a larger volume as the number of pores/defects is reduced with the addition of PVAy, resulting in an increase in σ. For the GO1 film σ = 11.74 ± 1.62 MPa however, addition of PVA0 yields an increase of 321% to 37.74 ± 6.75 MPa (GO1/PVA0). For GO1/PVA1 σ = 62.85 ± 7.27 MPa, corresponding to a substantial increase of 535% over GO1. The UPy dimers between the GO1 and PVA1 create strong interfacial interactions that greatly increase the energy required to break the 3D network. The UPy content in GO1/PVA1 is optimal to maximise σ where the hydrogen bonding is most cooperative. GO1/PVA5 and GO1/PVA10 have σ = 43.16 ± 1.31 MPa and 46.55 ± 2.89 MPa, respectively, most likely due to an increase in hydrogen bonding between PVAy chains hindering cooperative interactions with the GO1. Despite this, σ still increases by close to 400% for both the GO1/PVA5 and GO1/PVA10 films when compared to GO1.In the case of GO0, σ is recorded from 23.75 ± 1.13 MPa for GO10 to 42.50 ± 3.80 MPa for GO10/PVA0. No further increase in σ is observed for GO10/PVA1 (31.55 ± 3.26 MPa), GO10/PVA5 (43.17 ± 3.52 MPa) or GO10/PVA10 (41.95 ± 5.79 MPa) however, all have increased σ when compared to GO10, most likely due to increased plastic deformation. It is clear that increasing y content on the PVAy  does not increase σ. This suggests that hydrogen bonding between adjacent GO10 sheets dominates hydrogen bonding between the GO10 and PVAy. Finally, σ decreases from 30.43 ± 7.37 MPa for GO50 to 21.03 ± 1.81 MPa, 20.39 ± 2.11 MPa, 28.27 ± 2.63 MPa and 19.41 ± 2.52 MPa for GO50/PVA0, GO50/PVA1, GO50/PVA5 and GO50/PVA10, respectively. GO50 has higher σ when compared to GO0, GO1 and GO10, suggesting the strongest hydrogen bonding between GOx sheets is present in the GO50 film. It is likely the PVAy disrupts the hydrogen bonding between the GO50 sheets, thus weakening interfacial interactions. As a result, stress cannot be dissipated between adjacent GO50 sheets resulting in a decrease in σ.An increase in εmax = 0.28 ± 0.06% with an increase of 154%, to 0.43 ± 0.07%, recorded for GO0/PVA0. PVA0 hydrogen bonds to GO0 through pendant hydroxyl groups and the interactions to the GO0 allow the GO0 sheets to slide over each other as the PVA0 chains disentangle. GO0/PVA1, GO0/PVA5 and GO0/PVA10 recorded εmax of 0.55 ± 0.11%, 0.51 ± 0.16% and 0.44 ± 0.11% respectively, all showing an increase over the GO0 film. The increase in εmax between GO0/PVA0 and GO0/PVA1, GO0/PVA5 and GO0/PVA10 is relatively small due to the lack of UPy functionalisation on the GO0 component.GO0 had εmax = 1.34 ± 0.66%, a 362% increase over GO1 that measured 0.37 ± 0.12%. In this case, the UPy on the GO1 binds to the hydroxyl groups of the PVA0 and the flexibility of the PVA0 enables extension through disentangling of the polymer chain. Similarly, for GO10/PVA0, εmax = 1.93 ± 0.39%, compared to 0.40 ± 0.07% for GO10. εmax decreased for GO1/PVA1 (1.02 ± 0.19%) compared to GO1/PVA0 whilst GO10/PVA1 (1.25 ± 0.33%) decreases compared to GO10/PVA0. It is expected that εmax would increase due to formation of UPy dimers between the GO1/GO10 and PVA1 components. The UPy six-carbon chain is longer than the hydroxyl groups present in PVA0 and so should extend further prior to failure.23 A decrease in ε suggests the UPy dimers between the GO1 and PVA1 create anchor points that prevent the PVA1 chains from disentangling. For GO10/PVA10, εmax = 2.39 ± 0.43% that corresponds to a 598% increase over the GO10 film (0.40 ± 0.07%). This represents the optimal GOx/PVAy composition for ε and is due to the cooperative effect of elongation of UPy dimers and disentangling of the PVA10 chains. This allows the GO10 sheets to slide over each other enabling higher elongation. Critically, optimal UPy content is different for maximising σ or εmax.For GO1/PVA0, UT) is a strong indicator of the overall ability of the films to absorb energy prior to failure. Calculated from the area under the stress–strain curve, UT depends on E, σ and ε. For GO0, UT = 0.89 ± 0.31 kJ m−3 that increases 281%, to 2.50 ± 1.19 kJ m−3, for GO0/PVA0. Subsequently, for GO0/PVA1, GO0/PVA5 and GO0/PVA10, UT = 3.86 ± 0.79 kJ m−3, 5.61 ± 2.06 kJ m−3 and 4.24 ± 1.79 kJ m−3 respectively. PVAy aids in construction of a cooperative 3D network through occupation of pores within the GO0 structure. This enables dissipation of energy throughout the structure by eliminating localised stress points to increase σ and εmax, resulting in an increase in UT.Tensile toughness  eclipse those reported previously.3,4,22Similar to GO0, GO1, GO10 and GO50, 4.E, σ, εmax and UT of 392% (GO1/PVA1), 535% (GO1/PVA1), 598% (GO10/PVA5) and 1789% (GO1/PVA10) compared to GOx analogues, respectively. Critically, the optimal UPy content for GOx and PVAy was different for E and σ (GO1/PVA1), εmax and UT (GO10/PVA5), confirming the energy dissipation mechanisms within the GOx/PVAy structures are complex and competing.In this work, it was shown that UPy reacts readily with PVA and GO at room temperature, as confirmed by FTIR. The resulting PVAy dissolves in GOx–DMSO dispersions and was found to self-assemble into a nacre-mimetic layered structure through vacuum-assisted filtration, thus producing the first GOx/PVAy films of their kind. The resulting films display substantial increases in The authors report no conflicts of interest.


The differential expression of circRNA in different biological samples renders it as an ideal biomarker for disease diagnosis and identification of tissue development. In addition, the gradual clarification of the mode of action of circRNA in disease makes it as a potential therapeutic target. The purpose of this study is to investigate the role and regulating mechanism of circular RNA has circ 003390 (circWEE1) on Endometrial cancer (EC) genesis. To estimate clinical values of circWEE1 on cell migration and proliferation in EC, and its possible mechanisms. The expression of circWEE1 and EIF4A3in EC cells have been evaluated using qPCR and Western blot. The expression of circWEE1 and EIF4A3 levels were increased in patients with EC. Over-expression of circWEE1 or down-regulation of miR-195-5p promoted cell migration and proliferation in EC. Next, we verified that eIF4A3 binds to the circWEE1 mRNA transcript, circWEE1 served as a sponge that directly targeted miR-195-5p. Bioinformatics prediction forecast that miR-195-5p directly targeted VEGF at 3’-UTR, which was confirmed by luciferase reporter assay. Our findings indicate that Circular RNA hsa circWEE1/EIF4A3 promoted cell migration and proliferation in EC via VEGF signaling by miR-195-5p, which could provide pivotal potential therapeutic targets for the treatment of EC.  We identified that circWEE1 acts as an oncogene and promotes EC cellproliferation and migration.We identified that circWEE1 acts as an oncogene and promotes EC cell proliferation and migration.We found that CircWEE1 can directly bind to miR-195-5p and regulate itsexpression.We demonstrated that VEGF is target genes of the circWEE1/ miR-195-5p axis in EC cell.We provide pivotal potential therapeutic targets for the treatment of Endometrialcancer.1.Endometrial cancer (EC), one of the most common primary woman tumors, harbors extremely high morbidity and mortality . Most ECVascular endothelial growth factor (VEGF), specific mitogen isolated from endothelial cells in the late 20th century, has been confirmed to have the ability of inducing both physiological and pathological angiogenesis . In the Eukaryotic translation initiation factor 4A (eIF4A) protein, belonging to the DEAD-box RNA helicase family, has RNA-dependent ATPase activity and ATP-dependent RNA helicase activity . Three tMicroRNAs (miRs) are a class of non-coding small RNA molecules with approximately 19–24 nucleotides in length . In 1993Circular Rs (circRs) are a class of circular non-coding RNAs generated by alternative splicing of precursor RNA, typically consisting of more than one exon . CircR wThe purpose of this study is to investigate the role and regulating mechanism of circular RNA has circ 003390 (circWEE1) on Endometrial cancer (EC) genesis and to estimate clinical values of circWEE1 on cell migration and proliferation in EC, and its possible mechanisms.2.2.1This study was approved by the Ethics Committee of the Forth hospital of Hebei Medical University (No. 2020KY065). All the serum samples were immediately snap frozen in liquid nitrogen and stored at −80°C for further using. Neuropathological evaluation was performed according to the WHO classification by two experienced clinical pathologists. Basic knowledge of patient with EC was showed at 2.22O. PCR started at 95°C for 15 min, followed by 40 cycles at 95°C for 10s, 60°C for 40s, and finished by 40s at 60°C in the last cycle.Total RNA was extracted using TRI-zol reagent . cDNA was synthesized using a High-Capacity cDNA Reverse Transcription kit . qRT-PCR was performed using a SYBR Green kit  in an ABI 7900 PCR Thermal Cycler. The total volume of the amplification reaction system was 20 µl, including 6 µl primers, 10 µl of SYBR Green PCR Master Mix, 1.5 µl cDNA, and 2.5 µl ddHThe primers used werecircWEE1: Forward (5´-GGATAAACCGTGGTAATTCTATG-3´) and Reverse (5´-GGCAAATGCTTTCGCAGTAG-3´);EIF4A3: Forward (5´-AAGGGAGAGATGTCATCGCAC-3´) and Reverse (5´-GCTTGAGTTTCACGAACCTGA-3´);GAPDH: Forward (5´-ATCTTCCAGGAGCGAGATCCC-3´) and Reverse 5´-TGAGTCCTTCCACGATACCAA-3´).The mRNA expression levels of genes were measured using the 2ΔΔ-Ct method. GAPDH was used as internal control. Microarrays (HLivH180Su14) were purchased from Shanghai Outdo Biotech Co. Ltd. .2.3The human EC cell lines  and normal human endometrial epithelial cells (hEM15A) were purchased from the Cell Bank of the Chinese Academy of Sciences. All cell lines were cultured in Dulbecco’s modified Eagle’s medium  containing 10% fetal bovine serum  at 37°C, 5% CO 2, and 95% air. All oligonucleotides sequences were synthesized by Gene-Pharma . KLE or HEC-1B cells were transfected using the Lipofectamine 2000 Kit .2.42 for 1 h. The absorbance was detected using a microplate reader  at 450 nm10 μL CCK-8 assay  was carried out to evaluate cell proliferation, added into each well and the culture plates were incubated at 37°C with 5% CO2.5The migratory and invasiveness were assessed using an 8-µm pore polycarbonate membrane Boyden chamber insert in a Corning Transwell apparatus . KLE and HEC-1B cells (5 × 104 cells) were plated into the upper compartment of chamber insets and lower compartments were filled with 500 µL DMEM containing 10% FBS. Cells were collected at 48 h post-transfection, washed with PBS and mechanically dissociated into a single-cell suspension. After 48 h of incubation, cells were fixed with 100% methanol, stained with 0.05% crystal violet, washed with PBS, invasion was observed under an inverted light microscope.2.6Renilla luciferase activity was normalized to that of the firefly luciferase activity.Using Lipofectamine 2000, cells were transfected with miR-195-5p, circWEE1 3`-UTR-wild type, and mutant plasmids . Next, cells were transfected with miR-195-5p, VEGF 3`-UTR-wild type, and mutant plasmids  Lipofectamine 2000. Cells were incubated at 37°C 5% CO2 condition and transfected for 48 h. Renilla luciferase activity was acted as the control. The activity was detected by using a luciferase assay kit . The 2.7The protein concentration was determined using the BCA Protein Assay Kit. Protein was separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% nonfat milk in TBST for 1 h and incubated with WEE1 , EIF4A3 , VEGF , PI3K , p-Akt , Akt  and GAPDH monoclonal anti-body  at 4°C overnight. Membrane was washed with PBS and incubated with the mouse ant-rabbit secondary antibody  at 37°C for 1 h. The immune complexes were detected using the ECL Western blot analysis Kit  and analzyed using Image Lab 3.0 .2.86 cells/well) were seeded into a 96-well plate at 37°C and Cells were washed with PBS. Cells were incubated at room temperature in the dark for 15 min, 5 mL Annexin V-FITC, and 5 mL PI. The apoptotic cells were measured using the FACScan flow cytometer  equipped with CellQuest Software (Becton Dickinson).Cells (1 × 102.9RNA binding protein immunoprecipitation (RIP) assay was performed using an EZMagna RIP kit . 500 μg of total cellular protein were incubated with RIP buffer and were incubated with Proteinase K and magnetic beads conjugated with anti-eIF4A3 antibody or control (IgG). The immune-complex was exposed to western blotting.2.10Five-week-old BALB/c male nude mice (n = 12/every group) were provided by the laboratory animal facility of Yangzhou Medical University. The mice were reared at a temperature 22 ± 2°C and relative humidity 50 ± 5%, with 12-h light and 12-h dark alternation and allowed free access to food and water. BALB/c nude mice were intraperitoneally injected with pentobarbital sodium (40 mg/kg) and inoculated subcutaneously from the right forelimb with 0.2 mL of KLE (2 × 106 cells/mL) as reference. Every three days, tumor volume was measured using vernier caliper. All methods were carried out in accordance with relevant guidelines and regulations. All protocol for animals were approved by ethics committee of Yijishan Hospital of Wannan Medical College.2.11Cell samples were fixed in 4% paraformaldehyde, incubated with 0.25% Tris-X100 for 10 min at room temperature for permeabilization. Cell samples were blocked with 5% BSA in PBST for 1 h at room temperature and incubated with anti- WEE1 or anti-EIF4A3 antibody , at 4°C overnight. After washing with TBST for three times, sections were incubated with secondary peroxidase-conjugated goat anti-rabbit-555 IgG  or goat anti-mouse-488 IgG  antibody for 2 h at room temperature. After washing with PBST for three times, sections were stained with DAPI for 15 min at darkness. Cell samples were observed using fluorescence microscope .2.12The tumor tissue samples were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. For the immunohistochemical examination, the tissue sections (5 μm) were incubated with the VEGF (1:100) and WEE1 (1:100) antibodies. Tissue samples were observed using fluorescence microscope .2.13The cells were fixed with 4% paraformaldehyde at room temperature. The cells were incubated with 70% ethanol for 1 h at 4°C. Next, the stock solution of RNA probes was incubated with hybridization buffer  overnight at 37°C, according to the manufacturer’s instructions for the RNA-FISH kit (RiboBio). After completing these steps, the nuclei were stained with DAPI . The cells were visualized and photographed using a laser confocal microscope  after adding the fluorescence-quenching agent.2.14All data are presented as mean ± standard deviation. Shapiro Wilk test was performed to check the normal distribution of the data. Statistical differences were analyzed using Student’s t-test or one-way analysis of variance (ANOVA) and Tukey’s post test. Significance level was set as p < 0.05.3.The expression of circWEE1 and EIF4A3 levels were increased in patients with EC. Over-expression of circWEE1 or down-regulation of miR-195-5p promoted cell migration and proliferation in EC. Next, we verified that eIF4A3 binds to the circWEE1 mRNA transcript, circWEE1 served as a sponge that directly targeted miR-195-5p. Bioinformatics prediction forecast that miR-195-5p directly targeted VEGF at 3’-UTR, which was confirmed by luciferase reporter assay.3.1To analyze the changes of Circular RNA in patients with EC, we used PCR to measure the expression of Circular RNA in patients with EC. As shown in Circular RNA hsa circWEE1 regulates miR-195-5pin vitro model. MiR-195-5p expression may be target spot for circWEE1  (The data from an RIP (RNA binding protein immunoprecipitation) assay using anti-eIF4A3 antibody indicated that circWEE1 can bind with eIF4A3 mRNA . Up-regu < 0.05) . Si-eIF4 < 0.05) . The inh < 0.05) .Figure 3.4http://www.targetscan.org) to explain binding sites. As shown in This study used bioinformatics method (3.5in vivo model (in vivo model (in vivo model (in vivo model (in vivo model (in vivo model (In vivo, U87 cells were transfected with the mock or circWEE1 plasmid or si- miR-195-5p. The results indicated that the overexpression of circWEE1 generated an outstanding increase tumor volume, tumor size, and tumor weight vo model . IHC assvo model . We founvo model . Next, svo model . IHC assvo model . Silencivo model , suggest4.EC in the United States, Europe, and other developed areas, is close to 50% of new gynecological malignancies . In 2018A large number of studies have shown that the expression levels of various miRNAs are abnormally changed in EC tissues or cells . Some miThe binding of VEGF to VEGFR-2 would promote VEGF-mediated endothelial cell proliferation, promote endothelial cell survival and anti-apoptosis, promote endothelial cell migration, and increase vascular permeability . These fBased on the ‘sponge’ effect of circRNA molecules on miRNA, their expression is associated with the occurrence and progression of various diseases, and significantly different expression can be observed in pathological samples . StudiesStudying the different functions of eIF4A1, eIF4A2 and eIF4A3 is a focus of recent eIF4A research . In cons5.We first identified that circWEE1 acts as an oncogene and promotes EC cell proliferation and migration. EIF4A3-induced circWEE1 cyclization and increased circWEE1 expression. CircWEE1 can directly bind to miR-195-5p and regulate its expression. We demonstrated that VEGF is target genes of the circWEE1/miR-195-5p axis in EC cells . Further


The results showed that higher net NH4+ and NO3− fluxes occurred at 2.1–3.0 mm from the root apex in T.ascendens and T. distichum. Compared to NH4+ or NO3− alone, more stable net NH4+ and NO3− fluxes were found under NH4NO3 supply conditions, of which net NH4+ flux was promoted at least 1.71 times by NO3−, whereas net NO3− flux was reduced more than 81.66% by NH4+ in all plants, which indicated that NH4+ is preferred by Taxodium plants. T. ascendens and T. mucronatum had the largest net NH4+ and total N influxes when NH4+:NO3− was 3:1. 15N Atom% and activities of N assimilation enzymes were improved by single N fertilization in the roots of T. distichum. In most cases, net H+ fluxes were tightly correlated with net NH4+ and NO3− fluxes. Thus, both N forms and proportions could affect N uptake of Taxodium. These findings could provide useful guidance for N management for better productivity of Taxodium plants.To understand the characteristics of net NH Spatial variability in the fluxes of NH4+ and NO3− has been explored along the roots in some herbaceous and woody plants [4+ influx happened at the root apex in rice (Oryza sativa L.) [Populus simonii [4+ influx appeared at 5 mm, 10 mm, and 5–20 mm from the root apex in lodgepole pine (Pinus contorta) [Populus popularis [Pseudotsuga menziesii) [3−, previous studies observed that the highest net NO3− flux occurred at 0–10 mm in P. contorta [P. simonii [P. popularis [3− influx increased to a maximum at 21 mm from the apex and then gradually declined [4+ and NO3− flux rates along the fine roots.Nitrogen (N) plays a significant role in plant growth and development since it is a crucial component of plants’ chlorophylls, nucleic acids, proteins, and secondary metabolites . Ammoniuy plants ,3,4,5. Ftiva L.)  and Popuontorta) , Populusopularis  and Dougnziesii) , respectcontorta , and at opularis . In rice4+ and NO3− fluxes are also affected by environmental factors such as N levels. A previous study in tea (Camellia sinensis) roots demonstrated increased net influxes of NH4+ and NO3− when the solution concentration increased from 0.2 mM to 1.2 mM under KNO3 and NH4Cl [Picea glauca revealed a converse result in most cases; the roots presented net NH4+ and NO3− influxes in 50 μM with net effluxes in 1500 μM solutions [4NO3 solutions, net NH4+ influxes increased gradually in P. popularis but decreased by degrees in Populus alba × Populus glandulosa [3− influxes than P. popularis in most cases [4+ and NO3−, and they are significantly related to the plant species. Additionally, previous studies revealed that interactions between NH4+ and NO3− exist on fluxes of both ions [4+ and NO3− are complicated among plants [4+ and NO3− negatively affect the uptake of each other, but NH4+ is preferred in C. sinensis [4+ influx was induced by the simultaneous provision of NO3−, and net NO3− influx was inhibited in the presence of NH4+ in roots of P. popularis and Populus asperata [4+ uptake remained unchanged in the presence or absence of NO3− [4+ and NO3− and their preferences may result in changes of NH4+ and NO3− fluxes under different proportions of NH4+ and NO3− supply. Nonetheless, little information is available on the fluxes of NH4+ and NO3− in plant roots under fluctuating proportions of both inorganic N forms.Apart from the spatial variation along the roots, NHolutions . When rooth ions ,9. The ig plants , and theg plants . It is dasperata ,11. More of NO3− . Overall4+ and NO3− are correlated with the plasma membrane PM-H+-ATPase activity that extrudes H+ from the cytosol to the outside at the expense of adenosine triphosphate (ATP) [3− is transported across the plasma membrane via NO3−/H+ symporters with the involvement of PM-H+-ATPase [4+ can increase the activity of PM-H+-ATPase [+-ATPase was positively associated with fluxes of NH4+ and NO3− [4+, NO3− fluxes, and H+ uptake rate have been observed in many plants [On the other hand, fluxes of NHte (ATP) . Previou+-ATPase . The con+-ATPase . Furtherand NO3− . The sigy plants ,15,16,17Taxodium species including T. ascendens, T. distichum, and T. mucronatum have been introduced from southeastern America to many countries owing to their economic and ecological benefits [Taxodium oil showed adequate bioassay for insecticidal activity [Taxodium plants have been selected as suitable species for afforestation in many challenging areas [Taxodium growth and development, less information is available on the fluxes of NH4+ and NO3− as well as their correlation with H+ flux in fine roots. In this study, a non-invasive micro-electrodes technique was employed to investigate NH4+, NO3− and H+ fluxes in fine roots of T. ascendens, T. distichum, and T. mucronatum under different N forms and their proportions. Our objectives were (i) to determine the distance from the root apex of Taxodium plants where there are greater net NH4+ and NO3− fluxes; (ii) to illustrate the characteristics of NH4+, NO3− and H+ fluxes and their interactions under different N forms and proportions.benefits . For insbenefits . Taxodiuactivity . Compounactivity . Moreoveng areas ,23. AlthT. ascendens, T. distichum, and T. mucronatum were selected. After being soaked in 3‰ 3-indoleacetic acid (IAA) solution for 2 min, they were repotted into a pot containing 1:1 volume of peat: perlite in a ventilated greenhouse at the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences , under normal growth conditions (approximately 25 °C) in a photoperiod of 14/10 h of light/dark. Two months later, cuttings with uniform size and development were selected for the NMT experiments.Semi-lignified cuttings  of T. distichum grown in the Institute of Botany, Jiangsu Province and Chinese Academy of Sciences. They were planted in black plastic pots (5 × 5 × 15 cm) filled with 1:1 volume of peat:perlite in a climate chamber . After 19 weeks of growth, the plants were transferred into black plastic boxes  containing 4 L of modified 1/4 Hoagland’s nutrient solution [15N Atom% and enzymatic activities.Seeds were collected from a healthy solution . All nut4+ and NO3− occur, a preliminary experiment was carried out at 14 positions, in turns, 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1, 2.5, 3.0, 5.0, 8.0, 15.0 and 30.0 mm away from the root apex. The measuring solution was 0.5 mM MES (2-(N-Morpholino) ethanesulfonic acid hydrate buffer.), pH 6.0, to which either 1.0 mM NH4Cl for NH4+ or 1.0 mM KNO3 for NO3− was added. After that, the position where the greater net uptake of NH4+ and NO3− occurred was detected to carry out the following experiments.To determine the positions along the root where the maximal influxes of NH4+ and NO3− under different N forms and proportions, the measuring solutions were designed as (1) NH4+ (NH4Cl): 0.1 and 1.0 mM, (2) NO3− (KNO3): 0.1 and 1.0 mM and (3) NH4+:NO3−: 1:3, 1:1, and 3:1 , containing 0.5 mM MES, pH 6.0.To investigate the net fluxes of NH15N Atom% and enzymatic activities under single N fertilization, 24 seedlings with similar performance (ca. 15 cm in height) were selected and divided into three groups (8 plants in each group). Three N treatments: 0 mM 15NH4Cl and K15NO3 , 1 mM 15NH4Cl and 1 mM K15NO3 in 1/4 modified Hoagland’s nutrient solution [2H4N4) was added into the nutrient solution to inhibit nitrification [15N Atom%, and the remaining 4 plants in each treatment were used for enzymatic activities.To explore the biomass, fication . After 34+, NO3− and H+ uptake by the fine roots under different treatments, ions flux alterations on the root surface were measured by using a non-invasive micro-test technology (NMT) system   a.4+ electrode, in sequence, 100 mM NH4Cl was used as a backfilling solution, followed by an NH4+ selective liquid ion exchange cocktail . Similarly, for the NO3− electrode, 10 mM KNO3 was used as the backfilling solution, followed by a NO3− selective liquid ion exchange cocktail . For the H+ electrode, 15 mM NaCl and 40 mM KH2PO4 were used as backfilling solution, followed by an H+ selective liquid ion exchange cocktail . Prior to the flux measurements, the microelectrodes were calibrated. For NH4+ calibration, 0.05/0.5 mM NH4Cl in addition to other compounds  were used in the measuring solution; for NO3− calibration, 0.05/0.5 mM KNO3 in addition to the compounds  were used in the measuring solution; for H+ calibration, pH 6.4/5.4 in addition to 0.5 mM MES were used in the measuring solution. Only electrodes with Nernstian slopes higher than 55 mV per tenfold concentration difference were used.The measurement procedures were described by Zhao et al. . Firstly4+/NO3− were recorded at each measurement point for 5 min. Not only eight biological repetitions (eight fine roots from four plants) but also 50 measurement time points in each repetition were considered.After that, fine white roots, 15–35 mm from the apex, were selected. They were fixed at the bottom of the petri dish filled with 10–20 mL measuring solutions for 20 min. After being equilibrated, the samples were transferred to another petri dish containing 5 mL fresh solution and then were put under the microscope. The tips of the microelectrodes were aligned and kept 30 μm away from the target point, which is a specific distance from the apex of the root. Net fluxes of NH15N Atom% (15N AT%) and the amount of plant N derived from 15N-labeled fertilizer (Ndff%) were detected [The roots were harvested and rinsed three times in distilled water. The detected .Activities of nitrate reductase , nitrite reductase , glutamine synthetase , glutamate synthetase  and glutamate dehydrogenase  in the roots were assayed [4+, NO3− and H+ fluxes along the root tip, imFluxes V2.0 (xuyue.net) was used to obtain the data at each measuring point. The positive values represent net influxes, and the net negative values represent net effluxes. To analyze data for the ion fluxes, biomass, 15N AT% and enzyme activities, one-way ANOVA (Duncan’s multiple range tests at 5% level) was performed with the SPSS 25.0 . GraphPad Prism version 9.1 was used to draw figures.In order to determine the NH4+ and NO3− were determined along the root tip up to 30.0 mm from the apex, and their fluxes were widely varied at different locations (4+ fluxes ranged from 17.02 (net influx) to 88.89 (net influx) pmol cm−2 s−1 in T. ascendens, and varied dramatically from −30.52 to 167.15 pmol cm−2 s−1 in T. distichum, and fluctuated between −90.94 and 46.31 pmol cm−2 s−1 in T. mucronatum when supplied as 1 mM NH4Cl , stable fluxes of NH4+ and NO3− were observed, and distinctly, NH4+ fluxes were much greater than NO3− fluxes in all Taxodium plants  than those in the other two species under 0.1 mM NH4Cl  in 1.0 mM than in 0.1 mM KNO3 in T. mucronatum , T. ascendens, T. distichum, and T. mucronatum showed great diversities in the fluxes of NH4+, NO3− and TN  consisting of various proportions of NH− and TN . The net species a. It wasectively a. Similacronatum a. A decroportion a.4+ influx, the net flux of NO3− was much lower, ranging from −100.53 to 84.85 pmol cm−2 s−1 under different proportions of NH4Cl and KNO3  increased by 1.68 times when the NH4+ proportion was raised from 1:1 to 3:1  than segment II (35–70 mm) in Populus × canescens [T. distichum had the greatest NH4+ and NO3− uptake rates among the three Taxodium species.Fine roots consist of four distinct regions, including root cap, meristematic, elongation, and maturation zones, characterized by different anatomical and functional features . These aot zones ,27,28. S species . For exa simonii , and at contorta  and P. popularis  respectit growth ,31. Simion zones . Moreoveoot apex . The net4+ and NO3− fluxes of fine roots [4+ and/or NO3− were determined when supplied N was elevated in P. popularis and P. alba × P. glandulosa [C. sinensis [P. glauca [Zea mays L.) [T. ascendens under NH4Cl and T. mucronatum under KNO3 solutions, most outcomes showed elevated NH4+ or NO3− uptake rates resulting from increasing NH4Cl or KNO3 supply. Consistently, higher 15N AT% and Ndff% were induced by 15NH4+ or 15NO3− treatment in the roots of T. distichum. Moreover, 15NH4+ or 15NO3− fertilization also brought about higher activities of NR, NiR, GS, GDH and GOGAT in the roots of T. distichum. These results suggest that N fertilization could be applied to stimulate NH4+ and NO3− absorption and assimilation capacities for Taxodium plants in practice.Generally, environmental N levels have a significant impact on the NHsinensis . However. glauca , wheat [. glauca , and cormays L.) . In our 4+ could lead to soil acidification [+ while absorbing NH4+, decreasing pH in the growth medium [3−, OH− could be released, contributing to the increase of pH [4+ and NO3− is expected to improve the N uptake of plants and the soil environment.Although an increasing N supply is likely to enhance N uptake in most cases, the provision of just NHfication . In mosth medium ,36. Evenh medium . For exah medium . In contse of pH . Thus, a4+ and NO3− was affected by each other when both N forms were provided [3− stimulated the uptake of NH4+, whereas the net fluxes of NO3− were inhibited by NH4+ in Taxodium plants. Similar results were found in the roots of corn, tea, wheat, rice and Brassica campestris [4+ and NO3− might interact with each other under coexistence N forms. These results might be related to cytosolic NH4+/NO3− thresholds [4+ may be required for plant development when NO3− was provided, while pre-existing NH4+ may reduce the thresholds of NO3− in the plant [4+ influx than NO3− was observed, it can be concluded that Taxodium plants show a preference for NH4+. It is noted that when NH4Cl or KNO3 was solely supplied, the fluxes of NH4+ or NO3− in the three species were erratic. However, stable net NH4+ and NO3− fluxes were observed when NH4+ and NO3− were both present in the solution, indicating a better balance in the mixed solution. In addition, this interesting phenomenon was reported in the study of C. sinensi, which might be the result of the competition between NH4+ and NO3−, and the underlying mechanism needs to be further studied [Many studies had demonstrated that the uptake of NHprovided ,7. In thmpestris ,9,24,33,resholds . In detahe plant . Conside studied .4+ in fine roots of Taxodium species was expected to occur when NH4+ and NO3− were supplied in different proportions [4+ influx was observed when NH4+:NO3− was 1:1, and the highest net NO3− influx occurred when NH4+:NO3− was 1.2:1 [Vaccinium corymbosum L.), the mRNA levels of ammonium transporter 3 (VcAMT3) involved in NH4+ uptake as well as nitrate transporter 1.5 (VcNRT1.5) and VcNRT2 involved in NO3− uptake was highest when the NH4+:NO3− ratio was 2:1 [T. aestivum L., Brachiaria brizantha, and Pseudostellaria heterophylla was found when the NH4+ and NO3− were supplied equivalently [4+:NO3− was 3:1, 1:3, and 3:1 for T. ascendens, T. distichum, and T. mucronatum, respectively, which could provide an applicable proportion of NH4+ and NO3− when producing special N fertilizer for the productivity of Taxodium plants. Additionally, the optimal equilibrium between NH4+ and NO3− supply largely differed between the three Taxodium species, implying that the induction of N transport systems require distinct NH4+ and NO3− ratios among these plants.Because of the greater N uptake in mixed treatments than in single N conditions, strong net uptake of NHportions . In the  was 2:1 . The higvalently ,41,42. I4+: NO3− , the NH4+ influxes were improved more than NO3−. This observation indicates a preference for NH4+ over NO3−, which is in good agreement with our previous outcomes. In most plant species, NH4+ is first absorbed into cells and then directly converted to amino acids, whereas cytosolic NO3− is assimilated at a higher energy cost. It is reduced to NO2− with the help of nitrate reductase (NR) and is further converted into NH4+ in plastids by nitrite reductase (NiR), which requires more energy than NH4+ for both transportation and further reduction [4+ and NO3− might result from the lower activity of NO3− transport systems affected by NH4+, which reduces the expression of the NO3−-related genes [4+: NO3− [4+ to a certain extent, which was mediated by the external concentration [4+ and NO3− when supplied at different proportions might be related to the distinct energetic and biochemical characteristics of uptake and assimilation pathway between NH4+ and NO3− in plant roots [In the present study, we found that with the change in the proportion of NHeduction . On the ed genes . In blue4+: NO3− . A previntration . Furtherntration ,8,10. Thnt roots ,24.+ fluxes was tightly associated with the variation in NH4+ or NO3−. Previous studies revealed that H+ fluxes might be correlated with the transport of NH4+ and NO3−, since NH4+ is transported into root cells through a symporter (co-transport with H+) and/or a uniporter, and NO3− is co-transported with H+ via a symporter into the cytosol [+-ATPase facilitates transport by pumping H+ into the apoplast during the uptake of NH4+ or NO3− in some parts of the roots [+-ATPase are determined by the transcript levels of corresponding mRNAs [+ fluxes under different N treatments were observed in the present study, H+ still plays an essential role in plant uptake of NH4+ and NO3−. Similar to a previous study in fine roots of P. popularis [+ uptake when two forms of N were supplied simultaneously. Intriguingly, fluxes of H+ fluctuated under both single N sources. Through our findings, we suspect that there may be an interaction between net H+ flux and net NH4+/NO3− flux in roots of Taxodium species. Similar results were observed by Garnett et al. [+ and NH4+/NO3− in Taxodium roots.In this study, the alteration of H cytosol ,13,14. Ahe roots ,45. The opularis , our datt et al. . The spe4+ and NO3− fluxes was observed along fine roots of Taxodium plants, and T. ascendens and T. distichum had higher fluxes of NH4+ and NO3− at 2.1–3.0 mm from the root apex. In most cases, net fluxes of NH4+ and NO3− increased with the elevated single N levels. NH4+ and NO3− affected each other when they were both supplied, and Taxodium plants preferred NH4+. Higher net N influxes were found when NH4+ and NO3− were simultaneously supplied than sole N treatments, especially in T. ascendens and T. mucronatum at 3:1 of NH4+:NO3−. Additionally, H+ fluxes were tightly correlated with net NH4+ and NO3− fluxes. These findings are valuable for understanding the characteristics of NH4+ and NO3− fluxes in the fine roots of Taxodium plants in the context of single and various ratios of N supply, and could provide a scientific basis for N management for silvicultural practice and better productivity of Taxodium plants.In summary, spatial variability of NH


In this work, we synthesized a novel pyridine derivative named compound H42 and investigated its anti-cancer activity in vivo and in vitro. We found that compound H42 inhibited ovarian cancer cell proliferation with IC50 values of 0.87 μM (SKOV3) and 5.4 μM (A2780). Further studies confirmed that compound H42 induced apoptosis, intracellular ROS production, and DNA damage. Moreover, compound H42 downregulated the expression of histone deacetylase 6 (HDAC6) with a distinct increase in the acetylation of α-tubulin and heat shock protein 90 (HSP90), followed by the degradation of cyclin D1, resulting in cell cycle arrest at the G0/G1 phase. Importantly, ectopic expression of HDAC6 induced deacetylation of HSP90 and α-tubulin, while HDAC6 knockdown upregulated the acetylation of HSP90 and α-tubulin. However, in the nude xenograft mouse study, compound H42 treatment can inhibit ovarian cancer growth without obvious toxicity. These findings indicated that compound H42 inhibited ovarian cancer cell proliferation through inducing cell cycle arrest at the G0/G1 phase via regulating HDAC6-mediated acetylation, suggesting compound H42 could serve as a lead compound for further development of ovarian cancer therapeutic agents.Ovarian cancer is the second leading cause of death of female gynecological malignant tumor patients worldwide. Although surgery and chemotherapy have achieved dramatic achievement, the mortality remains high, resulting in the demand for new specific drug discovery. Disrupting ovarian cancer growth  Globally, an estimated 313, 959 new ovarian cancer cases and approximately 207, 252 ovarian cancer deaths occurred in 2020, ranking second in the incidence and death causes among female gynecological malignant tumors . For allEpigenetic gene regulation, mainly including DNA methylation, chromatin remodeling, histone modification, and non-coding RNA regulation, has traditionally been considered closely related to the development and progression of human cancers . It shouHDAC6, as the largest member of the HDAC family, has attracted much interest  because in vivo and in vitro, suggesting it has the potential to be further developed as an agent for ovarian cancer treatment.In this study, we synthesized compound H42 and further evaluated its anti-cancer activity against ovarian cancer. The results showed that compound H42 inhibited ovarian cancer cell proliferation in both A2780 and SKOV3 cells. It induced apoptosis, intracellular ROS production, and DNA damage, along with related-protein expression change. However, compound H42 downregulated the expression of cyclin D1 through regulating HDAC6-mediated acetylation of HSP90, thus leading to G0/G1 phase cell cycle arrest. In addition, compound H42 could repress cancer progression in a human ovarian cancer cell xenograft model without obvious toxicity. These results indicated that compound H42 exerted potent anti-cancer activities 1HNMR and 13C NMR were obtained on the DPX-400 NMR , high-resolution mass spectra (HR-MS) data were signed by the Q-Tof type high-resolution mass spectrometer , and the melting point of the target compound was determined with the X-5 precision melting point analyzer . The NMR spectra are shown in For chemical synthesis, all solvents and reagents were purchased from commercial companies. Chemical reactions were monitored by thin-layer chromatography (TLC) and ultra-high performance liquid chromatography-mass spectrometry . In addition, silica gel (200–300 mesh) and silica gel (100–200 mesh) are used for column chromatography. Spectra data of The 2-aminopyridine (1 mmol) was weighed in a 250-ml round-bottomed flask and dissolved slowly by adding THF (150 ml). N-bromosuccininimide (NBS) (2 × 0.5 mmol) was added in fractions under stirring at room temperature for 0.5 h. The reaction was monitored by TLC (oil ether: ethyl acetate = 5:1). When the reaction was completed, the reaction system was quenched by adding water, extracted three times with ethyl acetate, combined with the organic phase, washed twice with saturated salt water (50 ml × 2), and the organic phase was removed by distillation under reduced pressure to obtain intermediate 2. As the reaction system is relatively simple, intermediate 2 can be directly added into the next step.Intermediate 2 (1 mmol) was placed in a 100-ml round-bottomed flask, dissolved by adding a mixture of dioxane–water (5:1) solvent. Sequentially, 4-methoxyphenylboronic acid (1.2 mmol) and anhydrous sodium carbonate (2 mmol) were added into the reaction system. Bis(triphenylphosphine) palladium dichloride (0.2 mmol) was added definitively under stirring at room temperature. Thereafter, the system was transferred to an oil bath at 110°C, heated under refluxing for 5 h under nitrogen protection, and monitored by thin-layer chromatography (petroleum ether-ethyl acetate = 5:1). When the reaction of raw material 2 was complete, then we stopped the reaction, and the reaction system cooled down to room temperature, quenched with water, extracted three times with ethyl acetate, combined with the organic phase, washed with saturated salt water, and distilled under reduced pressure to remove the organic phase. Eventually, column chromatography (petroleum ether: ethyl acetate = 3:1) was performed to obtain intermediate 3 as a pale yellow solid.Then, intermediate 3 (1 mmol) was placed in a 250-ml round-bottomed flask and dissolved by adding tetrahydrofuran solvent. NBS (2 × 0.5 mmol) was added successively, and the reaction was carried out for 0.5 h at room temperature. After the reaction was completed, the organic phase was extracted three times with water and ethyl acetate, combined with the organic phase, washed with saturated salt water, and removed by decompression distillation. Intermediate 4, due to the reaction system being simpler, can be directly added in the next step.Intermediate 4 was dissolved in a 100-ml round-bottomed flask by adding dioxane–water (5:1) mixture. Then, 4-isopropyl benzene boric acid (1.2 mmol), anhydrous sodium carbonate (2 mmol), and  palladium dichloromethane complex (0.2 mmol) were added. The reaction system was transferred to an oil bath at 110°C, heated and refluxed for 5 h under nitrogen protection, and was stopped by detecting the disappearance of raw material 4 by TLC. The reaction system was cooled to room temperature, quenched with water, extracted three times with ethyl acetate, combined with the organic phase, washed with saturated salt water, and distilled under reduced pressure to remove the organic phase. Last but not the least, column chromatography (petroleum ether: ethyl acetate = 2:1) was used to obtain intermediate 5.Afterward, intermediate 5 (1 mmol) was placed in 25-ml × 3 round-bottomed flasks and then separately dissolved by adding super dry THF. Sodium hydroxide (2 mmol) was added under stirring at room temperature. After stirring for 0.5 h, methyl 4-bromobenzoyl bromide (1.1 mmol) was added. Finally, the reaction was conducted overnight at room temperature. After the reaction system was completed, the organic phase was extracted three times with water and ethyl acetate, washed with saturated salt water, and removed by decompression distillation. In closing, intermediate six was acquired by column chromatography (petroleum ether: ethyl acetate = 5:1).N-hydroxy-4-{[(5-(4-isopropylphenyl)-3-(4-methoxyphenyl)pyridin-2-yl)amino]methyl}benzamide.Intermediate 6 was placed in a 25-ml round-bottomed flask and dissolved in DCM-MEOH (1:2) solvent. 50% aqueous hydroxylamine (30 mmol) was added under stirring at room temperature for 0.5 h. Then, 10 eq of sodium hydroxide was added. After 10 min, the reaction of raw material 6 was detected by TLC and then, the organic solvent was removed by distillation under reduced pressure. The residue was dissolved by adding water, and dilute hydrochloric acid was added to adjust the pH to 5–6 in an ice bath. The filtered solid was washed with water mixed with petroleum ether-ethyl acetate (1:1) twice and finally dried to obtain white solid end-product 7, 2. Drugs were prepared at a stock concentration of 10 mM with dimethyl sulfoxide . N-acetyl-cysteine was purchased from Dalian Meilun Biotechnlogy Co., LTD, and MG132 was obtained from MedChemExpress.Human ovarian cancer-derived cell lines A2780 and SKOV3 were purchased from Procell Life Science and Technology Co., Ltd.  with STR identification. A2780 and SKOV3 cells were cultured in RPMI 1640  and McCoy’s 5A , respectively, supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin at 37°C and 5% CO3 per well) were seeded in 96-well plates for 24 h, the culture medium was replaced with fresh medium containing serially 2-fold diluted drugs. Then, the cells were incubated for another 72 h, and the cell viability was measured daily for up to 3 consecutive days using CCK-8 assay. The CCK-8 solution (10 μl) was added to each well, and the absorbance at 450 nm was measured using a microplate reader after the plates incubated at 37°C for 1 h. The half-maximal concentration (IC50) was determined using GraphPad Prism 9.3.After the cells  according to the manufacturer’s instruction. Briefly, the cells seeded in 24-well plates were treated with vehicle or drugs for 48 h, EdU solution was added to the medium and incubated for 2 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. Then, click additive solution was added to the cells and incubated for 30 min at room temperature. The medium was discarded, and the cells were stained with Hoechst 33342. Images were acquired using a fluorescent microscope at 346 nm and 590 nm.Annexin V-FITC Apoptosis Detection Kit  by flow cytometry  according to the manufacturer’s instruction with analysis by FlowJo V10.Cells were inoculated in 6-well plates, and the culture medium was replaced with fresh medium containing indicated concentration drugs and incubated for 72 h. The cells were collected and apoptotic cells were detected using the Cells were seeded in 60-mm dishes and incubated for 24 h. After the supernatant was replaced with fresh medium without FBS, the cells were incubated for 24 h. The medium was discarded, fresh medium with FBS and indicated concentration drugs were added, and the cells were incubated for another 24 h. Then, the cells were harvested and stained with the staining solution for 30 min at room temperature in the dark according to the manufacturer’s instruction of the cell cycle detection kit . Cell cycle distribution was detected by flow cytometry  and analyzed by ModfitLT 5.Cells were inoculated into 6-well plates and cultured for 24 h. The cell scratch was made on the plate using a 200-μl pipette tip, and the supernatant was replaced with fresh medium without FBS but containing the indicated concentration of drugs. Then, images were acquired with an inverted microscope to record the scratch area. After 24 h of culture, the scratch area was photographed again. At least five fields were captured in each group. The relative wound healing ratio (%) = / × 100%.Cells in 200 μl medium without FBS were added to the upper chamber, and 600 μl medium with FBS and indicated concentration of drugs were added to the lower chamber, which was cultured for 24 h. After the supernatant was removed, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min. The cells in the upper chamber were wiped with a cotton swab lightly, and the migration cells in the lower chamber were observed and counted via an inverted microscope.Reactive Oxygen Species Assay Kit . Briefly, the cells were plated into a 6-well plate and cultured for 24 h. The medium was replaced with fresh medium containing the indicated concentration of drugs and incubated for 48 h. The medium was discarded, and DCFH-DA (10 μM) was added to the cells for 20-min incubation at 37°C. Then, intracellular ROS was measured by flow cytometry .Intracellular ROS was measured using the Cells were inoculated into 6-well plates and treated with indicated concentration of drugs for 48 h. The cells were harvested, mixed with low-melting point agarose, and layered onto microscopic slides, which were covered by a glass coverslip and incubated at 4°C for 15 min to allow gels to be solidified. The slides were placed in lysis solution for 2 h at 4°C to allow DNA unwinding. Then, the slides were placed in electrophoresis buffer, which were conducted for 30 min at 300 mA. After rinsing with distilled with water, the slides were dipped in 75% ethanol and dried for 15 min at 37°C. DNA was stained for 10 min at room temperature. The slides were rinsed with distilled water and imaged by a fluorescent microscope.Cells were plated on glass coverslips placed in a 24-well plate. After culturing for 24 h, the cells were treated with indicated concentration of drugs and incubated for another 48 h. Then, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS. After blocking with 5% BSA in PBS for 30 min, the cells were incubated with phosphor-H2A antibody  for 1 h, washed with PBS three times, and incubated with fluorescent secondary antibody  for 1 h. The slides were mounted with DAPI, and the cells were imaged using a fluorescence confocal microscope at 346 nm and 488 nm.Cells were washed with cold PBS twice and lysed with RIPA with protease and phosphorylase inhibitor . Proteins were boiled and electrophoretically separated with 10% or 12% polyacrylamide gel at 80–120 V. The proteins were transferred to 0.22-μm or 0.45-μm PVDF membranes , which were blocked with 5% BSA in TBST for 2 h. Then, the membranes were incubated with a primary antibody at 4°C overnight. After washing with TBST three times, the membranes were incubated with HRP-linked secondary antibodies  and washed with TBST three times and visualized using ECL reagents  by ChemiDOC Imaging System .Antibodies were used against acetyl-α-tubulin , α-tubulin , HDAC6 , caspase-3 , caspase-7 , cleaved caspase-9 , PARP , CDK4 , CDK6 , cyclin D1 , cyclin D3 , pCNA, p-c-Jun , p-p38 , Wee1 , p-CDC25c , γH2AX , p-CHK2 , p-CHK1 , E-cadherin , N-cadherin , Snail , GAPDH , β-actin , Bcl-2 , p-Erk1/2 , H3 , acetyl-H3K9 , HSP90 , and acetyl- HSP90 .After the cells were treated by compound H42 or control for specific time, the total RNA was extracted by TRIzol according to the instructions, followed by cDNA synthesis using the RevertAid First-Strand cDNA Synthesis Kit . The mRNA level of cyclin D1 was measured by real-time PCR with 18s as the control. The sequence of primers was as follows: forward primer of cyclin D1: 5′-CACGCGCAGACCTTCGT-3′, reverse primer of cyclin D1: 5′-ATGGAGGGCGGATTGGAA-3’; forward primer of 18S: 5′-CGGCGACGACCCATTCGAAC-3′, and reverse primer of 18S: 5′-GAATCGAACCCTGATTCCCCGTC-3’.A2780 cells were infected with lentiviruses carrying HDAC6 (shHDAC6) or empty vectors . After screening, shHDAC6 A2780 cells were obtained. Over-HDAC6 was produced by transfecting A2780 cells with the HDAC6 overexpression plasmid .6) suspended in 50 μl medium were subcutaneously injected into the right axilla of each nude mouse. When tumor volume was greater than 100 mm3, the mice were randomly divided into two groups (N = 5 per group): 1) mice that were given H42 at 50 mg/kg once a day; 2) mice that were given PBS once a day. Tumor volume and body weight were measured every 2–3 days. The mouse state was monitored every day. At 19 days after administration, all mice were euthanized and tumor weight was measured.A total of 10 female Balb/c nude mice (4–5 weeks old) were gifted by GemPharmatech Co., Ltd. and housed in the Animal Experimental Center of Henan Province. SKOV3 cells  of 28.43 ± 2.13 μΜ (24 H), 8.54 ± 0.93 μΜ (48 H), and 5.40 ± 0.53 μΜ (72 H) inhibited SKOV3 cell proliferation with an IC50 of 3.16 ± 0.17 μΜ (24 H), 0.94 ± 0.03 μΜ (48 H), and 0.85 ± 0.02 μΜ (72 H), respectively  was employed to monitor the phosphorylation of H2AX (γH2AX), a sensitive marker of DNA double-strand breaks (DSBs), in the nucleus of H42-treated A2780 and SKOV3 cells. Obviously, it was shown that H42 significantly increased the number of cells with γH2AX foci in the two ovarian cancer cells . In addivia arresting cells at the G0/G1 phase.To determine the underlying mechanism by which compound H42 inhibits ovarian cancer cell proliferation and apoptosis, the cell cycle distribution was analyzed in both cell lines after H42 treatment. As compared to the vehicle (ratio of the G0/G1 phase is 28.63% of A2780 cells and 19.35% of SKOV3 cells), treatment of H42 for 24 h led to an increase of the G0/G1 phase of the cell cycle to 52.56% and 70.27%, respectively, indicating that H42 could efficiently increase the percentage of cell cycle arrest at the G0/G1 phase , while wIt is reported that cyclin D1 played a critical role in cell cycle control  and tumovia inhibiting HDAC6-mediated deacetylation.It has been demonstrated that downregulation of HDAC activity gives rise to G1 cell cycle arrest, and the benzamide group in compound H42 was similar to the zinc-binding group in HDAC6 inhibitors. We wonder whether compound H42 functions through HDAC6? To confirm the effect of H42 on HDAC6, A2780 cells were treated with compound H42 for 48 h, and the expression levels of HDAC6, acetylated α-tubulin, and HSP90 were detected by Western blot. With the increasing concentration of H42, acetylated α-tubulin and HSP90, which were mediated by HDAC6, accumulated significantly . Meanwhivia regulating the function of HDAC6, which further interrupted the binding of HSP90 with cyclin D1, promoting the degradation of cyclin D1, resulting in cell growth arrest at G0/G1.To further determine the relationships between compound H42-induced cyclin D1 degradation and the HDAC6 signaling pathway, A2780 cells were transfected with the HDAC6 overexpression plasmid and the related-protein expression was analyzed. The results showed that overexpression of HDAC6 induced deacetylation of HSP90 and upregulation of cyclin D1 , which win vivo, SKOV3 cells (2 × 106) were injected subcutaneously into the right axilla of female Balb/c nude mice to construct an ovarian cancer xenograft tumor model. When the tumor volume was greater than 100 mm3, the mice were randomly divided into two groups: one group of mice received H42 at 50 mg/kg once a day, while the control group only received PBS. The mice were treated for 19 days, and the mouse state, body weight, and tumor volume were monitored. During the treatment, there was no significant change in the body weight, and the mice were in good mental state (in vivo without obvious toxicity.To further investigate the anti-tumor activity of compound H42 al state , suggestal state . Then, aal state . Additioal state . The afoHDAC6 is a widely studied member of the HDACs, which is known to anchor in the cytoplasm and catalyze the deacetylation of α-tubulin, HSP90, and so on . We founROS also represented an important signaling molecule participating in multiple cellular processes. The stimuli of oxidative stress and free radicals can facilitate the formation of DSBs and mitochondrion-mediated intrinsic pathway of apoptosis . So, we It is reported that HDACI treatment often induced the upregulation of P21 , which cIn this work, the A2780 cell line derived from ovarian endometrioid adenocarcinoma with wild-type TP53 and the SKOV3 cell was a TP53-mutant cell line derived from the ascites of serous ovarian cystadenoma. Compound H42 inhibited cell proliferation in the 2 cell lines regardless of the status of TP53, suggesting that H42 will be effective against a broad spectrum of tumor types, including tumors without p53 function.via HDAC6-mediated cyclin D1 degradation and suppressed ovarian cancer progression in nude xenograft mice, indicating compound H42 has a preclinical value in ovarian cancer therapeutics, warranting further investigation.Compound H42 inhibited ovarian cancer cell proliferation


Additionally, a significant increase in DNA double-strand breaks (DSB) and a decrease in migration ability were observed. Our study revealed that combining radiation and the PI3K-α isoform improved radiosensitivity that resulted in a significant delay in tumor growth and improved survival rate.Non-small cell lung cancer (NSCLC) is a malignant lung tumor with a dismal prognosis. The activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is common in many tumor types including NSCLC, which results in radioresistance and changes in the tumor microenvironment. Although pan-PI3K inhibitors have been tested in clinical trials to overcome radioresistance, concerns regarding their excessive side effects led to the consideration of selective inhibition of PI3K isoforms. In this study, we assessed whether combining radiation with the administration of the PI3K isoform-selective inhibitors reduces radioresistance and tumor growth in NSCLC. Inhibition of the PI3K/AKT pathway enhanced radiosensitivity substantially, and PI3K-α inhibitor showed superior radiosensitizing effect similar to PI3K pan-inhibitor, both  Non-small cell lung cancer (NSCLC) is a major cause of cancer death . NSCLC aThe phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway has been identified as one of the critical signaling pathways involved in cancer cell growth, survival and proliferation. Furthermore, this pathway is also known to be involved in the mechanisms of tumor-specific radioresistance . InhibitThere is growing evidence supporting the use of PI3K inhibitors as radiosensitizers. However, the pan-PI3K inhibitor that suppresses all PI3K isoforms can cause side effects such as hyperglycemia, rash, neutropenia, diarrhea, neuropsychiatric effects, nausea and fatigue . The useIn this study, we hypothesized that the combination of radiation and PI3K isoform inhibitors will increase radiosensitivity and suppress tumor development in NSCLC. The goal of this study was to develop a new radiation-PI3K isoform-selective inhibitor combination therapy to overcome radioresistance and improve the response rate of tumors to the radio therapy.2, humidified environment.LLC1 (mouse lung carcinoma cell line) and A549 (human lung carcinoma cell line) were used in this study. Both LLC1 and A549 cells were plated in Dulbecco Modified Eagle Medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) and then cultured at 37°C in a 5% COin vitro experiment, NSCLC cell lines were irradiated with a single dose of 2 Gy using X-rad 320  for colony formation assay. 4 Gy and 6 Gy irradiation was applied with two repeated doses and three repeated doses of 2 Gy, respectively. Radiation fraction was operated at 320 kVp and 12.5 mA with 2.0 mm Al filtration and a dose rate of 4.76 cGy/sec.In the Four isoform-selective PI3K inhibitors were used in this study. Pictilisib (GDC-0941) is a potent inhibitor of PI3Kα/δ with modest selectivity against p110β (11-fold) and p110γ (25-fold). Taselisib (GDC-0032) is a PI3K inhibitor with potent inhibitory activity against p110α(PI3K-α inhibitor). Idelalisib  is a p110δ selective inhibitor (PI3K- δ inhibitor). Duvelisib (IPI-145) is a p100δ selective inhibitor that inhibits p110δ, P110γ, p110β and p110α(PI3K- γ/δ inhibitor). All inhibitors were purchased from Selleckchem .The markers of the PI3K-AKT signaling pathway, phosphorylated AKT and double-stranded DNA break marker γ-H2AX were analyzed using Western blot. Protein was extracted from whole-cell lysates using a Cell Extraction Buffer  containing Halt™ Protease and Phosphatase Inhibitor Cocktail . Protein concentration was measured by BCA protein assay Kit . For Western blot analysis, protein extracts were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 3% bovine serum albumin (BSA) in tris-buffered saline with 0.1% Tween 20 (TBST) for 1 hour. The membranes were incubated overnight at 4°C in primary antibodies and washed three times for 10 minutes with TBST. Next, the blots were incubated in the horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature and washed with TBST three times for 10 minutes. After the last wash, the membranes were incubated in enhanced chemiluminescence (ECL) solution  and exposed to X-ray films. The detailed information on antibodies used in Western blotting is provided in The effect of the PI3K-isoform inhibitors on cell proliferation was measured using the 3--2,5-diphenyltetrazolium bromide  colorimetric assay. Approximately 1000–2000 cells per well were seeded on 96-well plates with 100 μL of DMEM growth medium and incubated overnight in a 37°C CO2 incubator. Then, 10% MTT solution in growth medium was added to each well. The plate was incubated in a 37°C CO2 incubator for 4 h. Thereafter, formazan crystals were dissolved in 100 μL of DMSO. Absorbance was measured at 590 nm using a microplate reader .After the irradiation of 2 Gy using X-RAD 320 , 2000 cells were plated on 4-well slide plates. After plating, the cells were treated with PI3K kinase inhibitors  for 12 hours. The cells were fixed by a 4% paraformaldehyde solution. The cells were permeabilized by incubating in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 for 10 min followed by washing three times with PBS for 5 min. Next, the cells were blocked with 1% BSA in PBS with 0.1% Tween 20 (PBST) for 1 h. The cells were incubated overnight at 4°C with anti-phospho-γ-H2AX antibody , and washed three times for 5 minutes with PBST. The cells were incubated with a fluorescein isothiocyanate-labeled secondary antibody for 1 h in the dark and mounted with VECTASHIELD solution . Observation phospho-γ-H2AX foci were observed and fluorescence images were acquired using the Zeiss LSM 700 confocal fluorescence microscope.2 and humidified environment. Colonies were counted by staining the cells with 0.5% crystal violet.The cells irradiated using X-RAD 320  were plated in 6-well plates in triplicate. After plating, the cells were treated with PI3K kinase inhibitors . The cells were incubated for 9–12 days at 37°C in a 5% COTo measure the cell migration, A549 cell lines were seeded into 6-well plates containing RPMI media with 10% FBS, 1% P/S and cultured to approximately 100% confluence. Next, the cell monolayers were denuded at the median axes with sterile 1 mL pipette tips. The scratch-wound healing by cell migration was monitored over time, and images were captured using a light microscope with a 10× objective lens at the 0 and 48 h time-points after the denudation.6 cells in 100 μL Dulbecco’s phosphate buffered saline) were injected subcutaneously into the mouse thigh. When tumor size reached approximately 100 mm3, the mice were randomly divided into four groups: vehicle control, radiation, GDC0032-treated and GDC0032+radiation-treated. GDC0032 was formulated in DMSO and intraperitoneally injected at a dose of 5 mg/kg one h after the irradiation. Mice in RT groups were irradiated with a single dose of 5 Gy fractions one time.The mouse xenograft model was established in the C57BL/6 strain. LLC1 cells , P < 0.01 (**) and P < 0.001(***)).The Student’s t-test was used to assess the difference in continuous variables of two groups. A repeated-measures ANOVA was used to compare changes in the tumor volume in each treatment group which was measured at eight different time-points . All the experiments were repeated at least three times. All the data were presented as the mean ± standard deviation. Data were plotted using GraphPad Prism 5.0 . Statistical analyses were performed using SPSS software, version 25.0 . A 2-sided To prove that the PI3K/AKT signaling pathway is activated upon irradiation of NSCLC tumor, we analyzed AKT activation at the protein level using Western blot. LLC1 cell xenografts in the mouse lungs were irradiated with doses ranging from 1 Gy to 8 Gy. After 24 h after irradiation, normal lung tissue and LLC1 tumors were analyzed by probing with anti-phospho-AKT and phospho-γ-H2A.X antibodies. . It was We next performed a colony formation assay to confirm the radiosensitivity of the NSCLC cell lines . A549 anP < 0.001). Similar effect was observed in the LLC1 cells . The mean rates of cell proliferation in A549 cells with four different PI3K inhibitors  were as follows: 67.54, 72.90, 82.93, 85.03 at 0.1 μM, 58.82, 63.72, 74.39, 65.67 at 1 μM, 45,65, 48.48, 59.62, 54.45 at 10 μM, respectively. The mean rates of cell growth in LLC1 cells with four different PI3K inhibitors were as follows: 83.85, 81.70, 92.60, 91.57 at 0.1 μM, 69.12, 75.52, 80.81, 82.22 at 1 μM, 56.89, 64.18, 51.33, 72.06 at 10 μM, respectively. Furthermore, PI3K inhibitors reduced phosphorylation of AKT in the A549 and LLC1 cells , 25% , 38%  and 37% , respectively. The rates were 10% , 14% , 36%  and 31%  in LLC1 cell line. Although combination of radiation with PI3K- δ isoform inhibitor (CAL101) and PI3K- γ/δ (IPI145) inhibitor also showed decreased survival rates, the PI3K α-isoform inhibitor (GDC0032) inhibited colony formation the most. Its inhibition rate was comparable to that of the PI3K pan-inhibitor (GDC0941). Also, the expression of cleaved-caspase 3, an important marker of cell apoptosis, was increased in combination therapy. In particular, the expression of cleaved-caspase 3 increased in PI3K- α isoform inhibition as much as in PI3K-pan inhibition. The AKT activation increased by irradiation was decreased by PI3K-isoform inhibitors   treatment. The combination of radiation with PI3K-α inhibitor improved NSCLC radiosensitivity and markedly delayed tumor growth  < 0.05) . We also < 0.05) . Moreovein vitro and in vivo [Radioresistance is one of the major causes of failure in cancer therapy. PI3K pathway has been proven to play a crucial role in the progression of and radioresistance in several types of cancer , 21. Man in vivo , 26–29.in vivo for the first time. The excessive activation of AKT reduced the expression of phospho-γ-H2A.X, which indicated the induction of radioresistance. Our results were consistent with other studies that reported that the activation of AKT may reduce the efficacy of radiotherapy by promoting DNA repair in NSCLCs [DSBs are the major cause of irradiation-induced cell death. DSBs are rarely induced endogenously and are primarily radiation-induced. DSBs start to appear at a dose of a few mGy and increase as the radiation dose increases. Radiation induces simple and complex DSBs with 3′ blocked ends . DSB repn NSCLCs , 40.in vivo and no weight loss or disease was seen in mice. PI3K has four isoforms of the catalytic subunit: p110α, p110β, p110δ and p110γ [PI3K inhibitors might serve as a potential remedy for radioresistance. However, in the relevant clinical trials, it has been reported that first-generation pan-PI3K inhibitors have limited efficacy and relatively high rates of side effects . Becausend p110γ , 45. Thend p110γ . It has nd p110γ , 48. p11nd p110γ . The rolnd p110γ , 51. Recnd p110γ , 53.However, the role of the PI3K isoforms in radioresistance in NSCLCs remained unclear. In this context, we explored the role of the PI3K inhibitors in irradiated NSCLC cells and tried to find the most effective isoform-selective inhibitor. In addition, we selected a concentration that showed 70% AKT activity inhibition for a particular dose of PI3K-isoform inhibitors to minimize side effects. We found that the PI3K signaling pathway modulates the radiosensitivity of NSCLC cell lines. Therefore, irradiation with PI3K isoform-selective inhibitors could delay the DSB repairs and increase radiosensitivity. In particular, PI3K-α inhibitor was as effective as PI3K-pan inhibitors, which implies that PI3K-α-isoform might play a key role in the radioresistance of NSCLC cells. Moreover, the expression level of the activated caspase-3, which indicates the occurrence of radiation-induced apoptosis, was elevated after the inhibition of PI3K-α than after the inhibition of other isoforms . Our resin vitro and in vivo. E-cadherin and Slug are associated with EMT and are known to be involved in modulating radioresistance [in vitro and in vivo modulation in the expression of EMT markers in LLC1 cells induced by the combination treatment of irradiation and PI3K-α-isoform inhibitor was comparable to that by the treatment with the PI3K-pan isoform inhibitor. When the A549 cells were treated only with the PI3K inhibitors, the expression of Slug was increased; however, its expression was decreased when the cells were treated with radiation as well. Additionally, in the wound healing assay, the reduction in the cell migration by the combination of PI3K-α isoform inhibition and radiation was comparable to that by the PI3K-pan inhibition. It suggested that the irradiation combined with the inhibition of PI3K-α isoform increases radiosensitivity and reduces metastatic capacity.The PI3K pathway plays an important role in EMT, and its activation was found to be increased in several invasive cancer cells , 61–63. sistance . The expsistance . It was sistance . Vimentisistance . In our Buparlisib (BKM120) is an oral pan class 1 PI3K inhibitor, which has been studied in preclinical and clinical studies. After establishing favorable pharmacokinetics with acceptable toxicity, the first phase I clinical trial of Buparlisib with palliative thoracic radiotherapy in NSCLC patients was conducted , 74. In in vivo tumor growth. Second, the reason why PI3K-α-isoform inhibition regulates EMT and radioresistance more effectively than the inhibition of other isoforms of PI3K is not explained in this study. Further studies elucidating the downstream factors are warranted to validate our findings. Nevertheless, our study found that inhibition of PI3K-α isoform influences radiosensitivity in NSCLCs and confirmed that it also suppresses EMT.Our study has several limitations. First, we could not verify the inhibitory effect of other isoform-inhibitions on the In conclusion, the combination of radiation with PI3K-α-specific inhibition shows antitumor efficacy in NSCLC by modulating tumor growth, cell survival and EMT. We propose that the PI3K-α isoform inhibition is a potentially effective strategy to overcome radioresistance safely and efficiently. In the future, relevant clinical studies are required to help understand the mechanism and clarify the actual benefit to cancer patients.This study was supported by a Dongin Sports research grant of Yonsei University College of Medicine (6–2017-0105).Research data are stored in an institutional repository and will be shared upon request to the corresponding author.None of the authors have any conflicts of interest to disclose.Supplement_data_rrac018Click here for additional data file.