Research progress on the role and intervention strategies of NRP1 in colorectal cancer / 药学学报

Colorectal cancer (CRC) is one of the most common malignant tumors in the world, and its incidence and mortality are among the top three of all malignant tumors. In recent years, CRC is becoming more common in younger patients. Currently, surgery is the main or first treatment of early stage CRC, however, up to 50% patients have recurrence and metastasis post-surgery. While chemotherapy and radiotherapy are often used as adjuvant treatment after surgery or as main treatment options for late stage CRC, they usually induce severe adverse effects. Safe and effective treatments for CRC are still lacking. Therefore, it is essential to discover new therapies for CRC. Neuropilin 1 (NRP1), as a transmembrane glycoprotein, is reported to highly express in CRC, and its overexpression is demonstrated to be closely related to the occurrence and development of CRC. NRP1 is involved in angiogenesis, tumor growth, autophagy, and lipid metabolism, which is expected to be a potential new target for the treatment of CRC. This paper reviews the role of NRP1 in CRC, including its molecular structure, expression in CRC, as well as its connection with autophagy and metabolism. The regulatory factors of NRP1 in CRC were introduced, including vascular endothelial growth factor (VEGF), semaphorin 3A (SEMA3A), transforming growth factor-β (TGF-β), etc. The potential intervention strategies of CRC targeting NRP1 were summarized in order to provide reference for the diagnosis and prevention of CRC.

Functional role and underlying mechanisms of neuropilin-1 in proliferation and migration of rat vascular smooth muscle cells / 中国胸心血管外科临床杂志

@#Objective    To investigate the role and potential mechanisms of neuropilin-1 (NRP1) in the pathogenesis of vein graft failure. Methods    The rat vascular smooth muscle cells (VSMCs) were transfected with NRP1-shRNA adenovirus and negative control adenovirus respectively. Cell counting kit-8, flow cytometry, Transwell and Western blot were used to investigate the effects of inhibition of NRP1 on VSMCs proliferation viability, apoptosis, migration capacity and its downstream signaling pathway protein expression. Results    The proliferation and migration of rat VSMCs could be inhibited after down-regulation of NRP1, and the increase of apoptosis was also observed. Moreover, inhibition of NRP1 significantly reduced Akt and NF-κB phosphorylation in rat VSMCs, but had little effect on activation of ERK1/2. Conclusion    NRP1 may promote vein graft hyperplastic remodeling by regulating the proliferation and migration of VSMCs through PI3K/Akt and NF-κB pathways, but further animal study is required.

Effect of NRP1 gene knockout on process of radiationinduced pulmonary fibrosis and its mechanism / 吉林大学学报(医学版)

Objective: To observe the effect of neuroopilin-1 (NRP1) gene on the process of radiation-induced pulmonary fibrosis (RIPF), and to explore its roles in the occurrence and development of epithelial-mesenchymal transition (EMT) mediated by Wnt/fi-catenin pathway tail identification was performed in and TGF-β1/Smads pathway, and extracellular matrix (ECM) deposition. Methods: The Cre-LoxP recombinase system was used to construct the transgenic C57BL/6J mice with NRP1 gene specific knockout in alveolar type II epithelial cells (AT-II) and the mice. A total of 160 mice were randomly divided into 4-week group, 8-week group, 16-week group and 24-week group. In each group, the mice were randomly divided into wild type (Con) group, wild type+irradiation (IR) group, NRP1 gene-specific knockout (KO-Con) group, NRP1 gene-specific knockout+irradiation (KO+IR) group according to the method of random number table; there were 10 mice per group. In KO-Con and KO + IR groups, the NRP1 gene was specifically knocked out in the AT-II cells by intraperitoneal injection of tamoxifen, and the mouse models of RTPF were established by 20 Gy total thoracic irradiation in IR group and KO+IR group. After the models were constructed, HE staining and Masson staining were used to verify whether the models were successfully constructed. Immunohistochemistry (IHC) method was used to detect the type I collagen (Col I) and crsmooth muscle actin α-SMA) protein expression levels; Western blotting method was performed to detect the NRP1, β-catenin, TGF-β1, and Smad2 protein expression levels in the lung tissue of the mice; Quantitative fluorensence real-time polymerase chain reaction (qRT-PCR) method was used to detect the expression levels of NRP1, Col I, α-SMA, β-catenin, TGF-βl, Smad2, E-cadherin, N-cadherin, and Vimentin mRNA in the lung tissue of the mice. Results: The results of HE and Masson staining showed the RTPF models were successfully established, and the lung tissue of the mice in IR group mainly showed the pathomorphology of radiation pneumonitis. Compared with Con group, the protein and mRNA expression levels of NRP1 in the lung tissue of the mice in IR group were gradually increased with the prolongation of time (P<0.05), and reached the highest at 24 weeks (P<0.01). Compared with Con group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 proteins and mRNA in the lung tissue of the mice in IR group and KO+IR group were increased gradually with the prolongation of time (P<0.05 or P<0.01). Compared with IR group, the expression levels of Col I, α-SMA, β-catenin, TGF-β1, and Smad2 protein and mRNA in the lung tissue of the mice in KO+IR group were significantly decreased (P<0.05 or P<0.01), but they were higher than those in Con group (P<0.05 or P<0.01). Compared with Con group, the expression levels of the epithelial cell marker E-Cadherin mRNA in the lung tissue of the mice in IR group and KO+IR group were gradually decreased with the prolongation of time (P< 0.01), and the expression levels of the interstitial cell markers N-Cadherin and Vimentin were increased (P<0.05 or P<0.01), but the expression levels of E-cadhern mRNA in the lung tissue of the mice in KO-IR group were significantly higher than those in IR group (P<0.05 or P<0.01), and the expression levels of N-Cadherin and Vimentin mRNA in the lung tissue of the mice in KO + IR group at each time point were lower than those in IR group (P<0.05 or P<0.01). Conclusion: Knockout of NRP1 gene can inhibit the occurrence and development of RTPF, and its mechanism may be involved in regulating the expressions of Wnt/fi-catenin and TGF-β1/Smads signaling pathways in the lung tissue and inhibiting the EMT process in the mice.

Application of NRP-1 targeting molecular probe in grading diagnosis of heterotopic brain glioma in nude mice / 中华行为医学与脑科学杂志

Objective To observe the expression of neuropilin-1 (NRP-1) in glioma cells of different grades,and evaluate the application value of a novel molecular probe(USPIO-PEG-tLyP-1)in the grading diagnosis of heterotopic glioma in nude mice by magnetic resonance imaging (MRI).Methods Expression levels of NRP-1 in glioma cell lines of different grades were detected by Western-Blot.USPIO-PEG-tLyP-1 was synthesized by carbon diimine method.The U87-MG tumor-bearing mice model (U87-MG group) and CHG-5 tumor-bearing mice model(CHG-5 group) were established with 10 mice in each group.Six tumorbearing mice with a tumor volume about 0.6 cm3 were selected from each group,and they were given with 2mg/kg molecular probes via tail vein respectively and was detected by MRI at 0 h,6 h,12 h and 24 h,then R2 values were calculated.After the imaging,tumor-bearing mice were sacrificed,and tumor tissue sections were made.The iron particles in the sections was detected by Prussian blue staining.The binding ability of molecular probes and tumor tissues in the two groups was compared.Results The expression of NRP-1 in U87-MG and CHG-5 cell lines was significantly higher than that in HA.In addition,the expression of NRP-1 in U87-MG was higher than that in CHG-5 cell(P＜0.01).MRI results showed that R2 values of tumor tissues in the two groups were compared,and the difference was not statistically significant before the injection of molecular probe(U87-MG group(10.35±0.52)vs CHG-5 group(9.86±0.43),t=1.779,P=0.106).The R2 value of tumor tissue in the U87-MG group was higher than that in the CHG-5 group after the injection of molecular probe (6 h:U87-MG group (11.63±0.85)vs CHG-5 group (10.51 ±0.49),t=2.796,P=0.019;12h:U87-MG group(14.23±0.68)vs CHG-5 group(12.29±0.28),t=6.462,P=0.000;24 h:U87-MG group (13.36±0.92) vs CHG-5 group(11.32±0.64),t=4.459,P=0.001).The results of Prussian blue staining showed that there were significantly more blue staining particles in tumor tissues of the U87-MG group than that of the CHG-5 group,and the difference was statistically significant(P＜0.01).Conclusion The NRP-1 targeted molecular probe can be used for grading diagnosis of high and low grade heterotopic brain glioma in nude mice.

Therapeutic Effects of Synthetic Antimicrobial Peptides, TRAIL and NRP1 Blocking Peptides in Psoriatic Keratinocytes / 전남의대학술지

Psoriasis is a chronic, recurrent, heterogeneous, cutaneous inflammatory skin disease for which there is no cure. It affects approximately 7.5 million people in the United States. Currently, several biologic agents that target different molecules implicated in the pathogenic processes of psoriasis are being assessed in diverse clinical studies. However, relapse usually occurs within weeks or months, meaning there is currently no cure for psoriasis. Therefore, recent studies have discovered diverse new potential treatments for psoriasis: inhibitors of bacteria such as Staphylococcus aureus, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and neuropilin 1 (NRP1). A promising approach that has recently been described involves modifying antimicrobial peptides to develop new cutaneous anti-bacterial agents that target inflammatory skin disease induced by Staphylococcus. Increased expression of TRAIL and its death receptors DR4 and DR5 has been implicated in the pathogenesis of plaque psoriasis. In addition, TRAIL has the ability to inhibit angiogenesis by inducing endothelial cell death and by negative regulation of VEGF-induced angiogenesis via caspase-8-mediated enzymatic and non-enzymatic functions. Since NRP1 regulates angiogenesis induced by multiple signals, including VEGF, ECM and semaphorins, and also initiates proliferation of keratinocytes through NF-κB signaling pathway in involved psoriatic skin, targeting NRP1 pathways may offer numerous windows for intervention in psoriasis. In this review, we will focus on the current knowledge about the emerging role of synthetic antimicrobial peptides, TRAIL and NRP1 blocking peptides in the pathogenesis and treatment of psoriasis.

Changes of vascular endothelial growth factor and neuropilin-1 in rats with early blast lung injury and its correlation with lung injury / 解放军医学杂志

[Abstract] Objective To explore the regular pattern of the changes of vascular endothelial growth factor (VEGF) and neuropilin-1 (NRP-1) of rat model in early (<48h) blast lung injury (BLI) and the correlation of such changes to the degree of lung injury. Methods The rat model of lung blast injury was established with reducing three-way pipe and fixed pressure pneumatic simulation device. Forty SD rats were randomly divided into 5 groups: control group (C), 1h after blast (B1h), 6h after blast (B6h), 24h after blast (B24h) and 48h after blast (B48h) group. The data were collected of concentrations of VEGF and NRP-1 in serum and lung tissue. The degree of lung injury was estimated. Results The levels of NRP-1 increased in both serum and lung tissue (P<0.01) and the level of VEGF decreased in lung tissue (P<0.05) in control group than in B groups. The concentrations of NRP-1 in serum and lung tissue were highest, and of VEGF in lung tissue were lowest in the group B6h. The lung injury score was positively correlated with the levels of NRP-1 in lung tissue (r=0.429, P=0.014). In group B6h, the lung injury score was negatively correlated with the levels of VEGF in lung tissue (r=–0.769, P=0.013). In group B48h, both the lung injury score and the wet and dry ratios (W/D) of lung tissue were positively correlated with the concentrations of VEGF in serum (r=0.777, P=0.012 and r=0.687, P=0.030, respectively). Pulmonary interstitial vascular rupture, alveolar hemorrhage, alveolar epithelial cell and capillary injuries, and accompanied by inflammatory cell infiltration were the basic pathological changes of BLI. Conclusion The levels of NRP-1 and VEGF in serum and lung tissue may vary with time and are correlated with the degree of lung injury in early BLI.

Regulation of neuropilin-1 in radiation-induced transformation of lung epithelial cells / 中华放射医学与防护杂志

Objective To investigate the effect of neuropilin-1 (NRP1) on radiation-induced epithelial-mesenchymal transition (EMT) by measuring the expressions of EMT-related transcription factors in the irradiated cells with different levels of NRP 1.Methods Human lung type Ⅱ epithelial cells (A549) were transfected with NRP1 over-expression lentiviral vector and NRP1 inhibition vector to construct two cell models of NRP1high-A549 and NRP1low-A549.A NRP1 knock-down cell model was also constructed by transferring siNRP1 into normal mouse lung epithelial MLE-12 cells that was validated at both protein and mRNA levels.A single dose of 10 Gy X-ray was delivered to these cell models,then total protein and RNA were extracted at 0,12,24 and 48 h after irradiation.The expressions of EMT-related transcription factors (Twist and ZEB1) and EMT markers (β3-catenin,N-cadherin,and Vimentin) in each cell model were detected by Western blot and qPCR.Results After 10 Gy irradiation,the expressions of NRP1 mRNA and protein were significantly increased in A549 and MLE-12 cells.The expressions of the mesenchymal markers (Vimentin and N-cadherin) and the transcription factors of ZEB1 and Twist were also significantly increased (A549:t=2.917,7.361,4.852,9.278,P＜0.01;MLE-12:t=9.652,31.357,30.985,17.266,P ＜0.01).The expressions of Vimentin and N-cadherin were significantly decreased in NRP1low-A549 (t =10.077,15.707,P ＜ 0.01) and siNRP1-MLE-12 cells (t =5.745,P ＜ 0.01),but the expression of epithelial marker (β3-catenin) was significantly increased in these cells.The expressions of N-Cadherin and Vimentin were significantly elevated (t =16.055,5.560,P ＜ 0.01),while β-catenin decreased significantly in NRP1high-A549 cells.After irradiation,the transcription factor of Twist in NRP1low-A549 group was significantly decreased (t=3.987,P＜0.01),while the transcription factors of ZEB1 and Twist in the NRP1high-A549 group increased in a time-dependent manner (t =11.289,2.903,P＜0.01).After irradiation,the transcription factor of ZEB1 decreased significantly in siNRP1-MLE-12 cells (t=13.449,P＜0.01),and the protein expressions of ZEB1 and Twist in siNRP1-MLE-12 cells were lower than those of control group in a time-dependent manner.Conclusions NRP1 promotes radiation-induced EMT in human and mouse epithelial cells through up-regulation of transcription factors of ZEB1 and Twist.

Efficacy of inverso isomer of CendR peptide on tumor tissue penetration

The dense extracellular matrix and high interstitial fluid pressure of tumor tissues prevent the ability of anti-tumor agents to penetrate deep into the tumor parenchyma for treatment effects. C-end rule (CendR) peptides can enhance the permeability of tumor blood vessels and tumor tissues binding to neuropilin-1 (NRP-1), thus aiding in drug delivery. In this study, we selected one of the CendR peptides (sequence RGERPPR) as the parent l-peptide and substituted d-amino acids for the l-amino acids to synthesize its inverso peptide (RGERPPR). We investigated the NRP-1 binding activity and tumor-penetrating ability of (RGERPPR). We found that the binding affinity of (RGERPPR) with NRP-1 and the cellular uptake was significantly higher than that of RGERPPR. Evans Blue tests revealed that (RGERPPR) exhibited improved tumor-penetrating ability in C6, U87 and A549 tumor-bearing nude mice. Using nude mice bearing A549 xenograft tumors as a model, we found that the rate of tumor growth in the group co-administered with (RGERPPR) and gemcitabine (Gem) was significantly lower than the gemcitabine-treated group with a tumor suppression rate (TSR%) of 55.4%. Together, our results demonstrate that (RGERPPR) is a potential tumor-penetrating peptide.

Expression of neuropilin-1 in human non-small cell lung cancer and its associations with clinicopathological features and prognosis / 肿瘤

Objective: To study the expression of neuropilin-1 (NRP-1) in nonsmall cell lung cancer (NSCLC) and the paracancerous normal lung tissues, and to investigate the association of NRP-1 expression with the clinicopathological characteristics and prognosis of NSCLC patients. Methods: Ninety-two patients with NSCLC who underwent complete resection and pathological diagnosis from June 2009 to June 2012 in Affiliated Hospital of Xuzhou Medical University were collected. The expression of NRP-1 in 92 NSCLC tissues and 88 paracancerous lung tissues was detected by immunohistochemistry. All patients were followed up by telephone after operation, the last followed-up time was June 2016. The correlations of NRP-1 expression with the clinicopathological features and prognosis of NSCLC patients were analyzed. Results: The high expression rate of NRP-1 in NSCLC was significantly higher than that in paracancerous normal lung tissues (P = 0.004). The expression of NRP-1 was significantly associated with tumor maximum diameter, lymph node metastasis, TNM stage and pathological grade (all P 0.05). The overall survival time of NSCLC patients with NRP-1 high expression was significantly shorter than that of patients with NRP-1 low expression (P < 0.001). COX multivariate regression analysis indicated that NRP1 expression and TNM stage were independent prognostic markers for NSCLC patients (both P < 0.05). Conclusion: The high expression of NRP-1 is associated with the poor clinicopathological characteristics and poor prognosis of NSCLC. NRP-1 may be a promising prognostic marker and potential therapeutic target for NSCLC patients (both P < 0.05).

Magnetic resonance study on the binding ability between targeted neuropilin-1 probe and ectopic glioma in mice / 中华行为医学与脑科学杂志

Objective To detect the binding ability of the molecular probe of neuropilin-1( NRP-1) to mouse ectopic glioma by magnetic resonance imaging ( MRI) . Methods Glioma model mice were pre-pared by glioma tissue transplantation.Thirty tumor bearing mice were randomly selected for tissue anatomy(n=12) and other 18 mice were randomly divided into 3 groups:the control group ( group A) ,the probe con-trol group (group B) and the probe group (group C),which were given 20 μl saline,20 μl USPIO-PEG, 20μl USPIO-PEG-tLyP-1 through the tail vein of the mice respectively.And at 0h,6h,12h,24h after admin-istration,T2WI and T2MAPPING sequences were detected by MRI. Then the tumor bearing mice were killed immediately and the glioma tissue was used to detect the iron content by Prussian blue staining to detect the binding ability of the glioma tissue with the new molecular probe. The biological toxicity of the new molecular probe was detected by pathological staining. Results The expression of NRP-1 in glioma tissues was signifi-cantly higher than that in the liver,kidney and brain(P<0.05).The 24h relaxation time ((14.19±0.87)ms) of the glioma tissue in the C group was significantly lower than that in the B group ((25.94±0.77)ms) (P<0.05) ,and the blue staining particles in the C group were more than those in the B group(P<0.05) . Conclu-sion In the animal experiment,the molecular probe with NRP-1 as the target has obvious targeting effect and good biocompatibility,which provides a clinical basis of glioma for further clinical diagnosis.

Expression and clinical value of NRP-1 in hepatocellular carcinoma / 国际肿瘤学杂志

Objective To determine the expression and clinical value of neuropilin-1 (NRP-1) in hepatocellular carcinoma (HCC).Methods One hundred and fifty-one cases of HCC tissues and 89 cases of healthy liver tissues were chosen to compare the expression of NRP-1 by immunohistochemistry.Then the relationships between different clinical factors and the expression of NRP-1 were analyzed by univariate and multivariate statistical analysis.Moreover,the survival rates were compared by survival analysis between different expressions of NRP-1 in HCC patients.Results Eleven cases were lost to follow-up or died for non HCC disease,and the effective cases in the final study were 140 cases.The positive expression rates of NRP-1 in HCC and normal liver tissues were 65.00％ and 35.96％ respectively,and the difference was statistically significant (x2 =18.843,P ＜0.001).According to the expression level of NRP-1,140 patients with HCC were divided into negative expression group (n =49) and positive expression group (n =91).Univariate analysis showed that the expression of NRP-1 in HCC was correlated with tumor number (x2 =8.025,P =0.005),TNM stage (x2 =26.467,P ＜ 0.001),differentiation degree (x2 =15.296,P ＜ 0.001),portal vein invasion (x2 =9.054,P =0.003) and hepatic vein invasion (x2 =5.928,P =0.015).Multivariate statistical analysis showed that TNM stage (OR =1.392,95％ CI:1.121-1.730,x2 =8.950,P =0.003),differentiation degree (OR =1.469,95％ CI:1.102-1.958,x2 =6.862,P =0.009),portal vein invasion (OR =1.829,95％ CI:1.157-2.893,x2 =6.665,P =0.010) and hepatic vein invasion (OR =2.161,95％ CI:1.172-3.987,x2 =6.084,P =0.014) were important factors for NRP-1 expression.The median survival time of NRP-1 negative HCC patients was significantly longer than that of positive group (44 months vs.23 months),and the difference was statistically significant (x2 =21.922,P ＜0.001).Conclusion NRP-1 is over-expression in HCC tissue and related to the malignant progress of HCC,and this suggests poor prognosis in patients with HCC.

Expressions of miR-9 and NRP1 in human NSCLC tissue and adj acent carcinoma tissue and their clinical significances / 吉林大学学报(医学版)

Objective:To detect the expression levels of neuropilin1 (NRP1)mRNA and miR-9 in non-small cell lung cancer (NSCLC)tissue samples, and to explore the correlations between the expressions of NRP1 mRNA, miR-9 and the clinicopathological characteristics of the patients with NSCLC.Methods:Informed consent was obtained from each patient before surgery.The tissue samples including 45 NSCLC tissue ,45 adjacent carcinoma tissue and 45 normal lung tissue were collected from China-Japan Union Hospital of Jilin University from 2010 to 2011.qRT-PCR was used to detect the expression levels of NRP1 mRNA and miR-9 in three kinds of lung tissue, and the correlation between the expressions of NRP1 mRNA, miR-9 and clinicopathological characteristics of the patients with NSCLC was analyzed.Results:Compared with normal tissue,the expression level of NRP1 mRNA in adjacent carcinoma tissue had no change (P>0.05),but the expression level of NRP1 mRNA in non-small cell lung cancer tissue was significantly decreased (P0.05),but the expression level of miR-9 in non-small cell lung cancer tissue was significantly increased (P 0.05),but was correlated to the sex (P<0.05). Conclusion:The expression level of miR-9 is up-regulated and the expression level of NRP1 mRNA is down-regulated significantly in non-small cell lung cancer tissue. The detection of the expression level of NRP1 mRNA contributes to j udge the histological subtype and lymph node metastasis of NSCLC.

NRP1 expression is associated with chemosensitivity and poor prognosis in advanced non-small cell lung cancer patients treated with ifrst-line platinum-based chemotherapy / 中国癌症杂志

Background and purpose:Neuropilin-1 (NRP1), a vascular endothelial growth factor (VEGF) receptor, plays an important role in tumor angiogenesis and tumor cell migration. The purpose of this study was to de-termine the correlation between NRP1 expression and sensitivity to ifrst-line platinum-based chemotherapy in patients with advanced non-small cell lung cancer (NSCLC), and between NRP1 expression and survival.Methods:NRP1 ex-pression in tumor tissues of 104 advanced NSCLC patients treated with ifrst-line platinum-based regimen was detected by immunohistochemisty.A chi-square test and logistic regression model were used to analyze the relationship between NRP1 expression and the chemotherapy response rate. Kaplan-Meier and Cox proportional hazard regression models were used to analyze the effect of NRP1 expression on patient survival.Results:Among the 104 patients, 56 (53.8%) had high expression of NRP1. High expression of NRP1 was not related to age, gender, histological type, degree of differentiation, performance status, and chemotherapy regimen. The chemotherapy response rate was significantly higher in patients with low NRP1 expression than in patients with high expression (43.8% vs23.2%,P=0.026). The low NRP1 expression was signiifcantly associated with longer progression-free survival (4.6 monthsvs3.0 months, P=0.001 for log-rank test,χ2=11.273) and overall survival (11.5 monthsvs9.2 months,P=0.000 for log-rank test,χ2=14.392) as compared with high NRP1 expression. Multivariate analysis showed that high expression of NRP1 was an independent predictor for the chemotherapy response rate and overall survival in patients with advanced NSCLC.Conclusion:NRP1 expression is associated with response rate and survival in advanced NSCLC patients treated with ifrst-line plati-num-based chemotherapy. NRP1 expression may be a potential biomarker for predicting chemosensitivity and prognosis in patients with advanced NSCLC.

Fabrication of molecular probe targeting NRP-1 and magnetic resonance molecular imaging of gliomas in vitro / 中华行为医学与脑科学杂志

Objective To synthetize a novel MR molecular imaging probe named USPIO?PEG?tLyP?1,and to evaluate its value in detecting U87 cells by MR imaging. Methods USPIO?PEG?tLyP?1 was synthetized by conjugating USPIO?PEG with tLyP?1. Neuropilin?1 expression levels of glioma cell lines were detected by Western blot. The cytotoxicity of USPIO?PEG and USPIO?PEG?tLyP?1 were assessed by MTT colorimetric assay. The uptake efficiency of USPIO?PEG?tLyP?1 was measured by Prussian blue staining, transmission electron microscope and MR imaging in vitro. Results The novel MR molecular imaging probe was synthetized with an average diameter of 43.84 nm. U87 glioma cell line was screened as test subject for the highly expression of NRP?1(P<0.05). USPIO?PEG?tLyP?1 group showed much more intracellular blue particles than USPIO?PEG group after Prussian blue staining. After incubation,USPIO?PEG?tLyP?1 mainly existed in lysosme,endoplasmic reticulum and mitochondria. In vitro MRI showed that USPIO?PEG?tLyP?1 significantly enhanced the negative contrast effect compared with USPIO?PEG(P<0.01). Conclusion The decoration of tLyP?1 enhanced targeting ability of USPIO?PEG to glioma cells and MR molecular imaging can be a promising method for early diagnosis of gliomas.

Expression of NRP-1 in gastric cancer tissue and its correlation with clinicopathological characteristics and prognosis / 重庆医学

Objective To detect the expression of NRP‐1 in gastric cancer tissue ,to analyze its relationship with clinicopath‐ological features ,and to explore its value in judging the prognosis of gastric cancer .Methods The clinical pathologic data and prog‐nosis situation in 168 cases of gastric cancer were retrospectively analyzed .The expression of NRP‐1 in gastric cancer tissue and normal tissue was detected by the immunohistochemical method .The relationship between the expression of NRP‐1 with the clinico‐pathological features and prognosis was investigated .Whether NRP‐1 serving as a reference indicator for judging the prognosis of gastric cancer was evaluated .Results (1) In 168 cases ,the NRP‐1 expression in gastric cancer tissue was higher than that in nor‐mal tissue (66 .7% vs .8 .33% ,P<0 .05) .(2) The NRP‐1 expression was related with the tumor size ,differentiation degree ,infil‐trative depth ,lymph node metastasis and TNM stage(P<0 .05) .(3)The median survival time in the patients with high NRP‐1 ex‐pression was shorter than that in the patients with low NRP‐1 expression (P<0 .05) .(4) The multiple factor analysis by COX pro‐portional hazard model showed that the NRP‐1 expression was an independent risk factor for the prognosis of gastric cancer(P<0 .05) .Conclusion (1)NRP‐1 plays an important role in the incidence and development process of gastric cancer and its expression is closely related with the malignant biological behavior of gastric cancer .(2)The high NRP‐1 expression prompts poor prognosis .(3) NRP‐1 may be expected to be regarded as one of the indexes for judging the biologic behaviors and prognosis of gastric cancer .

Effects on proliferation and apoptosis of T-cell leukemia cells by silencing NRP-1 / 中国免疫学杂志

Objective:To investigate the effect on proliferation and apoptosis of T-cell leukemia cells by silencing NRP-1 ( Jurkat cells).Methods:The lentivirus plasmid which expresses NRP1 gene specific shRNA was constructed in our preliminary ex-perimental.We transfected the lentivirus plasmid to human T-cell Lymphoma cells.The proliferation of Jurkat cells different groups and effect on cell proliferation after chemotherapy drug EPI-treated were found by CCK-8 kit.The proliferation level and apoptosis rate of the cells were detected by flow cytometry and Annexin-V-FITC/PI method.Results:The proliferation level of NRP-1 /shRNA interference group was decreased significantly in 48 h,72 h,96 h,which was compared with the control groups.The apoptosis rate of the NRP-1/shRNA interference group was increased compared with control groups.The chemotherapy drug sensitivity of epirubicin ( EPI ) test results showed that EPI concentration was 0.025,0.05,0.1,0.2,0.4 μg/ml,the NRP-1/shRNA interference group of cell growth inhibition rate was increased,the corresponding control group difference had statistical significance(P<0.05).We choose the drug con-centration of the EPI IC50 for next experiments.NRP-1/shRNA interference group cell apoptosis rate increased significantly after induction,compared with the control groups difference was statistically significant ( P<0.05 ).Compared with control group, the expression level of Bcl-2 protein was decreased and the expression level of bax protein was increased significantly after EPI induction.The percentage of cells at G0/G1 phase increased significantly,while those at S phase decreased significantly.Conclusion:Plasmid shRNA-NRP1 inhibited the expression of NRP1 in Jurkat cells and decreased the proliferation level of Jurkat cells and promote their apoptosis and enhance their drug sensitivity;the molecular mechanism may relate to down-regulation of Bcl-2 and up-regulation of Bax.and arrested the cell cycle at G0/G1 phase.

Regeneración axonal posterior a lesiones traumáticas de médula espinal: Papel crítico de galectina-1

Al producirse una lesión de médula espinal (LME), un sinnúmero de proteínas inhibidoras de la regeneración axonal ocupan el sitio de lesión en forma secuencial. La primer proteína en llegar al mismo se conoce como semaforina 3A (Sema3A), siendo además una de las más potentes por su acción de inhibir la regeneración axonal. A nivel mecanístico la unión de esta proteína al complejo-receptor neuronal neuropilin-1 (NRP-1)/PlexinA4 evita que se produzca regeneración axonal. En este trabajo de revisión se discutirá la acción de galectin-1 (Gal-1), una proteína endógena de unión a glicanos, que selectivamente se une al complejo-receptor NRP-1/PlexinA4 de las neuronas lesionadas a través de un mecanismo dependiente de interacciones lectina-glicano, interrumpiendo la señalización generada por Sema3A y permitiendo de esta manera la regeneración axonal y recuperación locomotora luego de producirse la LME. Mientras ambas formas de Gal-1 (monomérica y dimérica) contribuyen a la inactivación de la microglia, solo la forma dimérica de Gal-1 es capaz de unirse al complejo-receptor NRP-1/PlexinA4 y promover regeneración axonal. Por lo tanto, Gal-1 dimérica produce recuperación de las lesiones espinales interfiriendo en la señalización de Sema3A a través de la unión al complejo-receptor NRP-1/PlexinA4, sugiriendo el uso de esta lectina en su forma dimérica para el tratamiento de pacientes con LME.

When spinal cord injury (SCI) occurs, a great number of inhibitors of axonal regeneration consecutively invade the injured site. The first protein to reach the lesion is known as semaphorin 3A (Sema3A), which serves as a powerful inhibitor of axonal regeneration. Mechanistically binding of Sem3A to the neuronal receptor complex neuropilin-1 (NRP-1) / PlexinA4 prevents axonal regeneration. In this special article we review the effects of galectin-1 (Gal-1), an endogenous glycan-binding protein, abundantly present at inflammation and injury sites. Notably, Gal1 adheres selectively to the NRP-1/PlexinA4 receptor complex in injured neurons through glycan-dependent mechanisms, interrupts the Sema3A pathway and contributes to axonal regeneration and locomotor recovery after SCI. While both the monomeric and dimeric forms of Gal-1 contribute to ’switch-off’ classically-activated microglia, only dimeric Gal-1 binds to the NRP-1/PlexinA4 receptor complex and promotes axonal regeneration. Thus, dimeric Gal-1 promotes functional recovery of spinal lesions by interfering with inhibitory signals triggered by Sema3A adhering to the NRP-1/PlexinA4 complex, supporting the use of dimeric Gal-1 for the treatment of SCI patients.

Biodistribution and imaging of 131I labeled anti-neuropilin-1 monoclonal antibody in malignant gliomas xenografts / 中华核医学与分子影像杂志

Objective To synthesize 131I labeled anti-neuropilin-1 monoclonal antibody A6 (131IA6) and evaluate its biodistribution and imaging in malignant glioma xenografts.Methods (1) A6 was labeled with 131I by Iodogen method under the optimum labeling conditions,then the labeling efficiency,radiochemical purity and stability were measured in vitro.(2) In vitro bioactivity,cellular uptake and receptor affinity of 131I-A6 with U87MG cells were measured.(3) The nude mice bearing human U87MG cells were randomly divided into 5 groups with 5 in each group.The nude mice were sacrificed by cervical dislocation and dissected at 24,48,72,96,and 120 h,respectively,after intravenous injection of 1.2 MBq 131I-A6.The biodistribution of the agent was measured as ％ID/g,and the ratios of tumor/blood (T/B) and tumor/muscle (T/M) were calculated.(4) SPECT/CT imaging was performed in 6 mice including 3 in the competitive inhibition control group at 24,48,72,96,and 120 h post injection.Two-sample t test was used for data analysis.Results (1) The labeling yield of 131I-A6 was (95.46±3.34)％,and the radiochemical purity was more than 95％.At 96 h of incubation in PBS,the radiochemical purity was more than 85％.(2)131I-A6 had rapid accumulation in U87MG cells and reached the peak of (15.80±1.30)％ at 1 h.When the probe was incubated with large excesses of non-radioactive A6,the uptake level of 131I-A6 in U87MG cells was significantly inhibited (t=2.862,P＜0.05).Kd of 131I-A6 binding to NRP-1 was (1.67±0.14) nmol/L in U87MG cells.(3) Biodistribution study showed that the uptake in blood,liver and tumor was (8.00±1.42),(7.68±1.56) and (6.00±1.24) ％ID/g at 24 h,respectively.The uptake in muscle,brain and bone was lower.The T/B and T/M were 0.78±0.10 and 3.20±0.30 at 24 h,and they reached the highest level of 1.87±0.50 and 7.13±0.24 at 120 h.(4) The SPECT imaging showed that the tumors could be visualized at 24 h and delineated more clearly at 120 h post injection of 131I-A6.Conclusions 131I-A6 can be easily synthesized by Iodogen method with high radiochemical purity.The specific tumor uptake of 131I-A6,which correlates with NRP-1 expression in gliomas,suggests that it may be a new promising tumor targeting radiotracer.

Construction and identification of recombinant adenovirus vector of human neuropilin-1 gene / 西安交通大学学报(医学版)

Objective To construct a adenovirus vector containing human NRP-1 gene and 3Flag gene to interaction between tumor and interstitial cell.Methods Plasmid containing NRP-1 gene was digested by AgeⅠand NheⅠ restriction endonuclease.Then the DNA was ligated into linearized pDC315-3Flag vector.After having been constructed,the pDC3 1 5-NRP-1-3 Flag plasmid was co-transfected with framework plasmid pBHGlox△E1 , 3Cre into HEK 293 cells to obtain the homologous recombinant adenovirus,which was then amplified and purified its titer tested.Expression of NRP-1 protein was detected using Western blot.Results Polymerase chain reaction and sequencing analysis confirmed that the shuttle plasmid pDC3 1 5-NRP-1-3 Flag and design were consistent. Cytopathic effect was observed by inverted phase contrast after transfecting HEK2 9 3 cells with shuttle plasmid pDC315-NRP-1-3Flag.95-130 ku was detected by Western lot after transfecting HEK293 cells with shuttle plasmid pDC315-NRP-1-3Flag and recombinant adenovirus,the size being consistent with the NRP-1-3Flag fusion protein (104 ku),with a titer of 2.00E+11PFU/mL.Conclusion The recombinant adenovirus vector for human NRP-1 gene was successfully constructed expressed in HEK 2 9 3 cells.

Correlation of Neuropilin-1 and VEGF expression with tumor angiogenesis in human gastric carcinoma / 中国肿瘤临床

Objective:To investigate the expression of Neuropilin-1 (NRP-1) and vascular endothelial growth factor (VEGF) in gastric carcinoma tissue and to examine their relationship with tumor angiogenesis and the biological behavior of gastric carcinoma. Methods: The expressions of NRP-1mRNA and VEGFmRNA were detected by the reverse transcription polymerase chain reaction (RT-PCR) method. Microvascular density (MVD) marked by CD105-positive vascular with SP immunohistochemical staining was also assessed in surgical specimens of gastric carcinoma (GC) and normal gastric tissues (NG). The correlation of NRP-1mRNA, VEGFm-RNA with clinical patholo?gical factors and the relationship among NRP-1mRNA, VEGFmRNA, and MVD were analyzed. Results:The expressions of NRP-1mRNA, VEGFmRNA, and CD105 in GC were significantly higher than those in NG (P<0.01). The expres-sions of NRP-1mRNA and VEGFmRNA were closely correlated with the depth of invasion and lymph node metastasis in GC (P<0.05). A positive correlation was observed between the expressions of NRP-1mRNA and VEGFmRNA (r=0.58, P<0.01). MVD correlated with NRP-1 (r=0.52, P<0.01) and with VEGF (r=0.74, P<0.01). Conclusion:Results indicated that the NRP-1 and VEGF expressions could be involved in the angiogenesis and tumor progression of human gastric carcinoma. The combined detection of NRP-1 and VEGF could also determine the invasion and progression of gastric carcinoma, which plays a particular role in guiding the anti-angiogenic ther-apy of cancer.