Conversely, graphene has a 2D layered structure, possessing an exceptionally large specific surface area and high carrier mobility. Likewise, other emerging graphene‐analogous 2D nanomaterials, such as graphitic carbon nitride (g‐C3N4), transition‐metal dichalcogenides (TMDs), hexagonal boron nitride (h‐BN), and black phosphorus (BP), show unique electronic, physical, and chemical properties, which, however, exist only in the form of a monolayer and are typically anisotropic, limiting their applications. Upon hybridization with fullerenes, noncovalently or covalently, the physical/chemical properties of 2D nanomaterials can be tailored and, in most cases, improved, significantly extending their functionalities and applications. Here, an exhaustive review of all types of hybrids of fullerenes and 2D nanomaterials, such as graphene, g‐C3N4, TMDs, h‐BN, and BP, including their preparations, structures, properties, and applications, is presented. Finally, the prospects of fullerene‐2D nanomaterial hybrids, especially the opportunity of creating unknown functional materials by means of hybridization, are envisioned.Fullerene has a definite 0D closed‐cage molecular structure composed of merely sp In particular, g‐C3N4, with a moderate band gap and indirect‐semiconductor nature, has become a promising metal‐free catalyst for water splitting and the degradation of pollutants due to the advantages of low cost and high specific surface area (2500 m2 g−1 for the monolayer) and has been applied in organic solar cells.3N4 exhibits a poor electrical conductivity and low photocatalytic activity owing to the rapid recombination of photogenerated electron–hole pairs. Thus, improvements in the photocatalytic activity of g‐C3N4 to extend this material to further applications are highly desired. To this end, the hybridization of g‐C3N4 with other functional materials, including fullerenes, has been developed in recent years and appears feasible since the polymeric nature of g‐C3N4 renders the chemical structure flexible.During the last decade, several other types of emerging graphene‐analogous 2D nanomaterials have been discovered.2, consisting of hexagonal rings with Mo and S atoms alternately located at the hexagon corners, is the most representative TMD with a direct band gap in monolayer form and high in‐plane carrier mobility, thus being suitable for versatile applications in electro and photocatalysis, photovoltaics, and photoelectric devices.2 in photocatalytic H2 production, since only its edges have a high catalytic activity, whereas its basal plane is inactive, a low photocatalytic activity has been achieved, even for single‐layer MoS2. Consequently, MoS2 is generally applied as a cocatalyst loaded on conventional metal‐based semiconductor photocatalysts, such as TiO2, CdS, and ZnO, to reduce the energy barrier of water splitting and to increase the stability of the photocatalysts. To enhance the photocatalytic H2 production activity of MoS2 itself, the construction of nanocomposites composed of MoS2 and other functional materials, such as graphenes and fullerenes, provides a practical solution.3N4 and the TMD MoS2, no Review has reported their hybridization with fullerenes, which is crucial for understanding such intriguing 0D–2D hybrid systems.The inorganic transition‐metal dichalcogenides (TMDs) represent another type of emerging 2D semiconducting nanomaterial and have been attracting widespread attention due to their intriguing electronic, optical, and mechanical properties.3N4, TMDs, h‐BN, and BP, all types of hybrids of fullerenes and 2D nanomaterials reported to date are exhaustively reviewed, with emphasis on the exclusive impact of fullerenes with unique electronic and chemical properties on the electronic and band structures of 2D nanomaterials. Upon attachment of fullerene molecules, noncovalently or covalently, the physical/chemical properties of 2D nanomaterials can be tailored, and in most cases, improved, and the mechanisms responsible for such improvements are discussed in detail. Furthermore, fullerene incorporation may induce new properties in 2D nanomaterials as a result of strong intermolecular interactions, thus extending the functionalities and applications of 2D nanomaterials. At the end, we envision the prospects of fullerene‐2D nanomaterial hybrids, especially the opportunity of creating unknown functional materials by means of hybridization.In this Review, we focus on the hybrids of 0D fullerenes and 2D nanomaterials, including their preparations, structures, properties, and applications. Based on known 2D nanomaterials, such as graphene, g‐C22.1TableNoncovalent hybridizations of fullerenes and graphenes have been fulfilled by depositing fullerene onto a graphene layer to form a graphene/fullerene bilayer film and by physically blending pristine fullerenes or fullerene derivatives and graphenes see Table1.2.1.160 onto a graphene film to form a bilayer hybrid; in this way, the interfacial electronic interactions and charge transfer between C60 and graphene can be investigated. In 2015, Heinz et al. fabricated a C60/graphene hybrid by depositing C60 onto single‐layer graphene and studied the ground and excited‐state charge transfers at the C60/graphene interface by using Raman spectroscopy and THz time‐domain spectroscopy. The authors concluded that at equilibrium the C60 layer functioned as an electron acceptor affording hole doping of the graphene with an injection of ≈0.04 holes per interfacial C60 molecule at the surface of the bilayer hybrid, as well as a downshift of the graphene Fermi level by 160 meV , which provided a unique surface with humps and valleys facilitating the assembly of Cop Figure2.23 Th60 films onto graphene sample surfaces using a spray coating method and investigated the electrical interaction and charge transfer kinetics at the graphene/adsorbate interface by using flicker noise, as an insightful probe, in combination with Raman spectroscopy and DC‐transport measurements.60 deposition, which was attributed to the carrier trapping/detrapping processes in the hybrid system, finally leading to an enhanced charge noise. Meanwhile, the charge exchange process was significantly suppressed with decreasing temperatures, indicating that the flicker noise acted as a powerful probe to elucidate the nature of the charge transfer in the graphene–fullerene hybrid device. Furthermore, the authors fabricated graphene field‐effect transistors to quantitatively evaluate the charge transfer between C60 molecules and graphene and found that a large number of carriers with a high Fermi energy for n‐doped graphene may overcome the energy barrier to occupy the LUMO of C60 molecules compared to that of p‐doped graphene.60 was estimated to be 0.6–1.3 × 1012 cm−2, ≈0.01 electrons captured by a single C60 molecule.In 2013, Qiu and co‐workers constructed a fullerene/graphene hybrid device by depositing self‐assembled C60 onto FLG films prepared by a chemical vapor deposition (CVD) technique. The electrical conductance of the C60/FLG system decreased when the system was under illumination and was attributed to a kind of negative photoconductivity.60/FLG system varied, FLG exhibited p‐type doped characteristics under low light intensities and presented n‐type doped behavior for high light intensities.Likewise, in 2014, Mendoza et al. constructed a fullerene/few‐layer graphene (FLG) bilayer system by thermally evaporating C60–graphene vertical heterostructure with a well‐ordered film morphology on the graphene, as confirmed by transmission electron microscopy (TEM).60 crystal and graphene lattice directions. The preferential growth of C60 crystals is prone to graphene's zigzag and armchair directions. Moreover, the C60–graphene vertical junction‐based transistors showed n‐type transport behavior. The energy barrier modulation at the C60–graphene Schottky junction can achieve a high on/off ratio above 3 × 103, which is difficult to obtain for conventional graphene transistors. Thus, graphene is demonstrated to offer an excellent substrate for the epitaxial assembly of organic semiconductors toward electronics applications.Later, in 2015, Bao et al. fabricated a C60 molecules at the graphene/Cu interface by annealing led to amorphous and crystalline structures.60 molecules imprinted a local strain/deformation on the graphene layer with various magnitudes controlled by the intermolecular distance.In the same year, Reinke and co‐workers reported that the intercalation of C60 molecules were encapsulated by two layers of graphene  results in the Fermi‐level energy of the system to align inside the broadened LUMO. When the gate voltage is changed, the charge transfer to the molecule increases through the Dirac  point of graphene to the electron‐doped side , resulting in a Fermi level deeper inside the LUMO.Bilayer films comprised of graphene and fullerene derivatives have also been studied. In 2011, Lofwander and co‐workers constructed a single‐molecule device consisting of graphene and a fullerene derivative, where the graphene was patterned to form a nanogap and can be used as electrodes for gate‐tunable molecular electronics, and a dumbbell fulleropyrrolidine consisting of a central wire of 1,4‐phenylenediamine attached to C60 end‐capped molecular wire comprised of two styrene units covalently connected via a paracyclophane moiety in the center, Weber and co‐workers fabricated graphene–fullerene–graphene junctions with ultraflat, transparent, and robust graphene nanogaps. The single‐molecule contacts were confirmed according to the measured I–V curves, which were similar to those obtained with the mechanically controlled break junction technique. Additionally, nonlinear I–V curves were obtained in a very low yield when molecules were applied at low concentration, thus ruling out the possibility of larger numbers in the junction.Similar single‐molecule junctions employing a fullerene derivative were later constructed by several other groups. In 2015, using a C60 bisadduct used as the junction and observed the redox‐dependent Franck–Condon blockade and avalanche transport phenomena. The C60 bisadduct was synthesized through a 1,3‐dipolar cycloaddition reaction between C60, 1‐pyrene carboxaldehyde, and N‐methylglycine.60 with pyrene anchor groups was verified to effectively modify the electron delocalization and energy levels, leading to HOMO‐dominated transport.f) to the microheater and measuring the thermovoltage (Vth) drop across the device at a frequency (2f) for different back gate voltages (Vg),kBT0 at the operating temperature, and (3) the tunnel couplings to the left and right electrodes need to be equal to achieve a maximum power factor.In 2016, Mol and co‐workers studied a similar graphene–fullerene–graphene single‐molecule transistor with a pyrene‐functionalized C2.1.260 into graphene nanosheets prepared by the exfoliation of a bulk graphite crystal and followed by a reassembling process.60 physical blend increased from 540 to 784 mAh g−1 after the incorporation of C60.60 hybrid, in 2015, Lee and co‐workers carried out DFT studies and found that charge transfer from the graphene to the C60 took place in the graphene–C60 hybrid system, leaving the graphene positively charged (+0.095 e) and the C60 negatively charged (−0.095 e). As a result, Li ions preferentially adsorbed on the C60 side, particularly in the midway region between graphene and C60 or between two C60 molecules.60 hybrid system (−2.285 eV) was higher than that on bare graphene (−1.375 eV), indicating that the Li adsorption on the graphene–C60 hybrid system is more stable.Noncovalent hybrids of fullerenes with graphene have also been facilely prepared by physically blending both components. In 2008, Honma and co‐workers reported a graphene–fullerene physical blend by incorporating C60, pristine single walled carbon nanotubes (SWCNTs), and GO sheets.60/SWCNTs/GO exhibited a surface roughness of only a few nanometers. After the thermal reduction of GO, the as‐prepared C60/SWCNTs/rGO composite was utilized as the active layer of a photovoltaic device  of 1.23 mA cm−2, an open‐circuit voltage (Voc) of 0.59 V, a fill factor (FF) of 0.29, and a power conversion efficiency (PCE) of 0.21%. Such a decent PCE was attributed to the presence of numerous clean p–n junctions distributed throughout the all‐carbon hybrid, facilitating the spontaneous dissociation and subsequent separation of the photogenerated excitons along the bicontinuous heterojunctions.60 with C70 in both the active layer and blocking layer, a nearly fourfold increase in Jsc from 1.23 to 4.95 mA cm−2 was achieved, leading to a dramatic increase in PCE from 0.21% to 0.85%.In addition to pristine graphene (pG) sheets, exfoliating graphite flakes to few‐layer graphene via chemical functionalization is currently commonly used as the most cost‐effective method of obtaining graphene due to its easiness, reliability, and scalability.ce Figure4, affo60 with rGO by the liquid–liquid interfacial precipitation method, resulting in rGO‐wrapped C60 wires.π–π interactions between rGO and C60. FET devices fabricated with rGO/C60 wires exhibited a p‐type semiconducting behavior both in vacuum and in air, indicating that the rGO, as a shell layer, can transport holes effectively. However, the pure C60 wires and rGO sheets showed n‐type and ambipolar behaviors, respectively, in vacuum. Furthermore, the photovoltaic devices based on rGO/C60 wires as the active layer exhibited a significant enhancement in short‐circuit current (JSC) from 0.07 to 0.19 mA cm−2 compared to the devices based on a pure rGO active layer. This result can be interpreted as the photoinduced electron transfer from rGO to C60 under light irradiation.Several efforts have since been made to prepare hybrid materials based on GO or reduced graphene oxide (rGO). In 2011, Shin and co‐workers reported the preparation of hybrids of C60/C70 with GO by a simple grinding method, and the material exhibited a solubility of 5.0 mg mL−1 in water when the weight ratio of the fullerenes to GO was 1:3 , a PTA–GO–fullerene nanofilm was further fabricated by a one‐step electrodeposition. Compared to either PTA or PTA–GO‐modified electrodes, the PTA–GO–fullerene composites showed improved catalytic activity for the oxidation of a variety of small biomolecules, including dopamine (DA), with enlarged peak currents and an apparent negative shift of peak potentials  were found to be sensitive to the direct oxidation of CJ and provided a short analysis time, convenience, and good accuracy.60–GO/GE was because the partially reduced C60 in the electropolymeric process exhibited a favorable conductivity as an effective electron‐transfer medium compared to that of the pure GO film.In 2013, Hu and co‐workers prepared a noncovalent hybrid of C:3 Figure5A.84 T60 composite by electrochemical reduction of a GO–C60 composite, which was then used as an immobilization matrix for GOx.i–t response of an rGO–C60/GOx‐modified electrode to different concentrations of glucose and in constantly stirred oxygen‐saturated phosphate buffer saline (PBS) with a working potential of −0.42 V showed that the amperometric reduction current decreased with the increase in glucose concentration up to 14.6 × 10−3m.i–t response current for the biosensor exhibited a linear pattern against the concentration of glucose in a range of 0.1–12.5 × 10−3m. Hence, the rGO–C60/GOx‐modified electrode showed good analytical ability when used as a sensor for glucose over a wide response range with a low limit of detection, fast response time (3 s), and high sensitivity.60‐wrapped GO by using a simple sonication method, and the material was used to modify a glassy carbon electrode.60 hybrid showed a good sensitivity with a lower oxidation overpotential toward DA than did those of the pristine GO and C60. The as‐prepared sensor for DA detection exhibited a linear response in a range of 0.02–73.5 × 10−6m and a detection limit of 0.008 × 10−6m (based on 3σ) with a sensitivity of 4.23 µA µm−1cm−2. The GO–C60 hybrid‐based sensor also showed a wide linear response and high selectivity along with good practicality toward the detection of DA in rat brain and commercial DA injection samples.Electrochemical sensing applications of noncovalent hybrids of fullerene–graphene have also been reported by other groups. Given that the direct electron transfer properties of enzymes are helpful for studying key biological reactions and for the construction of biosensors and biofuel cells,60/graphene hybrid by using a simple solution reaction of GO and C60 in a lithium hydroxide (LiOH) saturated solution.60 particles on the GO surface served as spacers in the C60/graphene hybrid to support the graphene sheets. The noncovalent C60/graphene hybrid was employed as electrodes in a supercapacitor affording a capacitance of 135.36 F g−1 at a current density of 1 A g−1, which was higher than that based on pure graphene electrodes (101.88 F g−1). Furthermore, a remarkable retention rate of 92.35% after 1000 charge/discharge cycles was obtained, indicating the potential of the C60/graphene hybrid as a supercapacitor electrode.In addition to the applications of fullerene–graphene hybrids in solar cells and electrochemical sensors, fullerene–graphene hybrids have also been applied in supercapacitors. In 2014, Qin and co‐workers constructed a C60 molecules by self‐assembly and the Langmuir–Schaefer deposition technique.60–GO hybrid deposited on hydrophobic substrates was characterized by X‐ray diffraction, Raman and X‐ray photoelectron spectroscopies, atomic force and scanning electron microscopies, and conductivity measurements, confirming that C60 indeed existed in the hybrid multilayer system and that intercalation of fullerene into the interlayer space of the organo‐GO nanosheets did not affect the structure of GO itself. More importantly, a considerable improvement of an order‐of‐magnitude increase in the electrical conductivity for the C60–GO hybrid was achieved because of the presence of C60 intercalated between the graphene layers.Apart from physical blending, a new approach for constructing fullerene–graphene noncovalent hybrids was developed by Gournis and co‐workers in 2015. The approach was based on a bottom‐up layer‐by‐layer process, which proceeded via the formation of a hybridized organo‐GO Langmuir film, followed by the intercalation of C2.1.360–graphene hybrid in which alkylated C60 molecules self‐organized into nanoscale flake‐like films on a CVD‐grown graphene layer.60–graphene hybrid devices exhibited a clear and highly reproducible change in conductance upon visible‐light illumination due to the hole generation in the alkylated C60 molecules and the transfer of the holes to the graphene, as deduced from the upshift in the Dirac point and the broadening of the resistance. Moreover, a hole‐doping effect was observed in the C60–graphene hybrid devices, with carrier mobility decreasing from 286 to 208 cm2 V−1 s−1 upon illumination with visible light.Different from the pristine fullerenes, fullerene derivatives bear versatile functional groups grafted via chemical functionalization, facilitating their assembly on different substrates, including graphene films. In 2012, Kim, Lee and co‐workers constructed a C60 derivative based on ‐phenyl‐C61‐butyric acid methyl ester (PCBM) to rGO via the noncovalent approach in which pyrene was used as an anchoring bridge to link the rGO and PCBM components  owing to the intramolecular π–π stacking interactions between the graphene sheet and pyrene–PCBM. Importantly, according to the proposed geometric configuration of rGO–pyrene–PCBM, the C60 moiety was far from the graphene sheet and was bridged to the graphene sheet via the pyrene anchor. The rGO–pyrene–PCBM hybrid was then applied as an electron‐extraction material in P3HT:PCBM bulk heterojunction polymer solar cell (BHJ‐PSC) devices . The configurations of the fullerene–graphene hybrids were studied by molecular mechanics/molecular dynamics (MD) calculations, predicting that fullerene derivatives favorably interacted with graphene through both the pyrene base and the C60 moiety  matrix for improved thermal and mechanical properties. By using GO as the starting material, they prepared the C60@graphene hybrid (C60: ≈47.9 wt% and graphene: ≈35.1 wt%) via a three‐step reaction, with highly active amino groups introduced to react with C60 and contribute to the subsequent in situ reactive compatibilization reaction to improve the dispersion of the hybrid. The incorporation of 2.0 wt% C60@graphene (relative to PP) remarkably increased the initial degradation temperature by ≈59 °C and simultaneously enhanced the tensile strength and Young's modulus by 67% and 76%. The improvement in the thermal stability of PP was speculated to be due to the free‐radical‐trapping effect of C60 and the thermal barrier effect of the graphene layers, while the mechanical enhancement was mainly attributed to the reinforcing effect of the graphene nanosheets and the effective load transfer from the polymer matrix to the graphene sheets.Guo and co‐workers developed a new strategy to synthesize a C2.1.460, endohedral fullerenes (EMFs) with metal ions or metallic clusters entrapped in the carbon cage have been attracting considerable attention during the last two decades due to the charge transfer from the internal species to the outer carbon cage.3N@hI‐C80 is so far the most abundant and has an excellent stability superseding other conventional EMFs.3N)6+@(C2n)6−, resulting from the six‐electron transfer from the endohedral species to the carbon cage, renders an enhanced chemical reactivity and adsorption energy on surfaces compared with that of hollow fullerenes.3N@C80 absorbed on pristine graphene nanoflakes (GNFs). The Sc3N@C80–graphene noncovalent hybrid model was constructed, for which the simulated graphene nanoflake, composed of 96 C and 24 H atoms (C96H24) with symmetric hexagonal units, served as a support for the deposition of fullerenes in the center.60, which adsorbed on GNFs with an energy of 0.76 eV, Sc3N@C80 adsorption on the GNF resulted in a higher binding energy of 1.00 eV for nearly degenerate isomers that have a pentagon and a hexagon facing the single‐layer graphene (SLG). Such a large binding energy was explained in terms of a higher dispersion interaction between Sc3N@C80 and the GNF, and the fact that charge separation in Sc3N@C80 favored binding with the GNF.Different from hollow fullerenes, such as C2.2TableAlthough the noncovalent approach has been extensively used to construct fullerene–graphene hybrids, being facile and effective in achieving improved properties relative to the single components of fullerene and graphene, the determination of their chemical structures is difficult, making the manipulation of their structures and properties unrealistic. Additionally, the noncovalent nature renders relatively weak intermolecular interactions between fullerenes and graphenes. Thus, the covalent hybridization of fullerene and graphene is highly desired to construct hybrids in a controllable manner.2.2.160–graphene, termed as periodic graphene nanobuds (PGNBs) in which C60 molecules covalently attach to a graphene monolayer and form a periodic lattice structure  hybrid, termed as NanoBuds, prepared in continuous aerosol  reactors by using particles grown in situ via ferrocene vapor decomposition or by using premade iron‐catalyst particles produced by a hot‐wire generator. The authors proposed that fullerenes were formed on the iron‐catalyst particles together with SWNTs during CO disproportionation.re Figure8.17 Si54 structure was proposed by removing a six‐membered ring from C60. Four different kinds of connection positions for the C54 structure on graphene were investigated. In two cases, where six carbon atoms of graphene formed covalent bonds with the fullerene, the graphene–fullerene nanobuds were ferromagnetic at their ground states with magnetic moments of nearly 6 μB; whereas, for the other two cases, nonmagnetic states were found since the connections of two atoms in different sublattices of graphene could not generate unpaired spins.Likewise, in 2011, Li et al. designed four kinds of all‐carbon graphene–fullerene nanobuds and studied their magnetic properties by spin‐polarized DFT.60 hybrid.60 onto the carbons at the edges of the defect did not have priority over attaching to a pristine graphene sheet because the distortion of the ideal π system in graphene perturbed the π–π stacking interactions with the fullerene and forced the fullerene to avoid the defective surface of graphene.Deymier et al. adopted the self‐consistent charge density functional tight‐binding (DFTB) and DFT methods to evaluate energy barriers for the formation of a single‐layer graphene−C180, C320, C540, and C720) as sandwich cores. According to their calculation results, dangling bonds on the reciprocal locations of both graphene and fullerenes promoted the formation of covalent bonds between the fullerene and graphene layers. Moreover, the thermodynamic stability of the fullerene–graphene hybrid was evaluated with potential energy profiles, and the total configuration energies for all systems were minimized and remained stable over a long period of time, suggesting good thermodynamic stability.In 2015, Kirca designed a novel fullerene–graphene hybrid composed of fullerene covalently sandwiched between parallel graphene sheets by using MD simulations.60 onto graphene nanosheets  was attributed to the improved electron transport because the introduction of the C60–graphene covalent hybrid could provide a direct conduction path for electron transport via percolation through the highly conducting 2D graphene sheets.The experimental synthesis of a fullerene–graphene covalent hybrid was not reported until 2011, when Dai et al. developed a simple lithiation method to covalently attach monosubstituted Cts Figure9A. In 60–graphene hybrid by ball milling in which C60 was directly bonded to the edges of graphene nanoplatelets via two C—C single bonds, as confirmed by high‐resolution transmission electron microscopy (HR‐TEM) , showing improved ORR electrocatalytic activity over the pristine graphite in terms of a higher limiting current (−3.4 mA cm−2) than those of the pristine graphite (−1.8 mA cm−2) and C60 (−2.2 mA cm−2). This was interpreted as the charge transfer from graphene to C60 enabling the graphene basal plane to be positively charged and thus to be more electrocatalytically active toward the ORR than the pristine graphite  Figure10A.104e Figure , I–IV.1060. After heating the pG/C60 mixture composed of a saturated solution of C60 in 1‐chloronaphthalene mixed with pG nanosheets at 300 °C for 2 h, C60 was impregnated into pG nanosheets with the evaporation of the solvent, affording the formation of the pG–C60 covalent hybrid , thermal gravimetric analysis (TGA), and HR‐TEM  onto the GO‐CCH scaffold ‐ethynylaniline via an aryl diazonium salt reaction, followed by cleavage of the trimethylsilyl groups using tetra‐ld Figure14.34 RFigure9 s−1 for C60•−, suggesting the efficient charge separation and formation of long‐lived charge separated species.Later, by using hydrolyzed PCBM as a fullerene precursor via a very mild process without the use of metal catalysts, Kumar et al. synthesized a novel fullerene–GO covalent hybrid (PCBGO) by grafting PCBM onto GO via flexible linkages Figure15.112 60 reacted with 2,5‐bis(trimethylsilyl)‐1,4‐phenylene bis(triflate) via a [2 + 2] cycloaddition yielding a C60‐aryne precursor  phthalocyanine (ZnPc), acting as electron donor, and fulleropyrrolidine, acting as electron acceptor, were covalently linked onto single‐layer graphene oxide (SLGO) simultaneously , as evidenced by the appearance of the cation (ZnPc•+) and anion (C60•−) radicals in the visible to near‐infrared (NIR) regions. In contrast, for the control samples, GO and binary hybrids ZnPc–GO and GO–C60, no cation and anion radicals were observed. Thus, the formation of the ternary hybrid is crucial for such a photoinduced electron transfer phenomenon.In addition to above‐mentioned binary fullerene–graphene hybrids, the inclusion of another functional material to form ternary hybrids with fullerene and graphene as building blocks has also been reported. In 2013, Souza and co‐workers reported a novel ternary covalent fullerene–graphene hybrid ZnPc–SLGO–Cly Figure17.19 T60 hybrid lubricating film by a multistep self‐assembly method on silicon surfaces.60, the GO films assembled on the silicon wafers were amine‐functionalized by (N‐[3‐(trimethoxysilyl)‐propyl]‐ethylenediamine) molecules, which contained primary and secondary amino groups capable of undergoing N—H addition reactions across the C=C bonds in C60. The successful grafting of C60 chemically to the amine‐functional GO by nucleophilic addition reactions was verified by FTIR, X‐ray powder diffraction (XRD), TGA, Raman spectroscopy and XPS. Colloidal probe measurements show that APTES‐a‐graphene–C60 exhibited a low nanofriction coefficient due to the existence of a more ordered C60 outer layer, which facilitated microsphere sliding because of the small contact area between the microspheres and films, in addition to the presence of the few‐layer graphene with excellent nanolubrication properties. Furthermore, the conclusion pointed out that the strong bonds, with stable and not easily damaged characteristics, between the carbon atoms of C60 facilitated the long‐lasting and effective lubrication.In 2014, Wang and co‐workers fabricated a graphene–C60 or graphene, rendering them stable in solution with the free movement of C60 coated on the graphene restricted.60 covalent hybrid incorporating folic acid (FA) and polyethylene glycol (PEG) onto GO via an imide linkage  effect of 44.4% compared with that for the Au nanoparticles of 30%. Furthermore, when the FA–GO–PEG/C60 is taken by tumor cells, it caused heating and significant intracellular reactive oxygen species (ROS) production under light irradiation, leading to a marked decrease in cell survival and to the elevation of oxidative stress  efficacy against cancer cells. FA–GO–PEG/C60 showed a combined PDT and PTT treatment with remarkably improved and synergistic effects compared to that of PTT or PDT alone, having potential for a synergistic phototherapy of cancer.Graphenes have been reported to act as an excellent drug carrier and photothermal agent, which can efficiently convert NIR light into heat and thus induce hyperthermia to cells and surrounding tissues.ge Figure18A. Th33N4 is the most stable allotrope of carbon nitrides under ambient conditions with the 2D, π‐conjugated plane of polymeric tri‐s‐triazine connected via tertiary amines  and the existence of contact resistance between the nanosheets.3N4 needs to be improved so as to extend its photocatalytic applications.g‐Ces Figure19.115 60, which is beneficial for a rapid photoinduced charge transfer with donor molecules and a relatively slow charge recombination, hybridizing g‐C3N4 with C60 has been implemented, and the effectiveness of such a strategy in improving the photocatalytic activity of g‐C3N4 has been examined. In 2014, Chai et al. prepared a g‐C3N4/C60 composite by simply blending g‐C3N4 powder into a C60 solution in toluene.3N4/C60 composites were characterized by XRD, scanning electron microscope (SEM), TEM, and FTIR. The photocatalytic activity of the g‐C3N4/C60 composite in the degradation of rhodamine B was further evaluated, indicating that the g‐C3N4/C60 composite with the optimal amount of C60 of 1 wt% had the highest photocatalytic performance. The improved photocatalytic performance of the g‐C3N4/C60 composite was attributed to the synergetic effect of g‐C3N4 and C60, which facilitated the separation of photogenerated electrons and holes.Considering the excellent electron‐accepting ability of C60/g‐C3N4 composite was prepared by Zhu et al. via the thermal treatment of dicyandiamide, a precursor for synthesis of g‐C3N4, at 550 °C in atmosphere in the presence of C60.60 into a matrix of g‐C3N4, the valance band of g‐C3N4 shifted to a lower energy position, affording a strong photooxidation capability under visible light. Under the optimal loading amount of C60 of 0.03%, the photocatalytic activity of the C60/g‐C3N4 composite for the degradation of phenol and methylene blue under visible light was found to be ≈2.9 and 3.2 times higher than that of pristine g‐C3N4. The enhanced photocatalytic activity of the C60/g‐C3N4 composite was attributed to the promoted generation of holes, and •OH originated from the strong interaction of the conjugated π‐bond between C60 and g‐C3N4, leading to a rapid photogenerated electron transfer rate and high charge separation efficiency of C60 in the C60/g‐C3N4 composite  remained at the interface between them. Interestingly, for different stacking patterns, g‐C3N4 and C60 were always linked by vdW forces and formed type‐II heterojunctions in most cases. The valence band maximum and conduction band minimum of these heterostructures were dominated by the unsaturated nitrogen (N2) atoms and C60 molecules, respectively, which strongly interacted with each other and resulted in a strong charge transfer between g‐C3N4 and C60 and a clear bending of the g‐C3N4 sheets. The unsaturated N2 atoms included at the interfaces have a significant influence on promoting the photocatalytic performance, while the existence of saturated nitrogen (N1 and N3) atoms lying at the interfaces would weaken the interfacial interactions between C60 molecules and the g‐C3N4 monolayers.A fundamental understanding of the corresponding mechanism behind the greatly enhanced photocatalytic activity of g‐C3N4 before and after C60 adsorption by using a larger model with 25 different adsorption configurations.60 preferred to stay upon the “junction nitrogen” with the carbon atom of fullerene being nearest to the monolayer. The adsorption of fullerene resulted in a change in the geometrical morphology of g‐C3N4 from flat to wrinkled via an irreversible process. The wrinkled structure was more stable by ≈5.6 eV compared with that of the flat one. The shortest distance between g‐C3N4 and C60 for all configurations was ≈3 Å, indicating the existence of vdW interactions. Furthermore, the analysis of the electronic properties indicated that the adsorption of C60 onto g‐C3N4 did not result in efficient carrier separation at interface. In fact, C60 in these systems mainly induced the geometrical distortion of g‐C3N4 monolayers, thus leading to the improved photocatalytic performance.Dai et al. reported a systematic theoretical study on the changes in the structure, stability, electronic properties, and light absorption of g‐C60/g‐C3N4 and C70/g‐C3N4 hybrids via a hydrothermal process.3N4. The photocatalytic disinfection activity of these hybrids on the inactivation of Escherichia coli (E. coli) O157:H7 showed that the bacterial cell could be damaged by the active oxygen radicals (•O2− and •OH) produced from the fullerene/g‐C3N4 hybrids under visible‐light irradiation.70/g‐C3N4 composite exhibited a higher photocatalytic inactivation activity for completely inactivating all E. coli O157:H7 cells within 4 h under visible‐light irradiation. The high catalytic performance was attributed to the effective transfer of the photoinduced electrons from the fullerene/g‐C3N4 hybrids under visible‐light irradiation.In 2017, Cai et al. synthesized C3N4 hybrids are based on noncovalent hybridization, for which C60 was blended physically into the pristine g‐C3N4 or its precursor in a low blending ratio (<2.0 wt%), and a large number of unreacted C60 are retained in the composites. On the other hand, the photocatalytic activity of the C60/g‐C3N4 composite was examined only via the degradation of dyes. These limitations were not broken until the recent success of our group in synthesizing the first fullerene–g‐C3N4 covalent hybrid via a facile solid‐state mechanochemical route by ball‐milling g‐C3N4 and C60 in the presence of LiOH as a catalyst . Under the optimized conditions, the g‐C3N4/C60‐12 wt% hybrid exhibited the highest H2 production rate of 266 µmol h−1 g−1, which was ≈4.0 times higher than that for the pristine g‐C3N4 photocatalyst )  to g‐C3N4 due to the lowering of the conduction band of g‐C3N4 and the inhibited recombination of photoinduced electron–hole pairs owing to the fast photoinduced electron transfer process from g‐C3N4 to C60  is the most representative TMD, consisting of hexagonal rings with Mo and S atoms alternately located at the hexagon corners. Monolayer MoS2 has the direct band gap of ≈1.8 eV and a high in‐plane carrier mobility, enabling broad applications in electro and photocatalysts, photovoltaics and photoelectric devices.As another type of emerging inorganic 2D semiconducting nanomaterials, TMDs have been attracting widespread attention due to their intriguing electronic, optical, and mechanical properties. Molybdenum disulfide  method  and RESET (−5.5 V) voltages and a high ON/OFF resistance ratio (≈4 × 103)  with exposed catalytically active edge sites, yielding a 3D architecture with a high surface‐to‐volume ratio beneficial to electrocatalytic applications.2 nanosheets were interfaced with nC60 clusters by a facile solution‐deposition method via immersing the MoS2/CFP samples into a C60 solution in chlorobenzene, and the nC60/MoS2 hybrid showed greatly improved hydrogen evolution reaction activity with an overpotential value of 172 mV at 10 mA cm−2, a Tafel slope of 60 mV dec−1, and a turnover frequency value of 2.33 H2/s per active site at −0.2 V versus RHE at a C60 deposition concentration of 0.5 mg mL−1. The improved activity of the nC60/MoS2 hybrid catalyst was explained by the interfacial doping of MoS2 induced by charge transfer from the nC60 clusters to MoS2, resulting in the relative positioning of their valence and conduction band edges.In 2016, Fang, Banerjee and co‐workers reported a stepwise sulfidation approach for the direct integration of vertically aligned high edge‐density MoS2–C60 hybrid onto a semiconducting SnO2 electrode via a two‐step methodology.2 and C60 yielded MoS2–C60 hybrid aggregates via self‐assembly, followed by electrophoretic deposition onto the SnO2 electrode in the second step. The time‐resolved microwave conductivity measurements for the MoS2–C60 hybrid revealed substantially increased transient conductivities under photoexcitation in comparison with the single components of MoS2 and C60, indicating that the nanosized heterojunction structure of MoS2 and C60 promoted charge separation. Furthermore, the C60 molecules on the surface of MoS2 effectively hindered the undesirable charge combination between an electron in the electrode and a hole in the MoS2 nanosheets. Moreover, the authors fabricated a WS2–C60 hybrid for comparison and found that, despite a more suppressed charge recombination in WS2–C60 than in MoS2–C60, the incident photon‐to‐current efficiency value of the device with WS2–C60 was smaller than that with MoS2–C60 owing to its inhomogeneous film structure.In 2018, Imahori et al. fabricated a MoS60 on WSe2, which served as a weakly interacting organic/2D vdW heterostructure system.60 molecules formed a monolayer that extended uniformly over WSe2 with large grain sizes (≈5 µm). The interplay and balance between the adsorbate–adsorbate and adsorbate–substrate interactions resulted in the formation of rotational arrays of self‐assembled 2 × 2 molecules. According to vdW‐corrected ab initio DFT simulations, the C60 molecules tended to be electronically coupled with a long‐range orientational ordering, as reflected in the high crystallinity of C60 on WSe2. Meanwhile, the electronic structure of the hybrid system showed a spatial delocalization of the molecular orbitals throughout the 2 × 2 superlattice.To thoroughly understand the basic physical and chemical phenomena that rule highly crystalline architectures comprised of crystals of organic molecules and 2D materials, Wang and co‐workers fulfilled the growth of high‐quality self‐assembled monolayers of C2–fullerene hybrid can promote the photocatalytic H2 production activity of MoS2 remains intriguing. In 2016, Huang et al. carried out first‐principles calculations based on density functional theory to investigate the effects of vdW interactions on changes in the electronic structure, charge transfer and photoactivity of three typical monolayer MoS2/fullerene  hybrids. For the MoS2/fullerene vdW heterostructures, strong noncovalent interactions between the MoS2 and fullerene were concluded. The band gap of the MoS2/fullerene vdW heterostructure was smaller than that of monolayer MoS2, and thus, the visible‐light absorption and photoinduced electron transfer can be enhanced. Furthermore, the amount of charge transfer at interface induced by vdW interactions was found to be dependent on the size of the fullerene.Whether the formation of a MoS2–C60 hybrid featuring a vdW heterostructure, as well as its application in photocatalytic H2 production. The MoS2–C60 hybrid was prepared via a facile and ecofriendly solid‐state mechanochemical route by ball‐milling a mixture of bulk MoS2 and C60. In the process of ball‐milling, the bulk MoS2 was exfoliated to few‐layer MoS2 nanosheets, and the basal plane was reduced into significantly smaller nanosheets; while the C60 molecules bonded to the edge of the cut MoS2 nanosheets in the form of vdW heterostructures  was revealed  in the raw mixture, the MoS2–C60 hybrid containing 2.8 wt% C60 exhibited a visible light photocatalytic H2 production rate of 6.89 mmol h−1 g−1 in the presence of the photosensitizer EY. This value was 9.5 times higher than that of the obtained MoS2 nanosheets. This result was because the C60 bonding resulted in the fast photoinduced electron transfer from MoS2 to C60, and this action was beneficial for the separation of electron–hole pairs by inhibiting their recombination.Experimentally, our group recently reported the first MoSes Figure24.67 U60/MoS2 and C60/WS2 hybrids were also studied theoretically by Cheng and co‐workers.60/MoS2, C60/WS2, C59B/MoS2, C59B/WS2, C59N/MoS2, and C59N/WS2, C60 preferred to sit at the S‐top site with a hexagon parallel to the monolayer. All of the interfacial interactions were found to be weak, such that the main electronic features of the components were preserved. Although both the C60/MoS2 and C60/WS2 systems behaved as type‐II heterojunctions, the built‐in potential in the C60/MoS2 hybrid was too small to achieve an efficient electron–hole separation, whereas the latter system had a large built‐in potential and was thus predicted to yield an excellent photovoltaic performance.The photovoltaic applications of C55.12‐hybridized wide band‐gap semiconductor with a structure analogous to graphene and belonging to a hexagonal system with sublattices occupied by equal numbers of boron and nitrogen atoms alternatingly arranged in a honeycomb configuration. Similar to graphene, the different h‐BN planes are dominated by weak van der Waals interactions, while the in‐plane nitrogen and boron atoms are combined by sp2 orbitals to form strong σ‐bonds, which are highly polarized because of the high asymmetry of the sublattices, resulting in the large band gap (5–6 eV) of h‐BN.2D h‐BN is a sp60 has a narrower band gap, hybridizing h‐BN with C60 has been regarded as an effective strategy for modulating the electronic properties of h‐BN. In 2005, Greber and co‐workers grew a C60 monolayer film on one monolayer of h‐BN on Ni(111), providing an atomically flat metal‐insulator interface. They observed that, between 150 and 250 K, the work function of C60 decreased, while the binding energy of the HOMO increased by ≈100 meV along with a strong charge in the LUMO of C60 by 0.4 ± 0.1 electrons. Such a charge redistribution was induced by the onset of a molecular rocking motion, and the magnitude of the charge transfer was an indication of strong electron–phonon interactions.Given that C5.22 V−1 s−1, makes BP an emerging member of 2D nanomaterials and renders its potential for application in transistors, biomedicine, and energy conversion and storage.3‐orbital hybridization, as well as the armchair and zigzag‐configurations along the x and y‐axial directions, respectively.BP with a century‐long history did not draw significant attention until the recent recognition of its anisotropic 2D layered structure.60 composite via a mechanochemical ball‐milling route in a comparative study with a BP–graphite composite synthesized under the same conditions. Prior to blending with BP, C60 or graphite was ball‐milled. Then, the mixture of BP and C60 (graphite) was sealed in an argon‐filled glove box, and high‐energy mechanical milling was performed under a 1.2 MPa Ar atmosphere with a rotation rate of 700 rpm for 12 h. According to their detailed characterizations of the BP–graphite composite, phosphorus–carbon bonds formed after ball‐milling and remained stable during lithium insertion/extraction. As a result, an excellent electrical connection between BP and the carbon material was established, contributing to a high initial discharge capacity of 2786 mAh g−1 at 0.2 C and an excellent cycle life of 100 cycles with 80% capacity retention. For comparison, within the BP–C60 composite, the content of the as‐formed phosphorus–carbon bonds was significantly lower than that for the BP–graphite composite, resulting in an inferior capability. Hence, the authors concluded that the carbon structures played essential roles in the formation of stable phosphorus–carbon bonds.Despite extensive reports on hybrids of BP with other 2D nanomaterials, especially graphenes, studies on hybrids of BP and fullerene have rarely been reported. In 2014, Cui and co‐workers made an attempt to synthesize a BP–C660 as the first fullerene in 1985 marks a milestone in nanoscience due to its unique spherical molecular structure merely composed of sp2‐hybridized carbon atoms. As the only soluble species among all known nanocarbons derived from the 0D structure, fullerenes have been popularly used as important building blocks useful for constructing versatile noncovalent/covalent nanohybrids with a variety of functional materials, including 2D nanomaterials, as elaborately reviewed in this paper.The discovery of C3N4, TMDs, h‐BN, and BP, which all exhibit similar electronic properties. Meanwhile, their intrinsic differences in lattice composition and interlayer distance bring about a clear diversity in specific functionalities and applications, greatly accelerating the field of 2D nanomaterials. Despite the tremendous progress in investigating the 2D nanomaterials themselves during the past decade, enhancing the existing properties for better performance and even exploring unknown functionalities toward extended applications are definitely necessary. To this end, hybridizing 2D nanomaterials with 0D fullerenes has been well established as a successful and powerful protocol based on the detailed analyses of versatile fullerene‐2D nanomaterial hybrid systems in this Review.In 2004, the paradigm‐shifting disclosure of graphene as the first and most representative 2D layered nanomaterial opened up a new era of nanoscience because of the material's exceptionally large specific surface area and high carrier mobility, which are achievable only in the 2D layered structure. The exceptional structure and property of graphene soon stimulated the recognition of other graphene‐analogous 2D nanomaterials, such as g‐CThere are yet considerable challenges in the emerging field of fullerene‐2D nanomaterial hybrids. First, most fullerene‐2D nanomaterial hybrids that have been reported so far were synthesized in the noncovalent style via the simple blending of two components, whereas only limited reports involved the successful construction of fullerene‐2D nanomaterial covalent hybrids, which are more promising in terms of strong intermolecular interactions between fullerene and 2D nanomaterial moieties. Clearly, the natural divergence in dimensions between 0D fullerene and 2D nanomaterials makes their covalent connection difficult. Therefore, prior to hybridization, chemically functionalizing the components of fullerene and/or the 2D nanomaterial appears critical. While the chemical derivatization of fullerenes has been well developed, chemical derivatizations of 2D nanomaterials are still in their infancy, and the development of novel derivatization approaches, especially for such less studied 2D nanomaterials as h‐BN and BP, is highly desired.Another pending problem is how to determine precisely the chemical structures of fullerene‐2D nanomaterial hybrids at the molecular level. In particular, for fullerene‐2D nanomaterial covalent hybrids, the additional pattern of covalent bonding between fullerene and the 2D nanomaterial, i.e., single‐bond addition or cycloaddition onto the fullerene cage, is essential for the disruption of the π‐conjugated system of the fullerene cage. Furthermore, since the properties of 2D nanomaterials are typically anisotropic, the addition site (surface vs edge) where fullerene bonds onto a 2D nanomaterial strongly affects the difficulty of the hybridization and the modulation extent of the 2D nanomaterial within the fullerene‐2D nanomaterial hybrid. For the latter point, the direct visualization of the fullerene addition sites within the hybrid is undoubtedly needed, and thus, further improvement of the resolution of HR‐TEM/scanning tunneling microscope (STM) for clearer morphological characterizations of the geometric structure of the hybrids is of high importance.As mentioned above, hybridizing 2D nanomaterials with fullerenes may not only fulfill the modulation of the physical/chemical properties of 2D nanomaterials but also induce new properties in the 2D nanomaterials, thus significantly extending the functionalities and applications of 2D nanomaterials. An intriguing question is whether the hybridization strategy can be used to further create unknown functional materials. For instance, on the basis of binary fullerene‐2D nanomaterial hybrids, by involving one or two other functional materials, which are either another 2D nanomaterial or even a 1D nanomaterial (such as a carbon nanotube or boron‐nitride nanotube), ternary or even quaternary hybrids are expected to form. However, due to a lack of experimental reports, whether the properties of such hybrids can surpass their binary counterparts is unknown. With the foreseeable development of chemical derivatizations of 2D nanomaterials, the syntheses of such unknown complex hybrids will likely come true in the near future, making the emerging fullerene‐2D nanomaterial hybrids even more exciting and promising for vast applications.The authors declare no conflict of interest.


Rationale: Emerging evidences have highlighted the critical roles of lncRNAs in human cancer development. The work sought to assess the biological role and potential underlying mechanisms of lncRNA-CF129 (CF129) which is significantly reduced in pancreatic cancer (PC).Methods: CF129 expression and its association with multiple clinicopathologic characteristics in PC specimens were analyzed. The role of CF129 both in vitro and in vivo was assessed, with RNA pull-down and immunoprecipitation assays being performed to detect the interaction between CF129 and p53 and E3 ligase MKRN1. Chromatin immunoprecipitation and luciferase assays were utilized to identify the interaction between p53 and FOXC2 promoter, HIF-1α/HDAC1 complex and CF129 promoter, FOXC2 and HIF-1α promoter, respectively.Results: CF129 levels were markedly lower in PC compared with paired non-tumor adjacent tissues. Low CF129 expression predicted short overall survival in PC patients. CF129 inhibited invasion and metastasis of PC cells in a FOXC2-dependent manner. In addition, CF129 regulates FOXC2 transcription through association with mutant p53. CF129 directly binds to p53 and E3 ligase MKRN1, and such an interaction leading to p53 protein ubiquitination and degradation. Furthermore, CF129 is a hypoxia-responsive lncRNA, which is transcriptionally downregulated by binding between HIF-1α/HDAC1 complex and CF129 promoter. Finally, it is revealed that HIF-1α is reciprocally regulated by FOXC2 in transcriptional level. Clinically, CF129 downregulation coordinates overexpression of FOXC2.Conclusions: Our study suggests that CF129 inhibits pancreatic cell proliferation and invasion by suppression of FOXC2 transcription, which depends on MKRN1-mediated ubiquitin-dependent p53 degradation. The HIF-1α/CF129/ p53/FOXC2 axis may function as a potential biomarker and therapeutic target. Pancreatic cancer is among the deadliest malignancies, with a mortality rate that ranks among the top four in worldwide. Unfortunately, there is currently no specific marker for early pancreatic cancer diagnosis, and as a result it is often diagnosed in advanced stages when there is already metastatic invasion and no opportunity for radical operation. Nevertheless, pancreatic cancer is resistance to chemotherapy and radiotherapy, which exaggerates the miserable prognosis. Because of complexities of signaling at genetic, epigenetic, and metabolic levels, the therapeutic options and progress in drug development are impeded in pancreatic cancers Long noncoding RNAs (lncRNAs) are RNAs that are over 200 nucleotides long and do not code for protein, even though they play essential roles in many biological contexts including epigenetic regulation, cell cycle control, nuclear import, differentiation, and other pathways in vivo and in vitro. Mechanical experiments further indicated that CF129 promotes an interaction of p53 and E3 ligase MKRN1, which consequently induces the ubiquitination and degradation of p53 protein in pancreatic cancers. We further revealed p53 promoted FOXC2 transcription, thereby, CF129 repressed FOXC2 expression through inducing p53 instability. Specifically, we showed CF129 was inhibited by the complex of HIF-1α and HDAC1 during hypoxia. In the meantime, FOXC2 regulates transcription of HIF-1α, by which CF129 can indirectly inhibit HIF-1α expression. Therefore, the present study demonstrates a lncRNA-interacted feedback of HIF-1α and FOXC2 which is involved in pancreatic cancer progression during hypoxia microenvironment.Many lncRNAs dysregulated in pancreatic cancer have been previously identified through microarray assay Figure ). Moreover, the downregulation of CF129 was further verified in 40 cases of PC tissues relative to paired NP tissues . Compared to immortalized human pancreatic duct cells (HPDC), CF129 was downregulated in most of the cell lines and PANC-1 and BxPC-3 cells were the lowest . Meanwhile, the secondary structure of CF129 is predicted by online database AnnoLnc Figure ). In addition, the protein-coding capacity of CF129 was evaluated by online prediction software, which validated CF129 as a non-coding RNA . Furthermore, results from nuclear/cytoplasmic RNA fractionation revealed that CF129 mainly presents in the nucleus . This result was further confirmed via fluorescence in situ hybridization (FISH) using a CF129 probe . We next evaluated the relationship between CF129 expression and the clinicopathologic characteristics of PC patients. The correlation analysis showed that low expression of CF129 was associated with large tumor size, poor tumor differentiation, lymphatic invasion, distant metastasis (Supplementary Table 1), and shorter overall survival . This suggested CF129 might be involved in PC development.Differential lncRNA expression between pancreatic cancer (PC) and paired noncancerous peritumoral (NP) tissues determined via microarray, revealing lncRNA-CF129 (CF129) to be among the most significantly decreased lncRNAs in PC samples , the PANC-1/BxPC-3 proliferation and colony formation were remarkably suppressed . Additionally, both the wound-healing and transwell assays demonstrated suppression of migration and invasion of PC cells due to CF129 overexpression .Since CF129 was downregulated in PC tissues, we explored its biology by overexpression of CF129. After transfection with CF129-containing vector (CF129-OE) . In addition, the number of mice with liver metastases and the visible metastasis loci in the LV-CF129 group was apparently less than those in the control group .To validate these findings, PANC-1 cells were transfected stably using lentivirus containing negative control (LV-NC) or CF129 sequences (LV-CF129) and further implanted into nude mice to construct xenograft tumor model. Relative to LV-NC mice, the tumors in LV-CF129 mice were smaller and weighed less . Contrary to CF129 overexpression, downregulation of CF129 remarkedly promoted proliferation, colony formation of PANC-1/BxPC-3 cells . In addition, migration and invasion were also significantly increased following transfection with siCF129 .To further elaborate impacts of CF129 on pancreatic cancer cells, three siRNAs targeting CF129 were designed and siCF129-1 and siCF129-2 were used for experiments , we further explore whether CF129 could regulate expression of FOXF1, FOXC2 or FOXL1. Interestingly, CF129 overexpression or knockdown dramatically inhibited or increased expression of FOXC2 mRNA, but not expression of FOXF1 and FOXL1 mRNA . Moreover, the luciferase reporter assay showed that siCF129 obviously increased the luciferase intensity of PANC-1 cells which were transfected with vector containing FOXC2 promoter . Coincidently, expression level of FOXC2 protein was significantly increased by siCF129 but decreased with CF129 overexpression . As that of CF129 overexpression, FOXC2 knockdown with siFOXC2 dramatically suppressed proliferation and invasion of PANC-1 and BxPC-3 cells . Furthermore, the co-transfection with FOXC2-OE effectively rescued FOXC2 expression, proliferation, and invasion of PANC-1 cells which were inhibited by CF129 overexpression . On the contrary, siFOXC2 dramatically depleted FOXC2 expression, proliferation and invasion of PANC-1 cells which were induced by siCF129 . These results intensively suggest that CF129 exerts function in pancreatic cancer cells by repressing FOXC2 expression in transcriptional level.Researches had demonstrated that lncRNAs regulate expression of neighborhood gene in cis. Since CF129 is adjacent to forkhead family of transcription factors including FOXF1, FOXC2 and FOXL1 . Moreover, the results of Jaspar revealed 3 putative binding sits for p53 in the FOXC2 promoter region . Meanwhile, knockdown of p53 dramatically inhibited FOXC2 expression both in mRNA and protein level . Therefore, we assumed that p53 may act as transcription factor to mediate the regulation of CF129 on FOXC2 expression. To further identify the association of CF129 and p53, we performed RNA pulldown and RNA immunoprecipitation (RIP) assays. The western blot results showed that p53 protein was accumulated by CF129 but not by antisense transcript . The qPCR results confirmed that CF129 was significantly enriched by anti-p53 antibody but not IgG . In addition, deletion-mapping analyses identified that the 195nt region at the 5' end of CF129 is required for its association with p53 . In addition, the ChIP assay verified the association of p53 and the chromatin fragments corresponding to the site P2/P3, rather than the site P1 within the FOXC2 promoter region . Furthermore, we used luciferase reporter assays to validate whether p53 activating transcription of FOXC2 promoter. FOXC2 promoter sequence containing wild-type (WT) or mutant-type P2/P3 (MUT) was inserted in the luciferase reporter plasmid promoter. We found that luciferase activity from the WT, but not the MUT, was markedly decreased by p53 knockdown .lncRNAs have recently been found to exert effects in part via interacting with transcription factors. To predict the putative transcription factor involved in regulation of CF129 on FOXC2, an intersection analysis of online database catRAPID and Jaspar was applied. The catRAPID analysis showed high affinity between CF129 transcript and p53 protein . Coincidently, the luciferase activity of WT was decreased by CF129 overexpression, but increased by CF129 downregulation . Coincidently, the siCF129- induced upregulation of FOXC2 was inhibited by the knockdown of p53 in BxPC-3 cells . Importantly, the enhanced proliferation and invasion abilities of CF129-knockdown BxPC-3 cells were reversed by co-transfection with sip53 . Since both BxPC-3 and PANC-1 cell lines are p53 mutant phenotype Moreover, the CF129 overexpression markedly decreased, while the CF129 knockdown increased the enrichment of p53 on FOXC2 promoter , while CF129 knockdown enforced expression of p53 protein . Nevertheless, neither siCF129 nor CF129-OE demonstrated obvious effects on mRNA expression of p53 . To further reveal whether CF129 regulated p53 stability, p53 protein was measured in cycloheximide (CHX) treated cells, to prevent protein synthesis. The results showed that p53 protein in pancreatic cancer cells was destabilized with CF129 overexpression , while knockdown of CF129 increased the stability of p53 protein .Researches had reported that lncRNA regulate protein degradation and thereby plays a critical role in tumorigenicity Figure ) or downregulation on p53 expression . Given that p53 is regulated by the ubiquitin-proteasome pathway Figure ), while knockdown of CF129 decreased ubiquitination of p53 , respectively. Together, these data suggest that CF129 inhibits p53 protein expression in post- transcriptional level by promoting ubiquitin- dependent degradation.Moreover, the proteasome inhibitor MG132 could abolish the regulation of CF129 overexpression . It has been reported that MKRN1 induces the ubiquitination and proteasomal degradation of p53 Figure ). A significant upregulation of p53 protein upon MKRN1 knockdown was detected in both PANC-1 and BxPC-3 cells . Based on the findings, we presumed that MKRN1 might be involved in the process about CF129- induced degradation of p53. RNA pulldown assays using lysates from PANC-1 cells confirmed the association between CF129 and MKRN1 . The association of CF129 and MKRN1 was further confirmed by RIP analysis with anti-MKRN1 antibody . Deletion-mapping analyses identified that the segment (5' 246-377nt 3') of CF129 were required for the association with MKRN1 . Coincidently, MKRN1 knockdown abrogated the decrease of p53 protein caused by the CF129 overexpression in PANC-1 and BxPC-3 cells . Vice versa, knockdown of CF129-induced p53 expression was rescued by overexpression of MKRN1 . We then further evaluated whether CF129 modulated the association between MKRN1 and p53. Co-IP assays revealed that overexpression of CF129 promoted the interactions between MKRN1 and p53 . On the contrary, the interaction between MKRN1 and p53 was reduced upon the CF129 knockdown . In addition, MKRN1 knockdown dramatically decreased p53 ubiquitination, which was increased after co-transfected with CF129 . In contrast, MKRN1-enhanced ubiquitination of p53 was reduced by CF129 knockdown . Interestingly, the knockdown of MKRN1 did not affect the interaction between CF129 and p53, whereas the knockdown of p53 also did not affect the association between CF129 and MKRN1 .We further explore the precise mechanism about how CF129 regulates the ubiquitin-dependent degradation of p53 protein. Interestingly, depending on the online prediction from catRAPID, Makorin Ring Finger Protein 1 (MKRN1), a E3 ligase, might be associated with CF129 , we next explored whether CF129 was transcriptionally regulated by HIF-1α during hypoxia. Coincidently, CF129 were significantly downregulated with treatment of hypoxia  or COCl2  in a time-dependent manner. Moreover, both qRT-PCR and FISH assays demonstrated that HIF-1α knockdown obviously increased CF129 expression which was repressed by the hypoxia  or COCl2 treatment . Coincidently, the ChIP assays validated an association between CF129 promoter and HIF-1α was enhanced by hypoxia or COCl2 but suppressed after transfection with siHIF-1α . Additionally, luciferase reporter plasmid containing CF129 promoter  or HRE-mutant CF129 promoter  was transfected into PANC-1 and BxPC-3 cells. The results verified the siHIF-1α enhanced luciferase intensity which was inhibited by hypoxia  or COCl2 treatment .Hypoxia is a common feature of solid tumors and plays key roles in tumor development. Recent results had shown lncRNAs were aberrantly expressed during hypoxia Figure ) or COCl2 . Since CF129 is a hypoxia-responsive lncRNA, we further identified the effects of CF129 on growth of PC cells during different oxygen concentration. Similar to 5% O2 , the growth of PANC-1 cells were significantly promoted by CF129 depletion but inhibited by CF129 overexpression during both 1% O2 and 0.5% O2 . Therefore, these data intensively indicate CF129 is a hypoxia-responsive lncRNA in pancreatic cancer.The expression of p53 and FOXC2 was further evaluated to validate the critical roles of CF129 during hypoxia. The western blot results showed that both siHIF-1α and CF129 overexpression could reverse p53 and FOXC2 expression which was induced by hypoxia . PANC-1/BxPC-3 CF129 expression during hypoxia was obviously upregulated by treatment of HDAC1 knockdown or deacetylase inhibitor trichostatin A (TSA) . Meantime, the ChIP assay displayed the hypoxia-enhanced binding of HDAC1 with HRE of CF129 promoter was significantly downregulated by siHDAC1 but not TSA . Nevertheless, the hypoxia-inhibited luciferase density of WT reporter was upregulated by siHDAC1 or TSA . Coincidently, we also revealed that hypoxia-induced p53 and FOXC2 was markedly repressed by siHDAC1 or TSA . Therefore, these results indicate that the downregulation of CF129 is regulated via the HIF-1α/HDAC1 complex in hypoxic contexts.Research has revealed that HDACs are involved in downregulation of noncoding RNA including lncRNA and microRNA under hypoxic condition Figure ), which implied HIF-1α might be regulated by FOXC2 in transcriptional level. As expected, both HIF-1α mRNA and protein expression was significantly reduced after knockdown of FOXC2  but increased by overexpression of FOXC2 . Coincidently, the ChIP assay demonstrated FOXC2 antibody induced obvious accumulation of HIF-1α promoter . To further validate whether FOXC2 binding on the HIF-1α promoter was functional, HIF-1α promoter containing wild-type (WT) or mutant (MUT) FOXC2-binding sequence was inserted into the luciferase reporter plasmid. Results showed the luciferase density of the WT reporter, but not the MUT reporter, was markedly induced by FOXC2 overexpression .Based on that FOXC2 is a transcriptional factor, DNA sequence analysis showed a putative HRE (-1649 to -1639nt) in the HIF-1α promoter region . Moreover, the ChIP and luciferase reporter assay demonstrated that CF129 overexpression significantly inhibited the accumulation and activation of CF129 on FOXC2 promoter . Meantime, the hypoxia- induced HIF-1α was dramatically attenuated by CF129 but rescued by the FOXC2 overexpression . Vice versa, the siCF129 markedly compensated the HIF-1α expression which was inhibited by siFOXC2 during hypoxia .Since FOXC2 is repressed by CF129, we further presumed whether HIF-1α can be reciprocally regulated by CF129. Results showed CF129 knockdown increased, while CF129 overexpression decreased both mRNA and protein level of HIF-1α in PANC-1/BxPC-3 cells . Moreover, the publicly available databases for pancreatic cancer  also displayed that FOXC2 was significantly higher in human PC tissues than that of NP tissues . Furthermore, a significant inverse correlation was observed between CF129 and FOXC2 transcript levels in 40 pancreatic cancer specimens  . Moreover, the online database (ChIPbase) demonstrated an obvious correlation between HIF-1α and FOXC2 mRNA expression in pancreatic adenocarcinoma (PDDA) . Kaplan-Meier and log-rank test analyses showed that pancreatic cancer patients with high FOXC2 expression had a shorter overall survival time than those patients with low FOXC2 expression . Based on our results, we depicted a schematic diagram to depict that CF129 played an important role in regulation of HIF-1α/FOXC2 pathway during hypoxia stress .To further assess the regulation of CF129 on FOXC2 expression, we measured FOXC2 mRNA level in the same cohort of the 40 pancreatic cancer tissues. The FOXC2 level was significantly higher in PC tissues than that in (NP) tissues  including FOXF1, FOXC2 and FOXL1, we further investigated whether CF129 exerts its function by regulating these FOX proteins expression in cis. Interestingly, knockdown or overexpression of CF129 obviously decreased or increased FOXC2 expression, without effects on FOXF1 and FOXL1 expression. FOXC2 has been reported to be overexpressed in numerous cancers and function as a central regulator for epithelia-mesenchymal transition (EMT) and metastasis, including breast cancer, lung cancer, colorectal cancer, et alMany studies have found that lncRNAs can exert their effects in part via interactions with specific proteins. Therefore, we further applied bioinformation analysis to define the putative protein which might be involved in the regulation of CF129-FOXC2 pathway. The intersecting analysis of catRAPID and ChIPBase demonstrated that p53 protein might be recruited by both CF129 transcript and FOXC2 promoter. Coincidently, the RIP and RNA-pull down assays revealed that CF129 can bind to p53. Moreover, the ChIP and luciferase reporter assays verified the accumulation and activation of p53 on FOXC2 promoter, which was impeded by CF129 overexpression. In addition, p53 knockdown obviously reduced FOXC2 expression which was inhibited by CF129. Therefore, these results indicate that p53 is required for the regulation of CF129 on FOXC2. The accumulation of mutant p53 protein inactivates its anti-proliferative properties but promote tumorigenesis and invasion through a gain-of-function activity The stability of p53 is carefully regulated via the ubiquitin-proteasome system, allowing levels of this protein to rise and fall in response to stressful conditions. lncRNAs are also known to play a role in regulating p53 Base on bioinformatic prediction from online database catRAPID, we revealed an association between CF129 and Makorin Ring Finger Protein 1 (MKRN1). MKRN1 is an E3 ligase for hTERT Hypoxia is common in solid tumors including those in pancreatic cancer, and it can modulate tumor progression as well as therapeutic responses Given the HIF-1α promoter containing potential binding sites for FOXC2, we further assumed whether HIF-1α can be reciprocally regulated by FOXC2. In agreement, our data showed that siFOXC2 dramatically inhibited HIF-1α expression in both mRNA and protein level. Moreover, the recruitment of FOXC2 on HIF-1α HREs was further identified by ChIP and luciferase reporter assays. Furthermore, CF129 dramatically inhibited HIF-1α expression in hypoxic conditions, and FOXC2 overexpression reversed this. In contrast, CF129 knockdown significantly increased HIF-1α expression which is inhibited by siFOXC2 during hypoxia. This suggests that FOXC2 mediates a reciprocal regulation of CF129 and HIF-1α. A result from Fan et al. reported that hypoxia/HIF-1α-induced lincRNA-p21 can disrupt the VHL-HIF-1α interaction and reciprocally enhances HIF-1α accumulation, by which promoting glycolysis under hypoxia In summary, we have shown that CF129 inhibits PC cell proliferation and invasion by suppression of FOXC2 transcription, which depends on MKRN1- mediated ubiquitination and degradation of mutant p53. Our findings suggest that the CF129/MRKN1/ p53/FOXC2 axis may be a promising target for PC therapy.In this study, 40 pancreatic cancer and corresponding non-tumor tissue samples were obtained from individuals undergoing operations at the Pancreatic Surgery Center, Union Hospital  from October 2015 to June 2016. If patients had previously undergone radiotherapy, chemotherapy or immunotherapy, they were not eligible for this study. All pancreatic cancer tissues were confirmed by the histopathological reports. Tumor staging was based on the revised international system. These clinical samples were used after obtaining informed consent from participants and their parents. All protocols were approved by the Union Hospital ethics committee.2, 1% O2, 5% O2, 5% CO2 and corresponding 94.9% N2 , 94% N2, 90% N2 ,respectively or treated with CoCl2 (400 μmol/L). Total RNA and protein were collected after 0h, 6h, 12h, 24h and 48h.The PANC-1 and BxPC-3 cell lines came from American Type Culture Collection . These cell lines were cultivated in RPMI-1640 (HyClone) tinting with 10% FBS (ScienCell) as well as penicillin/streptomycin in a standard 37℃ incubator. To induce hypoxia, cells were cultured with 0.1% OSupplementary Table The siRNA targeting CF129, FOXC2, p53, MKRN1, HIF-1α, HDAC1 and appropriate negative controls were obtained . The plasmids containing CF129, FOXC2 and MKRN1 and the matched negative control plasmid were synthesized from GeneChem Co. . Transfections were performed with lipofectamine 2000 (Invitrogen) based on provided instructions. The siRNAs were transfected at a final concentration of 50nM. For plasmids, 1.6μg was transfected into samples in 12-well plate, while 0.2μg was used for 96 well plate. Protein and total RNA were extracted at 48 h post-transfection for downstream analyses.The siRNA sequences used in this study are all shown in Supplementary Table An RNAiso Plus kit (TAKARA) was used for total RNA extraction based on provided protocols. The PrimeScript® RT Master Mix Perfect Real Time kit (TAKARA) was used to synthesize DNA. PCR amplifications were performed with the SYBR Premix Ex Taq II (TAKARA). The expression levels of LncRNA-CF129, FOXC2, p53, MKRN1, HIF-1α and HDAC1 were normalized to GAPDH as reference genes.Primer sequences used in this study are all shown in An MTT assay was employed to assess the proliferation of target cells. In 96-well plate, stable transfected cells (2000/well) with eight vices well for each sample. The proliferation was observed for 1 to 5 days. At certain hour every day,20μl MTT (5 mg/mL) was added to appropriate wells, and after 4h it was replaced with 150μl DMSO (Sigma). Finally, ELISA reader was used to determine the absorbance at 570nm.PANC-1 and BxPC-3 migratory abilities were measured using a wound healing assay. Stable transfected cells were allowed to form a confluent monolayer in 12-well plates. We observed the healing of the wound for 0h, 24h and 48h after would scratching with microscope. To invasion assay, the Matrigel Invasion Chamber of pore size 8μm  coated with Matrigel  was used. 5×10 cells in serum-free media were placed in the upper chamber with 250μL serum-free medium, while 700μL medium with 30% FBS was added to the lower chamber. After 24h, the cells were fixed and stained with removed in upper chamber. Finally, cells on the lower chamber were counted via microscopic assessment of five fields.Stable transfected cells in 6-well plates (1000/well) were cultured for 8 days. Wash cells with PBS and Fix cells with % polyformaldehyde for 20 minutes, and followed 1% Crystal Violet for 20 minutes after discarding solution. Finally, we count the number of cells in each well under the microscope.lncRNA CF129 was assessed via fluorescence in situ hybridization, a kit from named FISH Tag™ RNA Multicolor Kit  was used. The prepared cell samples were fixed with 5% formaldehyde for 25 minutes. Samples were then combined overnight at 42°C with the CF129 probe. Cells were then washed, nuclei were stained using DAPI, and stained cells were imaged via an LSM 5 Pascal Laser Scanning Microscope (Zeiss).The Pierce™ Magnetic RNA-Protein Pull-Down  and MAXIscript®  kits were used for this assay based on provided protocols. Briefly, PANC-1 nuclear extracts were combined with biotinylated CF129 or antisense- CF129, after which streptavidin beads were added to the samples. Pulled down RNA was then isolated by washing these beads, boiling them in SDS buffer, and subsequent western immunoblotting.A Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit  was used based on provided protocols. Briefly, 500uL lysis buffer supplemented with protease and RNase inhibitors was used to lyse cells in 6-well plates. Lysates were spin at 16,000 xg for 10 minutes, and supernatants were used for precipitation with antibodies against p53, MRKN1, and anti-mouse IgG or anti-rabbit IgG for 3 h at 4°C with gentle agitation. Beads were then washed three times in a wash buffer, twice in PBS, and RNA was isolated using the RNAiso Plus (TAKARA), for qRT-PCR as described above.Protein samples were electrophoretically separated on 10% polyacrylamide gels, followed by transfer to PVDF membranes (Millipore). Blots were then probed using the following primary antibodies: p53 , FOXC2 , MKRN1 , HIF-1α , HDAC-1 , GAPDH .For co-IP analysis, anti-MKRN1, anti-p53 or normal mouse/rabbit IgG were used as the primary antibody, and then the antibody-protein complex was following incubated with Protein A/G PLUS-Agarose (Santa Cruz Biotechnology). The agarose-antibody- protein complex was collected and then analyzed by Western blot.The EZ-ChIPTM Chromatin Immunoprecipitation Kit  was used based on provided instructions. All the procedures were performed according to the manufacturer's instructions. Rabbit anti-HIF-1α/HDAC-1 was used and corresponding rabbit-IgG as controls. PCR was used to amplify bound DNA, which was then assessed via electrophoretic separation on a 2% agarose gel.To investigate whether FOXC2 is transcriptionally regulated by p53, PANC-1 cells were transfected with FOXC2 promoter (P2/P3-WT or P2/P3-MUT) reporter constructs, and further co-transfected with sip53. After a 48-hour culture, the Dual-Luciferase reporter assay system was used to assess promoter activity, with Renilla luciferase used for normalization.To determine how HIF-1α and HDAC1 affect the activity of HRE on the CF129 promoter under hypoxia condition. PANC-1 and BxPC-1 cells expressing pGL3-based construct containing the HRE of CF129 were transfected with siNC and siHIF-1α, and further treated with and without TSA. Twenty-four hours later, cells were cultured with or without CoCl2 for an additional 24h. The reporter activity was measured as above.PANC-1 cells that had been lentivirally transduced to express negative control (LV-NC) or CF129 sequences (LV-CF129) were injected into the dorsal flanks of 4-week-old male nude BALB/c mice. . (n=7 per group). Tumor growth in these animals was monitored every 3 days, and mice were euthanized 21 days after implant at which time tumor weights were determined.In other experiments, mice instead received tail vein injections of stably transfected PANC-1 cells, and upon sacrifice the frequency and number of liver metastases in these mice was determined. The total RNA of tumors was extracted and analyzed by qRT-PCR. Studies on animals were conducted with approval from the Animal Research Committee of the Academic Medical Center of Huazhong University of Science and Technology.SPSS v13.0 and GraphPad Prism were utilized for all data analyses. Data are given as Mean ± SD. Comparisons between 2 groups were made with Student's t-tests, while survival was assessed via Kaplan-Meier approach with a Log-rank test. The Wilcoxon signed-rank test was used to assess significant differences of CF129, p53 and FOXC2 between samples. χ2 tests were utilized to analyze the relationships between lncRNA-CF129 levels and clinical characteristics. P<0.05 was the threshold of significance.Supplementary figures and tables.Click here for additional data file.