miR-106b-25 cluster is composed of miR-106b,miR-93 and miR-25,and is a paralogue of miR-17-92 cluster.Some studies have shown that the members in miR-106b-25 cluster abundantly expressed in many cancers.Over-expressions of these miRNAs promote the growth of tumor cells by negatively regulating p21 and p57,and suppress the apoptosis of tumor cells through inhibition of Bim.Moreover,high expression of miR-106b-25 cluster might endue tumor cells with resistance to inhibitory effect of cell growth induced by TGF-β signaling.

ObjectiveTo confirm the effect of miR-93 inhibitor in glioma cell growth and invasion.MethodsMalignant glioma cells were transfected with miR-93 inhibitor by lipofectamin to downregulate their overexpression of miR-93.Real time-PCR was taken to measure miR-93 expression after transfection.The cell cycle kinetics and cell growth rate were detected by flowcytometry and MTT assay,the cell proliferative ability was evaluated by soft agar assay,and the invasive ability was detected by transwell assay.ResultsThe highlevel expression of miR-93 was downregulated effectively in glioma cells after transfecting the miR-93 inhibitor.Meanwhile,the cell cycle progress was delayed,S phase cells were reduced,the speed of growth was slowed,cloning formation ability was receded,the number of cells through the matrigel was reduced,and invasive ability was significantly repressed.ConclusionDownregulation of miR-93 expression could inhibit the proliferative ability and invasive ability of glioma cells.

BACKGROUND: The observation of vascular endothelial growth factor gene expression of cerebrovascular diseases and ultrastructure of cells may be helpful to understand angiogenesis and its relative cellular factors involved in the pathogenesis at cellular and molecular levels. OBJECTIVE: To investigate the method of culture of human cerebral cap illary endothelial cell by separation of capillary fragment in vitro, and to ob serve vascular endothelial growth factor gene expression and ultrastructure of cells. DESIGN: A randomized controlled research on technique and method. SETTING: The neurosurgery department of a general hospital of a military area command of Chinese PLA and the neurosurgery department of a college hospital. PARTICIPANTS: Eighteen patients with arteriovenous malformation of brain(Spetzler Ⅱ-Ⅲ grade), as confirmed by aortocranial angiography before operation, in the Department of Neurosurgery, General Hospital of Shenyang Military Area Command of Chinese PLA were included. The material was obtained from fresh integrated specimen of arteriovenous malfor mation of brain with surrounding fresh brain tissues during the opera tion. Capillary endothelial cell was separated by homogenate, filtration and enzymatic digestion techniques. Cells grew well in culture flask and were divided into 4 groups(hypoxia state for 2, 4, 8 hours groups and control group). Each group contained four flasks.METHODS: Simulation of anoxia condition: volume faction 0.95 N2 and volume fraction 0.05 CO2. Expression of factor Ⅷ related antigen in cells was detected by immunohistochemistry. mRNA expression of vascular endothelial growth factor on endothelial in every group was observed by RT-PCR, protein content of vascular endothelial growth factor in supernatant detected by enzyme-linked immunoadsordent assay, and cellular ultrastructural change observed by transmission electron microscopy.MAIN OUTCOME MEASURES: mRNA expression of vascular endothelial growth factor on endothelial cell and protein content of vascular endothelial growth factor in supernatant in control group and every hypoxia groups; cellular ultrastructural changes.RESULTS: Under phase contrast microscope, cultured living cells had mono-layer pebble-like typical character. More than 90% of were factor Ⅷrelated antigen(FⅧ-RA) staining positive. mRNA and protein expression of vascular endothelial growth factor in hypoxia 4 hours group was 0.98 ±0. 19,( 180. 77 ± 20. 15) ng/L, which was significantly higher than in control group [0, (26. 20 ± 6.33) ng/L, P ＜ 0.01 ]. Eight hours later, expression decreased [(0. 35 ±0.07), (31.68 ±8.34) ng/L]; swollen mitochondrion, dilated endoplasmic reticulum, and lysosome vesiculation were found.CONCLUSION: Humane cerebral capillary endothelial cell can be cultured by separation of capillary fragment, which is easy to operate and the cellular purity is reliable. In the early stage of ischemia and hypoxia, expression of vascular endothelial growth factor is not enough to maintain cellular ultrastructure integrity. Cells may be injured along with the prolong of hypoxia.Zhao MG, Tang T, Gao YZ, Pu PY, Wei XZ. Culture of human cerebral capillary endothelial by separation of capillary fragment and the observation of vascular endothelial growth factor gene expression and cell ultrastructure. Zhongguo Linchuang Kangfu 2005; 9(21):211-3 (China) [www. zglckf. com]

Objective To investigate the gene expression of vascular endothelial growth factor(VEGF) and ultrastructural changes in cultured endothelial cells from human cerebral microvessels under hypoxic conditions.Methods Human cerebral microvessels were isolated from freshly obtained specimens of normal brain adherent to resected cerebral arteriovenous malformations.The expression of factor Ⅷ-relative antigen(FⅧ-RA) in cultured cells was observed with immunocytochemistry.The level of VEGFmRNA in cells and released VEGF protein in cell supernatant were determined by RT-PCR analysis and ELISA respectively when they were exposed to hypoxic conditions(95% N_2,5% CO_2;two hours,four hours,eight hours) or maintained in basal condition.Ultrastructural changes in cells were also observed by electron microscopy.Results In inverted microscope the cultured cells showed contact inhibition and a rounded cobblestone appearance.More than 90% of them were stained strongly with antibodies against FⅧ-RA.Significant VEGF mRNA and protein accumulated when these cells were exposed to hypoxia for 4 hours.However,their VEGF expression was down-regulated after hypoxia for 8 hours and a number of vesicles and swollen mitochondria were present in the cytoplasm.Conclusion The level of VEGF expression may havesignificant relationship with ultrastructural changes in human cerebral endothelial cells under hypoxic conditions.

Objective:This study aimed to explore the effect of Yes-associated protein (YAP) on the growth of the human glioma cell line LN229. Methods:YAPsiRNA was transfected into LN229 cells to knock down the YAP expression. The downregulation of the YAP expression was identified through Western Blot analysis. Colorimetric assay using methyl-thiazolyl-tetrazolium was applied to evaluate cell proliferation ability. Cell invasive activity was examined using Transwell assay. Flow cytometry and AnnexinV were used to detect cell cycle and apoptosis, respectively. The relevant molecules regulating proliferation, invasion, cell cycle progression, and apoptosis were examined through Western Blot analysis. Results:The YAP expression was downregulated after YAPsiRNA was trans-fected into LN229 glioma cells. Reduced YAP expression could arrest the cell cycle at G0/G1 phase, inhibit cell proliferation and inva-sion, and promote apoptosis. The expression of the proliferating cell nuclear antigen (Ki-67), matrix metallopeptidase-9 (MMP-9), cy-clin D1, and Bcl-2 were downregulated. Conclusion:The downregulation of YAP in LN229 cells suppresses cell proliferation and inva-sion, as well as promotes cell apoptosis. This study provides a novel evidence for further study on Hippo-YAP signal pathway in molec-ular pathology of glioma.

cells, the expression of PTEN was up-regulated while pAkt down-regulated. Conclusions AS-miR-221 and AS-miR-222 may enhance the radiosensitivity of MCF-7 breast cancer cells by up-regulating the expression of PTEN.

objective To identify SEPT7as one of the target genes of miR-19a and miR-19b.MethodsmiR-19a inhibitor and miR-19b inhibitor mediated by lipofectamine2000,were transfected to SNB19 glioma cells for knocking down miR-19a/19b overexpression.Real time PCR was conducted to detect the expression of miR-19a/miR-19b in transfected cells.The expression of SEPT7was determined by Western blot analysis.RT-PCR was used to detect the mRNA expression of SEPT7; and Luciferase reporter assay was used to identify the direct regulation of miR-19a/19b on SEPT7.ResultsIn SNB19 glioma cells transfected with miR-19a/19b inhibitor,the expression of miR-19a/miR-19b was significantly reduced,whereas the protein expression of SEPT7 was upreguhtted; no significant change of SEPT7 mRNA level was found and luciferase activity became stronger as compared to control cells.ConclusionSEPT7 is the target negatively regulated by miR-19a and miR-19b.

Objective To investigate the effect of knocking-down Yes-associated protein (YAP)on the biologi-cal characteristics of SNB19 glioblastoma cell.Methods The expression of YAB in SNB19 was knockdown by YAB small interfering RNA (YABsiRNA).The downregulation of YAP expression was identified by Western blot analysis. The proliferative ability of cell was determined by methyl thiazoyl terazolium (MTT).The invasive ability of cell was examined by Transwell assay.Flow cytometry and Annexin V staining were used to detect the cell cycle and apoptosis respectively.The results were analyzed by the statistical software SPSS18.0.Results The expression of YAP in the cells transfected with YAPsiRNA was significantly reduced.The cell proliferation activity of SNB19 cells was inhibited, which decreased from (100.00 ±0.00)%to (52.32 ±3.10)%(F=33.00,P<0.01).The cell cycle was arrested in G0-G1 phase (F=8.76,P<0.01).The cell invasive ability was attenuated apparently,which decreased from (163.20 ±10.10)to (37.71 ±2.52)(F=282.05,P<0.01).The apoptosis ratio of the tumor cell which transfected with YAPsi-RNA was increased from (3.56 ±0.35)%to (18.99 ±0.66)%,(F=931.99,P<0.01).Conclusion Knocking-down YAP expression in glioma cells could inhibit the proliferative activity and invasive ability of SNB19 cell and could induce cell apoptosis.YAP could be served as a potential target for the gene therapy of glioma.

Objective To study the inhibitory effect of knocking down miR-30a-5p on the U87 human glioma xenograft growth and its possible mechanism.Methods Nude mice bearing subcutaneous U87 human glioblastoma were established and separated into three groups (eight for each group) by randomized digital table method,including control group,scr-ODN treated group and AS-miR-30a-5p treated group.After relevant subcutaneous injection treatment,tumor size was measured every other day until the observation period ended.Researchers executed the animals after the treatment,stripped tumor tissues and extracted RNA and protein.Real-time PCR was conducted to detect the expression of miR-30a-5p.The histopathological characteristics and proliferation and apoptosis biological characters (including SEPT7,PCNA,cyclin D1,MMP-2,apoptosis related factor P53,bcl-2 and caspase3) were evaluated by HE and immunohistochemical staining,Westem blot analysis respectively,and the cell apoptosis was detected by TUNEL method.Results In AS-miR-30a-5p treated group,the tumor growth was delayed and the final tumor volume was smaller than that in the control and scr-ODN treated group (F =7.167,P ＜0.05),and the expression of miR-30a-5p was knocked down.The expression of PCNA,cyclin D1 were significantly downregulated while P53,SEPT7 and caspase3 up-regulated.Apoptotic index was increased significantly.Conclusion As-miR-30a-5p suppresses the growth of U87 human gliomas xenografts significantly.Malignant phenotype of tumors are reversed to a considerable degree.Therefore,miR-30a-5p can be a candidate for targeted therapy of human glioma.

Objective Increasing evidence suggest that aberrant activation of PI3K/Akt is involved in many human cancers, and that inhibition of the PI3K/Akt pathway might be a promising strategy for cancer therapy. The study is to evaluate the effects of combined therapy of PI3K inhibitor (LY294002) and Ad-PTEN in athymic mice xenogeneic transplant model of human glioma and to reveal the possible mechanisms involved.Methods Twenty-four athymic mice were randomly divided into 4 groups (DMSO、Ad-vector plus DMSO、LY294002 alone and Ad-PTEN plus LY294002), and were treated, respectively. Athymic mice xenogeneic transplant model was established by inoculation (sc) with LN229 glioma cells. Body mass (BM) and diameter of tumor mass were measured. Furthermore, The protein expressions of PTEN、p-Akt、CyclinD1、Caspase-3、MMP-2、p-FAK in tumor tissues were analyzed with immunohistochemistry.Results The tumor-inhibiting rate of was significantly higher in Ad-PTEN plus LY294002 than in the LY294002 alone (92.46 vs 65.59%)( P <0.05).The protein expressions of PTEN and Caspase-3 were significantly higher, while PCNA、CyclinD1、bcl-2 and MMP-2、p-FAK was significantly lower in Ad-PTEN plus LY294002 group than in the other three groups ( P <0.05).Conclusions LY294002 plus Ad-PTEN achieve better outcome than either alone in treating glioma possibly through enhancement of the inhibitory action of PI3K/Akt pathway and Ad-PTEN pathway.