The expression of S100A8/S100A9 is inducible and regulated by the Hippo/YAP pathway in squamous cell carcinomas.

BACKGROUND: S100A8 and S100A9, two heterodimer-forming members of the S100 family, aberrantly express in a variety of cancer types. However, little is known about the mechanism that regulates S100A8/S100A9 co-expression in cancer cells. METHODS: The expression level of S100A8/S100A9 measured in three squamous cell carcinomas (SCC) cell lines and their corresponding xenografts, as well as in 257 SCC tissues. The correlation between S100A8/S100A9, Hippo pathway and F-actin cytoskeleton were evaluated using western blot, qPCR, ChIP and Immunofluorescence staining tests. IncuCyte ZOOM long time live cell image monitoring system, qPCR and Flow Cytometry measured the effects of S100A8/S100A9 and YAP on cell proliferation, cell differentiation and apoptosis. RESULTS: Here, we report that through activation of the Hippo pathway, suspension and dense culture significantly induce S100A8/S100A9 co-expression and co-localization in SCC cells. Furthermore, these expressional characteristics of S100A8/S100A9 also observed in the xenografts derived from the corresponding SCC cells. Importantly, Co-expression of S100A8/S100A9 detected in 257 SCC specimens derived from five types of SCC tissues. Activation of the Hippo pathway by overexpression of Lats1, knockdown of YAP, as well as disruption of F-actin indeed obviously results in S100A8/S100A9 co-expression in attached SCC cells. Conversely, inhibition of the Hippo pathway leads to S100A8/S100A9 co-expression in a manner opposite of cell suspension and dense. In addition, we found that TEAD1 is required for YAP-induced S100A8/S100A9-expressions. The functional studies provide evidence that knockdown of S100A8/S100A9 together significantly inhibit cell proliferation but promote squamous differentiation and apoptosis. CONCLUSIONS: Our findings demonstrate for the first time that the expression of S100A8/S100A9 is inducible by changes of cell shape and density through activation of the Hippo pathway in SCC cells. Induced S100A8/S100A9 promoted cell proliferation, inhibit cell differentiation and apoptosis.

Multiphosphorylation and cellular localization of poly(rC) binding protein 1 during mitosis in hela cell.

OBJECTIVE: To monitor the phosphorylation modifications and cellular localization of poly(rC)-binding protein-1 (PCBP1) during the cell cycle progression of Hela cells. RESULT: Hela cells highly synchronized at five different phases from interphase to mitosis were obtained. Using mitotic phosphoprotein-specific monoclonal antibody MPM-2, the exclusive occurrences of multiphosphorylation statuses of PCBP1 in mitosis were confirmed by a series of spots with increasing acidic pI (isoelectric point) in two rounds of 2D western blotting on the same membrane, and a visible molecular mass shift that can be eliminated by the treatment with λ phosphatase in 1D western blotting. Immnuofluorescence revealed the localization shift of PCBP1 during cell cycle, with accumulations in nucleus as a patch pattern in interphase, and a dispersive distribution without the area of the condensed chromosomes during mitosis. CONCLUSIONS: These observations of mitosis-specific multiphosphorylations and localization shifts of PCBP1 suggest that the versatile PCBP1 was regulatable in a phosphorylation modification- and temporospatial-dependent manner in mitotic regulatory networks.

Serum translationally controlled tumor protein is involved in rat liver regeneration after hepatectomy.

AIM: The translationally controlled tumor protein (TCTP) has been reported to promote progression of many physiological processes. However, whether TCTP is involved in liver regeneration has been rarely studied. This study aimed to investigate the potential role of serum TCTP in liver regeneration after two-thirds partial hepatectomy. METHODS: The synthesis rate and accumulated expression of TCTP was assessed by phosphor imaging and Western blot analysis, respectively. The mRNA expression of tctp was analyzed by quantitative real-time PCR. The effect of serum TCTP on hepatocyte proliferation was investigated by bromodeoxyuridine incorporation, liver/body weight ratio, albumin concentration, and histological examination of liver following treatment of rat with anti-TCTP antibody or prokaryotic TCTP protein before hepatectomy. The MTT assay was used to examine effect of TCTP on hepatocyte proliferation in vitro. RESULTS: The results showed that the expression of intracellular and serum TCTP protein was significantly increased in rats after two-thirds partial hepatectomy. In vivo bromodeoxyuridine labeling assay suggested that treatment with anti-TCTP antibody before hepatectomy significantly decreased hepatocyte proliferation and liver/body weight ratio. The prokaryotic TCTP had a potential promoting effect on hepatocyte proliferation both in vivo and in vitro, although prokaryotic TCTP given to rats prior to hepatectomy did not increase the proliferation ratio or liver/body weight ratio. Furthermore, anti-TCTP antibody pretreatment decreased the expression of cyclin E, cdk2, and interleukin-6 in rat liver. CONCLUSION: These findings suggest serum TCTP is involved in rat liver regeneration through promoting hepatocyte proliferation.

RORα inhibits adipocyte-conditioned medium-induced colorectal cancer cell proliferation and migration and chick embryo chorioallantoic membrane angiopoiesis.

Lipid metabolic disturbances are related to many diseases, such as obesity, diabetes, and certain cancers. Notably, lipid metabolic disturbances have been reported to be a risk factor for colorectal cancer. Nuclear receptors act as ligand-dependent transcription regulators and play key roles in the regulation of body lipid metabolism and the development of many cancers. Retinoic acid receptor-related orphan receptor α (RORα) is a nuclear receptor and can regulate several lipid metabolism genes in certain cancers. Herein, we demonstrate that the conditioned medium from adipocytes has a proproliferative and promigratory effect on colorectal cancer cells and enhances angiogenesis in chicken embryonic chorioallantoic membranes. In addition, the conditioned medium leads to a decrease in the expression of RORα and its target genes. Meanwhile, RORα and its target gene expressions are lower in human colorectal tumor tissue compared with control colorectal tissue. Activation of RORα inhibits the effect of conditioned medium on the proliferation and migration of colorectal cancer cells as well as the angiogenesis in chicken embryonic allantoic membranes. In colorectal cancer cells, the putative ligand of RORα, cholesterol sulfate (CS), prevents cell cycle progression at the G1/S boundary and concurrently modulates the expression of cell cycle-regulatory genes in colorectal cancer cell. CS inhibits angiogenesis in chicken embryonic chorioallantoic membranes and concurrently decreases the mRNA expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α as well as the secretion of VEGF. In addition, lipogenic gene expression is higher in human colorectal tumor tissue compared with control colorectal tissue. CS inhibits the expression of lipogenic genes in colorectal cancer cells. These results suggest that RORα could represent a direct link between local lipid metabolism of colorectal tissue and colorectal cancer. Therefore, the reduction of the expression of RORα could represent a potential warning sign of colorectal cancer.

Chronological protein synthesis in regenerating rat liver.

Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration.

miR-137 inhibits proliferation of melanoma cells by targeting PAK2.

MicroRNAs (miRNA) are key players in a variety of cancers including malignant melanoma. miR-137 has been reported to be a tumor suppressor in melanoma and several targets have been identified for this miRNA. We previously developed a novel proteomics technology, (35) S in vivo/vitro labelling analysis for dynamic proteomics (SiLAD). Because of its high sensitivity in analysing protein expression rates, SiLAD has the potential to unravel miRNA effects on mRNAs coding for proteins with long half-lives or high abundance. Using SiLAD, we discovered that miR-137 significantly downregulated the expression rate of p21-activated kinase 2 (PAK2) in melanoma cells. Bioinformatics analysis predicted PAK2 as a direct target of miR-137, which was confirmed by luciferase reporter assay and Western blot analysis. We found that overexpression of miR-137 inhibited the proliferation of melanoma cells, which could be phenocopied by knockdown of PAK2 using siRNAs. Furthermore, overexpression of PAK2 restored miR-137-mediated suppression of cell proliferation. These findings indicate that miR-137 could inhibit proliferation through targeting PAK2 in melanoma cells.

S100A7 acts as a dual regulator in promoting proliferation and suppressing squamous differentiation through GATA-3/caspase-14 pathway in A431 cells.

S100A7 is expressed in many squamous cell carcinomas (SCCs), such as SCC of the skin, and well-differentiated SCC always displays stronger staining of this protein. A431 cells, an epidermal cancer cell line, were selected as a cell model to investigate the roles and mechanism of S100A7 in SCC of the skin. In this study, we demonstrated that the overexpression of S100A7 in A431 cells significantly promoted cell proliferation in vitro and tumor growth in vivo, whereas it suppressed the expression of GATA-3, caspase-14 and three squamous differentiation markers, keratin-1, TG-1 and involucrin. Conversely, the overexpression of caspase-14 not only significantly decreased cell proliferation and delayed tumor growth but also markedly induced the expression of three squamous differentiation markers, whereas S100A7 and GATA-3 were not influenced. Further evidence showed that silencing GATA-3 greatly inhibited the expression of caspase-14 and three differentiation markers, while the expression of S100A7 was not changed; contrary results were obtained when overexpressing GATA-3. Importantly, restoring the expression of GATA-3 and caspase-14 in A431-S100A7 cells could bypass the ability of S100A7 to increase cell viability and repress squamous differentiation. These data suggested that S100A7 expression in SCC may play an important role in the maintenance of SCC cell dedifferentiation, at least in SCC of the skin.

Sequence-dependent dsDNA-templated formation of fluorescent copper nanoparticles.

There are only a few systematic rules about how to selectively control the formation of DNA-templated metal nanoparticles (NPs) by varying sequence combinations of double-stranded DNA (dsDNA), although many attempts have been made. Herein, we develop a facile method for sequence-dependent formation of fluorescent CuNPs by using dsDNA as templates. Compared with random sequences, AT sequences are better templates for highly fluorescent CuNPs. Other specific sequences, for example, GC sequences, do not induce the formation of CuNPs. These results shed light on directed DNA metallization in a sequence-specific manner. Significantly, both the fluorescence intensity and the fluorescence lifetime of CuNPs can be tuned by the length or the sequence of dsDNA. In order to demonstrate the promising practicality of our findings, a sensitive and label-free fluorescence nuclease assay is proposed.

Solvatochromism, Reversible Chromism and Self-Assembly Effects of Heteroatom-Assisted Aggregation-Induced Enhanced Emission (AIEE) Compounds.

Two compounds, 9,10-bis[2-(quinolyl)vinyl]anthracene (BQVA) and 9,10-bis[2-(naphthalen-2-yl)vinyl]anthracene (BNVA), have been synthesised and investigated. Both of them have aggregation-induced enhanced emission (AIEE) properties. Heteroatom-assisted BQVA shows solvatochromism, reversible chromism properties and self-assembly effects. When increasing the solvent polarities, the green solution of BQVA turns to orange with a redshift of the fluorescence emission wavelengths from λ=527 to 565 nm. Notably, BQVA exhibits reversible chromism properties, including mechano- and thermochromism. The as-prepared BQVA powders show green fluorescence (λem=525 nm) and the colour can turn into orange (λem=573 nm) after grinding. Interestingly, the orange colour can return at high temperature. Based on these reversible chromism properties, a simple and convenient erasable board has been designed. Different from BQVA, non-heteroatom-assisted BNVA has no clear chromic processes. The results obtained from XRD, differential scanning calorimetry, single-crystal analysis and theoretical calculations indicate that the chromic processes depend on the heteroatoms in BQVA. Additionally, BQVA also exhibits excellent self-assembly effects in different solvents. Homogeneous nanospheres are formed in mixtures of tetrahydrofuran and water, which are then doped into silica nanoparticles and treated with 3-aminopropyltriethoxysilane to give amino-functionalised nanoparticles (BQVA-AFNPs). The BQVAAFNPs could be used to stain protein markers in polyacrylamide gel electrophoresis.

Identification and characterization of novel NuMA isoforms.

The large nuclear mitotic apparatus (NuMA) has been investigated for over 30years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two "hotspot" exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA's various functions.

Hsp70 and Hsp90 oppositely regulate TGF-ß signaling through CHIP/Stub1.

Transforming growth factor-ß (TGF-ß) signaling plays an important role in regulation of a wide variety of cellular processes. Canonical TGF-ß signaling is mediated by Smads which were further regulated by several factors. We previously reported that E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70-interacting protein, also named Stub1) controlled the sensitivity of TGF-ß signaling by modulating the basal level of Smad3 through ubiquitin-mediated degradation. Here, we present evidence that Hsp70 and Hsp90 regulate the complex formation of Smad3/CHIP. Furthermore, we observed that over-expressed Hsp70 or inhibition of Hsp90 by geldanamycin (GA) leads to facilitated CHIP-induced ubiquitination and degradation of Smad3, which finally enhances TGF-ß signaling. In contrast, over-expressed Hsp90 antagonizes CHIP mediated Smad3 ubiquitination and degradation and desensitizes cells in response to TGF-ß signaling. Taken together, our data reveal an opposite role of Hsp70 and Hsp90 in regulating TGF-ß signaling by implicating CHIP-mediated Smad3 ubiquitination and degradation. This study provides a new insight into understanding the regulation of the TGF-ß signaling by chaperones.

A highly sensitive "turn-on" fluorescent sensor for the detection of human serum proteins based on the size exclusion of the polyacrylamide gel.

A highly sensitive "turn-on" fluorescent sensor based on the size exclusion of the polyacrylamide gel was developed for the on-gels detection of human serum proteins after PAGE. The possible mechanism of this fluorescence sensor was illustrated and validated by utilizing five kinds of colloidal silver nanoparticles with different particle size distribution and six kinds of polyacrylamide gels with different pore size. It was attributed to that silver nanoparticles (<5 nm in diameter) had been selectively absorbed into the gel and formed the small silver nanoclusters, resulting in the red fluorescence. Using this new technique for the detection of human serum proteins after PAGE, a satisfactory sensitivity was achieved and some relatively low-abundance proteins (e.g. zinc-alpha-2-glycoprotein), which are the significant proteinic markers of certain diseases can be easily detected, but not with traditional methods. Furthermore, it was also successfully applied to distinguish between serums from hepatoma patient and healthy people. As a new protein detection technique, the colloidal silver nanoparticles based "turn-on" fluorescent sensor offers a rapid, economic, low background, and sensitive way for direct detection of human serum proteins, showing available potential and significance in the development of nanobiotechnology and proteome research.

Dual-modal imaging and photodynamic therapy using upconversion nanoparticles for tumor cells.

Here we synthesized silica-coated NaYF4:Yb,Tm@NaGdF4 nanoparticles with hypocrellin photosensitizers covalently incorporated inside the silica shells, combining dual modal imaging and photodynamic therapy (PDT) functions together. Under excitation at 980 nm, the tumor-targeting specificity of the as-prepared nanomaterials efficiently enhanced as folic acid (FA) was conjugated. The internalization of UCNPs@SiO2@hypocrellin A-FA in HeLa cells and HEK-293 cells was observed by confocal microscopy and in vitro magnetic resonance imaging (MRI), which demonstrated that the as-prepared nanocomposites have the ability to target folate receptor (FR) (+) cells. Moreover, magnetic resonance (MR) measurements also demonstrated that the as-prepared nanocomposites could be used as a contrast agent for MRI. All these results showed the feasibility and potential of the as-prepared nanocomposites for simultaneous imaging and PDT application.

High-confidence de novo peptide sequencing using positive charge derivatization and tandem MS spectra merging.

De novo peptide sequencing holds great promise in discovering new protein sequences and modifications but has often been hindered by low success rate of mass spectra interpretation, mainly due to the diversity of fragment ion types and insufficient information for each ion series. Here, we describe a novel methodology that combines highly efficient on-tip charge derivatization and tandem MS spectra merging, which greatly boosts the performance of interpretation. TMPP-Ac-OSu (succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide) was used to derivatize peptides at N-termini on tips to reduce mass spectra complexity. Then, a novel approach of spectra merging was adopted to combine the benefits of collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation. We applied this methodology to rat C6 glioma cells and the Cyprinus carpio and searched the resulting peptide sequences against the protein database. Then, we achieved thousands of high-confidence peptide sequences, a level that conventional de novo sequencing methods could not reach. Next, we identified dozens of novel peptide sequences by homology searching of sequences that were fully backbone covered but unmatched during the database search. Furthermore, we randomly chose 34 sequences discovered in rat C6 cells and verified them. Finally, we conclude that this novel methodology that combines on-tip positive charge derivatization and tandem MS spectra merging will greatly facilitate the discovery of novel proteins and the proteome analysis of nonmodel organisms.

Study of methylation of histone H3 lysine 9 and H3 lysine 27 during X chromosome inactivation in three types of cells.

Histone methylation is one epigenetic modification of an inactive X chromosome (Xi). Histone H3 lysine 9 dimethylation (H3K9me) and histone H3 lysine 27 trimethylation (H3K27me) are both associated with the chromatin of gene-silenced regions in the X chromosome and with X inactivation. Studies have shown that H3K9me is supposedly an early mark on the X chromosome during inactivation. Here, we examined the distribution and enrichment profiles of H3K9me and H3K27me by indirect immunofluorescence. We found that H3K9me appears to have a broad distribution throughout the whole genome, but is specific, to a certain extent, to the Xi in WI38 cells. In contrast, H3K27me is highly specific to the entire Xi, which differs significantly from other areas of the nucleus. Thus, H3K27me is more suitable as an epigenetic mark than H3K9me. The chromatin immunoprecipitation analyses also showed that H3K27me predominates on the inactive genes of the X chromosome. Additionally, we compared the levels of H3K9me and H3K27me in four X-linked genes and two autosomal genes between the normal cells (WI38) and the tumor cells (HeLa). The results revealed that the methylation levels of the inactive genes (POLA and OCRL) in tumor cells (HeLa) were lower than those in normal cells (WI38) and that the methylation levels of the Xi inactivation-avoidance genes (SMCX and ZFX) and autosomal genes (Myc and ß-actin) varied widely in tumor cells (HeLa). These events may be significant for cancer cell development and contribute to the characteristics of tumor cells.

Novel application of Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE).

We have developed a novel application for DNA oligonucleotide-stabilized Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE). Oligonucleotide-stabilized Ag nanoclusters were used as fluorescent probes for direct detection of proteins after native PAGE. Some relatively low-abundance proteins, such as α-1-antichymotrypsin (ACT) and α-2-glycoprotein 1, zinc (ZAG) were easily detected by oligonucleotide-stabilized Ag nanocluster-based fluorescent imaging and identified by MS and MS/MS techniques, without the need of expensive antibodies or tedious immunoassay procedures. The pH condition for the oligonucleotide-stabilized Ag nanocluster solution was optimized and the possible mechanism of interaction between proteins and DNA oligonucleotide-stabilized Ag nanoclusters was analyzed. As a novel fluorescent detection method it is simple, fast, nontoxic and sensitive, and it shows great analytical potential in proteome research and in biochemistry.

The application of amine-terminated silicon quantum dots on the imaging of human serum proteins after polyacrylamide gel electrophoresis (PAGE).

Novel amine-terminated silicon (Si) quantum dots (QDs) were synthesized and applied for the detection of human serum proteins on gels directly after polyacrylamide gel electrophoresis (PAGE). The diameter of these stable amine-terminated Si QDs was in the range of 0.5-2.0 nm. In this study, the fluorescent imaging conditions, such as the buffer solution, pH value, buffer concentration and quantity of Si QDs, were optimized and the possible mechanisms of Si QDs-protein interaction were analyzed. The mode of Si QDs and human serum albumin association was found to occur by hydrogen bond interactions; this was probably attributed to the interaction between the amino group of amine-terminated Si QDs and the carboxyl group of proteins. Meanwhile, human serum proteins separated by native 1D and native 2D electrophoresis were detected by Si QD-based fluorescent imaging. Some proteins, such as isoform 1 of α-1-antitrypsin, complement C3 (Fragment) and hemopexin, which were identified by mass spectrometry (MS), were easily detected by using Si QDs, but not with CBB-R250 staining. The Si QDs-based fluorescent imaging technique with high resolution is a sensitive and dependable method for direct detection of human serum proteins, and has enormous potential in clinical diagnosis.

Selective expression of S100A11 in lung cancer and its role in regulating proliferation of adenocarcinomas cells.

S100A11, one secreted protein, is overexpressed in certain cancers. We investigated S100A11 expression in various subtypes of lung cancer and explored its role in cell proliferation. S100A11 mRNA level was examined in 45 pairs of frozen lung cancer tissues by reverse transcriptase PCR (RT-PCR). The specific expression and subcellular distribution of S100A11 were examined in 78 paraffin-embedded lung cancers, 2 benign lung diseases as well as 22 healthy lung tissues by immunohistochemistry. S100A11 protein level was further analyzed in the sera of 86 lung cancer patients and 50 healthy individuals by enzyme-linked immunosorbent assay. We found that both mRNA and protein levels of S100A11 were overexpressed in adenocarcinomas (ADC) and squamous cell carcinomas (SCC) compared with paired non-cancerous lung tissues, while S100A11 was detected downregulated in small cell lung cancers (SCLC). Further immunohistochemistry staining was positive for S100A11 only in non-small cell lung cancer (NSCLC) (ADC, SCC, large cell carcinomas, et al.), but not SCLC. Conclusively, we found S100A11 protein level increased in the sera of NSCLC patients. Furthermore, when S100A11 expression was knocked down in lung adenocarcinoma cells A549 and LTEP-a-2, the cell proliferation was significantly inhibited in vitro and in vivo.

The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis.

In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-α-2-glycoprotein and α-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution.

Transplanting neurofibromatosis-1 gene knockout neural stem cells improve functional recovery in rats with spinal cord injury by enhancing the mTORC2 pathway.

The poor survival and low efficiency of neuronal differentiation limits the therapeutic effects of transplanted neural stem cells in the treatment of spinal cord injury. Neurofibromatosis-1 (NF-1) is a tumor suppressor gene that restricts the rapid and abnormal growth and differentiation of neural cells. In the present study, lentiviral vectors were used to knock out NF-1, Ricotr (the core member of mTORC2) or NF-1+Ricotr in neural stem cells in vitro, and the NF-1, Ricotr or NF-1+Ricotr knockout neural stem cells were transplanted at the lesion site in a rat model of spinal cord injury (SCI). We first demonstrated that targeted knockout of NF-1 had an antiapoptotic effect and improved neuronal differentiation by enhancing the mTORC2/Rictor pathway of neural stem cells in vitro. Subsequently, transplanting NF-1 knockout neural stem cells into the injured site sufficiently promoted the tissue repair and functional recovery of rats with spinal cord injury by enhancing the survival and neuronal differentiation of grafted neural stem cells. Collectively, these findings reveal a prominent role of NF-1 in neural stem cell biology, which is an invaluable step forward in enhancing the benefit of neural stem cell-mediated regenerative cell therapy for spinal cord injury and identifies the transplantation of NF-1 knockout neural stem cells as a promising strategy for spinal cord injury.