EGFL7基因沉默对裸鼠乳腺癌血管生成的影响

Objective To investigate the effect of short hairpin RNA (shRNA) targeting at epidermal growth factor-like domain 7 (EGFL7) gene on angiogenesis of breast carcinoma and its mechanism in nude mice. Methods shRNA targeting at EGFL7 gene was constructed and transfected into SGC-7901 cells (pshEGFL7 group) , meanwhile , the cells transfected with vector plasmids were as a control group. Positive clones were selected and the transplanted tumor animal models constructed in nude mice , and the growth and volume of tumors were observed. After 8 weeks , EGFL7 mRNA and protein in transplanted tumor tissues were detected,and graded. Moreover, Anti-CD34, VEGF and TSP1 were stained by the immuno- chemistry method,and MMP-2 and TIMP2 mRNA were detected by RT-PCR. Results EGFL7 mRNA was down regulated significantly in the pshEGFL7 group. In the psh EGFL7 group, the tumor volume was ( 1.86 ± 0. 65) cm3, MVD was 20. 84 ± 6.38; while in the control group , tumor volume was (4.86 ± 1.15) cm3, MVD was 39.48 ± 9.01, In the EGFL7 group ,TSP1 protein presented positive , and VEGF protein presented weakly positive or negative.The expression of MMP-2 mRNA decreased ,TIMP2 mRNA increased in the pshEGFL7 group,and there were significant differences compared with the control group , P ＜ 0.01. Conclusion RNA interference targeting at EGFL7 gene can balance TSP1/VEGF, through regulating the expression of MMP-2/TIMP2 to impair angiogenesis of breast cancer in nude mice.     

Resveratrol inhibits angiotensin II-induced ERK1/2 activation by downregulating quinone reductase 2 in rat vascular smooth muscle cells

Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells (VSMCs) and repress mRNA and protein expression of quinone reductase 2 (NQO2).This study further explored the potential mechanisms whereby resveratrol inhibits the proliferation of rat VSMCs.Lentiviral vectors that incorporated NQO2 small interfering RNA (siRNA) were constructed and transduced into rat VSMCs.The cell proliferation was detected using the bromodeoxyuridine (BrdU) assay.Cultured rat VSMCs were stimulated with angiotensin II and the level of reactive oxygen species (ROS) was measured using a ROS assay kit.A realtime quantitative PCR was used to detect NQO2 mRNA levels.Extracellular signal-regulated kinase (ERK1/2) and NQO2 protein expression were determined by Western blotting analysis.The inhibitory effect of resveratrol (10 and 50 μmol/L) on the proliferation of rat VSMCs in the NQO2 siRNA group was significantly weaker than that in the normal and scrambled siRNA group (P < 0.01).The ROS level in the NQO2 siRNA and resveratrol (50 μmol/L) treatment groups were lower than that in the normal and scrambled siRNA groups (P < 0.01 in both).Compared with the normal and scrambled siRNA group,the phosphorylation of ERK1/2 was significantly decreased in the NQO2 siRNA and resveratrol (50 μmol/L) treatment group (P < 0.01 in both).In conclusion,high concentration of resveratrol inhibits angiotensin II-induced ERK1/2 phosphorylation and subsequent proliferation by down-regulation of NQO2 in cultured rat VSMCs.     

Construction, Stable Expression of Survivin shRNA Vector

Objective: To construct survivin shRNA expression vector carting enhanced green fluorescent protein gene, transfect it into GBC-SDH cells via electroporation, and get GBC-SD cells which are stable expressing survivin shRNA. Methods: The siRNA sequence targeting survivin mRNA was synthesized and cloned into pEGFP-H1. The constructed plasmid and pEGFP-H1 were transfected into GBC-SD cells respectively via liposome, and the transfecting effect was detected with Flow Cytometry. Then the transfected cells were selected with G418. Results: The recombinant plasmid was successfully constructed, named pEGFP-survivin. The gene transfection efficiencies in pEGFP-H1-transfected group and pEGFP-survivin- transfected group were the 80.29%±2.71% and 83.85%±2.34%(P>0.05), which was successful to get the cells that are stable expressing shRNA, named GBC-SD/EGFP and GBC-SD/survivin. Conclusion: Survivin shRNA expression vector was constructed successfully and got GBC-SD cells which are stable expression shRNA.     

O-linked N-acetylglucosamine transferase (OGT) is overexpressed and promotes O-linked protein glycosylation in esophageal squamous cell carcinoma

The aim of this present study was to explore the expression and clinical significance of O-linked N-acetylglucosamine(O-GlcNAc) transferase(OGT) and enzymatic O-linked glycosylation(O-GlcNAcation) through the addition of O-linked-β-N-acetylglucosamine in esophageal squamous cell carcinoma.OGT expression and O-GlcNAcation in 40 samples from patients with esophageal squamous cell carcinoma was detected by immunohistochemical staining with anti-OGT antib ody and O-GlcNAc-specific antibody RL 2,respectively.The relationship between pathological and clinical factors of patients was analyzed.We found that the expression of OGT was higher in esophageal squamous cell carcinoma samples compared to the normal tissues.RL 2 antibody level was positively correlated with OGT expression,and the metastasis of lymph node,which means the level of O-GlcNAcation was high and related to the metastasis of lymph node in esophageal squamous cell carcinoma.In conclusion,OGT activation is the main reason for promoting the level of O-GlcNAcation in esophageal squamous cell carcinoma.O-GlcNAcylation may play an important role in esophageal squamous cell carcinoma.     

Impact of CCAAT enhancer binding protein α on the differentiation and apoptosis of acute myeloid leukemia HL60 cells

Objective To investigate the effect of CCAAT enhancer binding protein α(C/EBPα) on differentiation and apoptosis in the acute myeloid leukemia HL60 cells in vitro and in vivo and its possible mechanism. Methods The C/EBPα expression plasmid pEGFP-C/EBPα and empty control plasmid were respectively transfected into HL60 cells with cationic liposome as transfected group and empty plasmid transfected group, and untreated HL60 cells served as control group. The cells stably expressing the C/EBPα gene were obtained by G418 selection. The morphological changes were observed under light microscope following WrightGiemsa staining. MTT assay was employed to evaluate cell proliferation, and flow cytometry(FCM) was performed to analyze cell apoptosis. Meanwhile, the expression of c-myc was respectively detected by RT-PCR and Western blot both at the mRNA and protein level. Twenty BALB/c nude mice were divided into 3 groups in a completely randomized design: 7 mice in transfected group, 7 mice in empty plasmid transfected group and 6 in control group. Three kinds of cells including pEGFP-C/EBPα-HL60 cells, pEGFP -HL60 cells and the control HL60 cells were injected into mice separately through the subcutaneous. The mice were sacrificed at 20 d after injection. The mass and size of subcutaneous xenograft tumors were measured and the cell apoptosis of subcutaneous tumor were detected by TUNEL. Results The pEGFP-C/EBPα-HL60 cell line stably expressing the C/EBPα gene was screened out. Compared to either empty plasmid transfected group or control group, the expression of C/EBPα could promote cellular differentiation of HL60. FCM showed higher apoptotic rate in transfected group[ (21.9±4.5)%,P＜0.05 ] ,while (5.4±1.4)% in control group and (5.0±1.3)% in empty plasmid transfected group. c-myc expression was significantly down-regulated by C/EBPα both at the mRNA and protein level. The mass and size of tumors in transfected group were smaller than those in empty plasmid transfected group and control group [ (5.35±1.12)g and(25±4)mm in control group, (5.12±1.31)g and ( 18±3)mm in empty plasmid transfected group ,while (3.26±0.72)g and ( 11±2)mm in transfected group, all P＜0.05]. More apoptosis cells were found in subcutaneous tumor of transfected group(both P＜0.05). Conclusion C/EBPα can not only inhibit the proliferation, but also induce massive apoptosis of HL60 cells, meanwhile C/EBPα is a tumor suppressor of acute myeloid leukemia.     

Electroacupuncture delays articular cartilage degeneration in osteoarthritis via ras-raf-mek1/2-erk1/2 signaling pathway北大核心

BACKGROUND: Previous studies have found that electroacupuncture can delay articular cartilage degeneration mediated by JAK-STAT signaling pathway through upregulating the expression level of transforming growth factor β1 as well as mRNA expression levels of STAT3, Smad3 and LepR. In the meanwhile, electroacupuncture can inhibit the mRNA expression of p38 and Fas mRNA mediated by MAPK signaling pathways, further inhibiting the apoptosis of chondrocytes. OBJECTIVE: To explore the effect of electroacupuncture on the degeneration of articular cartilage in rats with knee osteoarthritis based on Ras-Raf-MEK1/2-ERK1/2 signaling pathway. METHODS: 120 male healthy Sprague-Dawley rats aged 2 months olds were selected and randomly divided into normal, model, 15-minite electroacupuncture and 30-minute electroacupuncture groups (n=30 per group). The rats in the latter three groups received the intra-articular injection of 4% papain bilaterally, and the remaining rats received no intervention. At 2 weeks after modeling, the latter two groups were respectively given 15-and 30-minute electroacupuncture, five times weekly for consecutive 12 weeks. The morphology of the cartilage was observed by hematoxylin-eosin staining, the expression level of interleukin-1β in the synovium was detected by ELISA assay, and the protein expression levels of Ras, Raf, MEK1/2, ERK1/2, C-MYC, C-FOS, and C-JUN were detected by western blot analysis. RESULTS AND CONCLUSION: Hematoxylin-eosin staining showed that: in the model group, the cartilage surface was rough, the cartilage layer became thinner, and the cartilage structure was damaged with incomplete tidal line; in the 15and 30-minute electroacupuncture groups, the cartilage structure was complete with clear layers and complete tidal line. ELISA showed that the expression level of interleukin-1β in the model group was significantly higher than that in the normal group (P < 0.01), and the level in the 15-and 30-minute electroacupuncture groups was significantly lower than that in the model group (P < 0.05). Western blot assay found that compared with the normal group, the protein expression levels of Ras, Raf, MEK1/2, ERK1/2, C-MYC, C-FOS, and C-JUN were increased in the model group. However, all above protein levels except ERK1/2 in the 15-and 30-minute electroacupuncture groups were significantly lower than those in the model group (P < 0.01, P < 0.05). To conclude, electroacupuncture inhibits the degeneration of articular cartilage in osteoarthritis via Ras-Raf-MEK1/2-ERK1/2 signaling pathway and downregulating the expression level of interleukin-1β. © 2017, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.