[Show abstract][Hide abstract] ABSTRACT: Glioblastoma is the most common primary central nervous system malignancy and its unique invasiveness hinders effective treatment. Its high invasiveness may be controlled partly by microRNAs (miRNAs, miRs) and their target genes. In the present study, we found that increased miR-223 expression and reduced PAX6 expression coexisted in glioblastoma as detected by quantitative PCR or tissue microarrays. We confirmed that miR-223 directly targets PAX6 through binding to its 3'-UTR using dual luciferase reporter assay. In U251 and U373 glioblastoma cells, overexpression of miR-223 decreased PAX6 mRNA and protein expression; however, inhibition of miR-223 increased PAX6 mRNA and protein expression. Moreover, overexpression of miR-223 led to effects similar to those of PAX6 knockdown: increased cell viability, increased percentage of cells in the G1 phase and increased cell invasiveness parallel with increased MMP2, MMP9 and VEGFA expression. In addition, inhibition of miR-223 resulted in effects similar to those of PAX6 overexpression: decreased cell viability, decreased percentage of cells in the G1 phase and decreased cell invasiveness parallel with reduced MMP2, MMP9 and VEGFA expression. The data presented here suggest that miR-223 promotes the growth and invasion of U251 and U373 glioblastoma cells by targeting PAX6, which serves as a tumor suppressor in glioblastoma exerting the functions of inhibition of cell cycle transition, and the expression of MMP2, MMP9 and VEGFA. In conclusion, the present study supports miR-223 and PAX6 as novel therapeutic targets for glioblastoma.
[Show abstract][Hide abstract] ABSTRACT: To investigate the effect of maslinic acid preconditioning against injuries of rat cortical neurons induced by oxygen-glucose deprivation (OGD).
The cortical neurons were isolated from Sprague-Dawley rat embryos at 15-17 days of gestation for primary culture. The cortical neurons were incubated with different concentrations (0.1, 1, and 10 micro;mol/L) of maslinic acid prior to OGD. The cell damage and viability were evaluated for lactate dehydrogenase (LDH) leakage and using MTT assay, respectively, and the expression of Bax protein was detected using Western blotting.
OGD significantly increased LDH release rate and decreased the viability of the cells. After preconditioning with maslinic acid (1 and 10 micro;mol/L), LDH leakage rate was decreased and cell the viability increased in cells exposed to OGD. Western blotting showed that Bax expression in the cells decreased as maslinic acid concentrations increased.
Pretreatment with maslinic acid can protect cultured embryonic rat cortical neurons against OGD-induced injury possibly in relation to decreased expression of Bax.
No preview · Article · Mar 2013 · Nan fang yi ke da xue xue bao = Journal of Southern Medical University
[Show abstract][Hide abstract] ABSTRACT: The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network, and the challenge to understand the network is to identify p53 downstream genes. In order to isolate and identify new p53 regulated genes, we constructed and characterized a normalized cDNA library from human brain glioma cell line U251 while exogenous p53 gene is overexpressed. The constructed cDNA library contained 1.3×10 6 directional recombinants, and its insert size ranged from 0.5 to 2.0 kb. Screening the cDNA library, we obtained two novel p53 downstream genes, PAP1 and PAP2. Polymerase chain reaction (PCR) analyses of the library for specific genes revealed the presence of cDNAs for p53 downstream genes such as p21, gadd45, and PCNA. These results demonstrate the sequence complexity and relatively low redundancy of our cDNA library. It is a valuable and unique resource for studying p53 gene expression, regulatory mechanisms and screening p53 downstream genes.
[Show abstract][Hide abstract] ABSTRACT: The present study was conducted to investigate the effect of hepatocyte growth factor (HGF) on cortical neurons exposed to oxygen-glucose deprivation/reperfusion (OGD/R). Primary cultured cerebral cortical neurons were prepared from Sprague-Dawley rats. The cells were used for experiments after culture for 12 d in vitro. To initiate OGD/R, the culture medium was replaced by glucose-free medium, and cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N(2) and 5% CO(2) at 37 °C for 2 h. Following this treatment, neurons were fed with glucose-supplemented (25 mmol/L) medium, and returned to the incubator under normoxic condition for 0-24 h. The cell viability was assessed by MTT assay, and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. The percentage of apoptotic cells was analyzed by flow cytometry and Hoechst 33258 staining. The expressions of c-Met mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. Oxygen-glucose deprivation for 2 h decreased the cell viability and increased LDH leakage rate in cultured cerebral cortical neurons. The cell viability declined and LDH leakage rate increased with the reperfusion time going on (0-24 h). To explore the influence of HGF on neurons under oxygen-glucose deprivation for 2 h/reperfusion for 24 h (OGD(2)/R(24)) condition, the cultures were pretreated with HGF at different concentrations (5-120 ng/mL) 2 h prior to OGD(2)/R(24). The results showed that OGD(2)/R(24) treatment significantly decreased the cell viability, increased LDH leakage rate and the percentage of apopototic cells. Pretreatment with HGF at 5 ng/mL and 10 ng/mL did not affect the decrease in cell viability resulting from OGD(2)/R(24). In the presence of 20 ng/mL HGF, the increase in cell viability in cortical neurons exposed to OGD(2)/R(24) began to appear, and 80 ng/mL of HGF exhibited the maximal effect. HGF at 5, 10 and 20 ng/mL did not affect the increase in LDH leakage rate in cortical neurons exposed to OGD(2)/R(24). In the presence of 40 ng/mL HGF, the decrease in LDH leakage rate in cortical neurons subjected to OGD(2)/R(24) began to appear, and 80 ng/mL of HGF displayed the maximal effect. In addition, HGF at 80 ng/mL significantly attenuated cell apoptosis resulting from OGD(2)/R(24). As detected by semi-quantitative RT-PCR and Western blot analysis, c-Met mRNA and protein were expressed in cerebral cortical neurons cultured for 12 d in vitro. c-Met mRNA and protein expressions in cortical neurons exposed to OGD(2)/R(24) were significantly upregulated and were not affected by pretreatment of HGF at 80 ng/mL. Treatment with c-Met inhibitor SU11274 (5 μmol/L) completely eliminated HGF-mediated protection of cortical neurons subjected to OGD(2)/R(24). The results suggest that HGF directly protects cortical neurons against OGD/R-induced cell injury in a dose-dependent manner, and HGF has a potent anti-apoptotic action on neurons exposed to OGD/R.
Preview · Article · May 2008 · Sheng li xue bao: [Acta physiologica Sinica]
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte growth factor (HGF) has been revealed to exert multipotent activities on a variety of cells. In this study, we investigated whether HGF had a direct neuroprotection on cultured cerebral cortical neurons subjected to hypoxia/reoxygenation (H/R) and explored the intracellular signalings mediated the effects. The decrease in cell viability and increase in number of apoptotic cells resulting from H/R were significantly prevented by HGF pre-treatment. HGF stimulated both ERK1/2 and Akt activities in cortical neurons. Inhibition of ERK activation completely abolished the protective effects of HGF, and inhibition of Akt activation reduced, but did not completely eliminate the HGF mediated neuroprotection. It is suggested that the neuroprotection of HGF depend on ERK1/2 pathway, and, to a lesser extent, PI-3K/Akt pathway. In addition, we found that pre-treatment with HGF remarkably attenuated the decrease in expression of Bcl-2 and Bcl-xL induced by H/R, but failed to affect the amount of Bax. It is likely that Bcl-2 and Bcl-xL contribute to the protective effects of HGF.
No preview · Article · Mar 2008 · Colloids and surfaces B: Biointerfaces
[Show abstract][Hide abstract] ABSTRACT: To explore the feasibility of a bone cancer pain model by injecting the Lewis lung carcinoma cells into the femur bone marrow cavity of C57BL/6 mice.
Sixty clear grade male C57BL/6 mice (body weight 18 approximately 20 g) were randomly divided into 4 groups(15 in each group). Cancer cell inoculated group: 2*10(6) Lewis lung carcinoma cells in 10 microL PBS were injected into the left femur bone marrow cavity, and the other 3 control groups were injected the heat inactivated Lewis cells, PBS, or a false operation respectively. Spontaneous lifting time and mechanical allodynia threshold of the mice hind paw were measured in the alternative days throughout the experiment. The structural damage of the femur was monitored by radiogram on the 7th,15th, and 23rd day respectively,and the pathohistological changes of the femur bones were observed by HE staining on the same days.
Those mice that received intra-femur innoculation of Lewis lung carcinoma cells gradually developed the spontaneous pain, which was began on the 11th day after the innoculation, and followed by mechanical allodynia. The course of flinch lasted in the later experimental session. The 50% Von Frey threshold was significantly decreased on the 13th day after the innoculation, and the mechanical allodynia lasted the whole experimental period. On the 23rd day after the innoculation, X-ray film showed that the medullary cavity of ipsilateral distal femur was filled with tumor cells, and the cortical bone became thick; furthermore, the tumor cells invaded the peripheral muscles.
Injecting the Lewis lung carcinoma cells into the femoral medullary cavity of C57BL/6 mice can successfully establish a murine bone cancer pain model, and the murine model shows much resemblance compared with the human bone cancer pain.
No preview · Article · Mar 2008 · Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
[Show abstract][Hide abstract] ABSTRACT: To explore the mechanisms of hypoxic preconditioning on protecting cultured astrocytes from hypoxia injury.
Cultured astrocytes were divided randomly into several groups: control(C), hypoxia(H) and hypoxic preconditioning (HP). Cells MTT metabolic activity, qualitation of apoptosis and modality to explore the protection effects of hypoxic preconditioning. Immunocytochemistry of Bcl-2 and Bax to explore the mechanisms of hypoxic preconditioning on protecting astrocytes from hypoxia.
Compared with H group there was marked increase of MTT metabolic activity in HP48 and HP72 groups. Immunocytochemistry of Bcl-2 and Bax showed that compared with H group, expression of Bcl-2 was increased in HP group, while expression of Bax was decreased in HP group.
Hypoxic preconditioning can protect astrocytes from hypoxia. One possible mechanism maybe concerned with inhibition of Bax and maintain of Bcl-2 to depress apoptosis procedure.
No preview · Article · Feb 2008 · Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of applied physiology
[Show abstract][Hide abstract] ABSTRACT: To observe the effect of thrombin on the cytotoxicity of astrocytes injured by hypoxia/reoxygenation(H/R) and to explore its relationship with inducible nitric oxide synthase (iNOS).
Primary astrocytes were cultured in DMEM with 10% approximately 15% calf serum and divided into 6 groups: a control group, a Tm control group, an H/R group, a Tm+H/R group, a hirudin (HR) control group, and a Tm+HR+ H/R group. The cell damage and viability were detected by the 3-(4, 5-dimethylthazol-2-yl)-2, 5 diphenyl tetrazo liumbromide (MTT) conversion method. The NO level in the cultured cell supernatant was assayed by Griess reagent. The flow cytometry was performed to evaluate the apoptosis rate of astrocytes. The iNOS mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemistry was used to observe the expression of iNOS protein.
The cell viability injured by H/R was lower than that of the control group, the NO production and apoptosis rate in the cell of H/R group were higher than those of the control group. Incubation of H/R cell with 10kU/L Tm enhanced the cytotoxicity of H/R stimulation compared with the cells injured by H/R. Hirudin can reverse the effect of thrombin. RT-PCR and immuneocytochemistry analysis demonstrated that the levels of iNOS mRNA and iNOS protein increased in the cells treated by H/R. Tm enhanced the expression of iNOS mRNA and iNOS protein in the cells treated by H/R. Hirudin blocked the effect of Tm.
Increasing the level of iNOS and enhancing the production of NO may be the mechanism of thrombinos cytotoxicity in astrocytes injured by H/R.
No preview · Article · Nov 2007 · Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
[Show abstract][Hide abstract] ABSTRACT: To explore the effect of thrombin precondition (TPC) on the rat cerebral astrocytes(As) cultured in oxygen-glucose deprivation (OGD).
Astrocytes were pretreated with thrombin (TB) at various concentrations (0.005 approximately 5.000 kU/L), and then insulted by OGD. The cell damage and viability were evaluated by the lactate dehydrogenase (LDH) effusion rate and the 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion method. Detection of apoptotic cells was determined by the flow cytometry technique. The glutamate uptake of astrocytes was studied with [3H]-glutamate incorporation.
OGD increased the LDH, decreased the cell viability, increased the number of apoptotic astrocytes, and decreased the glutamate uptake (P<0.01). While preconditioned with thrombin at the same condition, the LDH decreased, the cell viability increased, the percentage of apoptotic cells decreased, and the glutamate uptake increased (P<0.05). The maximum protective effect of thrombin was observed at 0.1 kU/L.
Low concentration of thrombin precondition (TPC) can protect the astrocytes from oxygen-glucose deprived injury, and attenuate its apoptosis in a dose-dependent manner.
No preview · Article · Oct 2007 · Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
[Show abstract][Hide abstract] ABSTRACT: The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network. p53 is a key molecular node in the network, which is activated in response to several cellular signals resulting in the maintenance of genetic stability. Several cellular signals may activate the p53 network. When the expression of P53 is elevated, P53-MDM2 module and the ubiquitin system can accurately regulate the expression level of P53. P53 can bind to specific DNA sequence, activate its downstream genes expression, and control cell-cycle arrest, DNA repair, and apoptosis. Elucidating the function of p53 gene network will help understand the interaction mechanisms of p53 and its downstream genes.
No preview · Article · Apr 2007 · Colloids and surfaces B: Biointerfaces
[Show abstract][Hide abstract] ABSTRACT: We have identified PAP1 gene, a novel member of the immunoglobulin superfamily (IGSF) from U251-pTet-p53 cell line, which carried a wild-type p53 transgene. The gene has been localised to chromosome 16p12-13. Alignment of the predicted protein sequence for Human, Pan troglodytes, Canis, Mus musculus and Gallus gallus revealed it was highly conserved. Its homologue, IGSF6, possible involves in mouse embryonic development. The presence of IGSF6 specific transcript was detected by Northern blot in the RNAs extracted from 11 to 14 day postconception. IGSF6 expression is different in mouse embryos of the different ages. In situ hybridization performed on mice embryos sections showed the differential presence of IGSF6 in developing lung and kidney. This structure and differential expression suggests a function involvement in embryonic development, perhaps involvement in cell proliferation.
No preview · Article · Oct 2006 · Colloids and surfaces B: Biointerfaces
[Show abstract][Hide abstract] ABSTRACT: To investigate the relationship between MICA*008/A5.1 allele and human cytomegalovirus (HCMV) infection in kidney transplanted donees of Hunan Han nationality.
The MICA*008/A5.1 allele based on 91 kidney transplanted donees and 81 unrelated normal individuals of Han nationality in Hunan Province were analyzed by PCR/SSP assay. At the same time, anti-HCMV antibody IgM was detected in the serum by ELISA method.
The positive rate of MICA*008/A5.1 allele was significantly higher in the control group (56.79%) than that in the kidney transplanted donee group (34.07%) (P <0.05). The infection rate of HCMV in those individuals whose genotype was MICA*008/A5.1 (-) was significantly higher than that in the MICA*008/A5.1(+).
The individual whose genotype is MICA*008/A5.1 (+) is not liable to HCMV infection, but the individual whose genotype is MICA*008/A5.1 (-) is liable to HCMV infection.
No preview · Article · Aug 2006 · Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences
[Show abstract][Hide abstract] ABSTRACT: To explore the effect of hepatocyte growth factor (HGF) on oxygen-glucose deprived injury and apoptosis of astrocytes.
The injury of primary cultured rat cerebral cortical astrocytes was induced by oxygen-glucose deprivation. Astrocytes were treated with HGF at various final concentrations of 20 - 100 ng/mL. The cell damage and viability were evaluated by the lactate dehydrogenase (LDH) released rate and the 3- (4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) conversion method. Detection of apoptotic cells was determined by the flow cytometry, and the ultrastructure was observed by the transmission electron microscope.
Oxygen-glucose deprivation increased the LDH release rate, decreased the cell viability and increased the number of apoptotic astrocytes. While exposed to HGF at the same condition, the LDH release rate decreased, the cell viability increased, and the percentage of apoptotic cells decreased (P <0.05). The maximum protective effect of HGF was observed at 60 ng/mL.
HGF can protect cultured astrocytes from oxygen-glucose deprived injury, and attenuate the apoptosis of astrocytes in a dose-dependent manner.
No preview · Article · Jun 2005 · Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences