The time-dependent variations of zebrafish intestine and gill after polyethylene microplastics exposure.

Microplastics (MPs) are common environmental contaminants that present a growing health concern due to their increasing presence in aquatic and human systems. However, the mechanisms behind MP effects on organisms are unclear. In this study, zebrafish (Danio rerio) were used as an in vivo model to investigate the potential risks and molecular mechanisms of the toxic effects of polyethylene MPs (45-53 µm). In the zebrafish intestine, 6, 5, and 186 genes showed differential expression after MP treatment for 1, 5, and 10 days, respectively. In the gills, 318, 92, and 484 genes showed differential expression after MP treatment for 1, 5, and 10 days, respectively. In both the intestine and the gills, Gene Ontology (GO) annotation showed that the main enriched terms were biological regulation, cellular process, metabolic process, cellular anatomical entity, and binding. KEGG enrichment analysis on DEGs revealed that the dominant pathways were carbohydrate metabolism and lipid metabolism, which were strongly influenced by MPs in the intestine. The dominant pathways in the gills were immune and lipid metabolism. The respiratory rate of gills, the activity of SOD and GSH in the intestine significantly increased after exposure to MPs compared with the control (p < 0.05), while the activity of SOD did not change in the gills. GSH activity was only significantly increased after MP exposure for 5 days. Also, the MDA content was not changed in the intestine but was significantly decreased in the gills after MP exposure. The activity of AChE significantly decreased only after MPs exposure for 5 days. Overall, these results indicated that MPs pollution significantly induced oxidative stress and neurotoxicity, increased respiratory rate, disturbed energy metabolism and stimulated immune function in fish, displaying an environmental risk of MPs to aquatic ecosystems.

[Expression and role of Pim1 in cultured cortical neurons with oxygen-glucose deprivation/reoxygen injury].

OBJECTIVE: To study the expression and effect of Pim1 in primary cortical neurons after hypoxic-ischemic injury. METHODS: Cortical neurons were isolated from 1-day-old C57BL/6 mice and cultured in neurobasal medium. On the 8th day of neuron culture, cells were subjected to oxygen-glucose deprivation/reoxygen (OGD/R) treatment to mimic in vivo hypoxic injury of neurons. Briefly, medium were changed to DMEM medium, and cells were cultured in 1% O2 for 3 hours and then changed back to normal medium and conditions. Cells were collected at 0 hour, 6 hours, 12 hours and 24 hours after OGD/R. Primary neurons were transfected with Pim1 overexpression plasmid or mock plasmid, and then were exposed to normal conditions or OGD/R treatment. They were named as Pim1 group, control group, OGD/R group and OGD/R+Pim1 group respectively. Real-time PCR was used to detect Pim1 mRNA expression. Western blot was used to detect the protein expression of Pim1 and apoptotic related protein cleaved caspase 3 (CC3). TUNEL staining was used to detect cell apoptosis. RESULTS: Real-time PCR and Western blot results showed that Pim1 mRNA and protein were significantly decreased in neurons after OGD/R. They began to decrease at 0 hour after OGD/R, reached to the lowest at 12 hours after OGD/R, and remained at a lower level at 24 hours after OGD/R (P<0.01). Overexpression of Pim1 significantly upregulated the protein level of Pim1. Under OGD/R conditions, the CC3 expression and the apoptosis rate in cells of the Pim1 group were significantly lower than in un-transfected cells (P<0.01). CONCLUSIONS: Hypoxic-ischemic injury may decrease Pim1 expression in neurons. Overexpressed Pim1 may inhibit apoptosis induced by OGD/R.

[Effect of irisin on hypoxic-ischemic brain damage in neonatal rats].

OBJECTIVE: To study the effect and mechanism of action of irisin on hypoxic-ischemic brain damage in neonatal rats. METHODS: A total of 248 7-day-old Sprague-Dawley rats were randomly divided into a sham-operation group, a model group, and low- and high-dose irisin intervention groups (n=62 each). The rats in the model and irisin intervention groups were given hypoxic treatment after right common carotid artery ligation to establish a model of hypoxic-ischemic brain damage. Those in the sham-operation group were given the separation of the right common carotid artery without ligation or hypoxic treatment. The rats in the high- and low-dose irisin intervention groups were given intracerebroventricular injection of recombinant irisin polypeptide at a dose of 0.30 µg and 0.15 µg respectively. Those in the model and sham-operation groups were given the injection of an equal volume of PBS. The water maze test was used to compare neurological behaviors between groups. TTC staining, hematoxylin-eosin staining and TUNEL staining were used to observe histopathological changes of the brain. Western blot was used to measure the expression of the apoptosis-related molecules cleaved-caspase-3 (CC3), BCL-2 and BAX. RESULTS: Compared with the sham-operation group, the model group had a significant increase in latency time and a significant reduction in the number of platform crossings (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significant reduction in latency time and a significant increase in the number of platform crossings (P<0.05). Compared with the sham-operation group, the model group had massive infarction in the right hemisphere, with significant increases in karyopyknosis and karyorrhexis. Compared with the model group, the high-dose irisin intervention group had a smaller infarct area of the right hemisphere, with reductions in karyopyknosis and karyorrhexis. The model group had a significantly higher apoptosis rate of cells in the right cerebral cortex and the hippocampus than the sham-operation group. The high-dose irisin intervention group had a significantly lower apoptosis rate than the model group (P<0.05). At 24 and 48 hours after modeling, the sham-operation group had a significantly lower level of CC3 than the model group (P<0.05). Compared with the model group, the high-dose irisin intervention group had a significantly lower level of CC3 and a significantly higher BCL-2/BAX ratio (P<0.05). The low-dose irisin intervention group had similar laboratory markers and histopathological changes of the brain to the model group. CONCLUSIONS: Irisin can alleviate hypoxic-ischemic brain damage in neonatal rats in a dose-dependent manner, possibly by reducing cell apoptosis in the cerebral cortex and the hippocampus.

[Investigation of Lentiviral Vectors Based Integrin ß8 RNAi System in Neonatal Rats' Brain].

OBJECTIVE: To construct lentiviral vectors expressing pSicoR-ß8 shRNA and evaluate its efficiency of RNA interference in neonatal rats' brain. METHODS: Plasmid vectors pSicoR-ß8 shRNA and pSicoR-control,as well as lentiviral packaging system pDM2G,g/p RRE and pRSV Rev were amplified respectively and plasmid DNA was identified by restriction enzyme digestion. Lentiviral packaging system and expressing vector pSicoR-ß8 shRNA/pSicoR-control were co-transfected into packaging cell line 293T. Lentiviral particles expressing ß8-shRNA or control sequence packaged and secreted by 293T were collected,concentrated by PEG-it,and viral titers were assayed by 50% tissue culture infective dose (TCID50). RNAi for integrin ß8 in neonatal rats' brain was performed by intraventricular injection of lentivirus expressing ß8-shRNA and rats received lentivirus expressing ß8-shRNA were served as control. Green fluorescent protein (GFP) expression after intraventricular injection of GFP-Lentivirus was observed under fluorescence microscope,ß8 mRNA and ß8 protein expression were detected by RT-PCR and Western blot respectively,all of which were performed to evaluate the RNAi efficiency and to choose the optimal time for intervention. RESULTS: Restrictive endonuclease digestion and agarose gel electrophoresis showed plasmids as same as the expected size. Lentiviral titers for LV-control after concentration was 1.0×108 PFU/mL,and for LV-ß8 shRNA 5.0×108 PFU/mL.One day after intraventricular injection of lentiviral vectors containing GFP sequence,lenticivirus genome was integrated into host cells and emitted green fluorescence. A relatively strong green fluorescence could be observed in brain slides 2 d,3 d and 5 d after intraventricular injection. Western blot and RT-PCR demonstrated a maximum inhibition happened 3 d after intraventricular injection of LV-ß8 shRNA,the inhibitory rate for ß8 mRNA and ß8 protein were 56% and 51%,respectively. CONCLUSION: Lentiviral vectors expressing ß8-shRNA are successfully constructed and lentiviral mediated ß8-RNAi is successfully applied for in vivo use.

[Expression rhythm of autophagic gene in neurons of neonatal rats with hypoxia/ischemia and its regulatory mechanism].

OBJECTIVE: To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia. METHODS: Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique. RESULTS: The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05). CONCLUSIONS: Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.

[Effect of telomerase activation on biological behaviors of neural stem cells in rats with hypoxic-ischemic insults].

OBJECTIVE: To investigate the effect of telomerase activation on biological behaviors of neural stem cells after hypoxic-ischemic insults. METHODS: The neural stem cells passaged in vitro were divided into four groups: control, oxygen-glucose deprivation (OGD), OGD+cycloastragenol (CAG) high concentration (final concentration of 25 µM), and OGD+CAG low concentration (final concentration of 10 µM). The latter three groups were subjected to OGD. Telomerase reverse transcriptase (TERT) expression level was evaluated by Western blot. Telomerase activity was detected by telomerase repeat amplification protocol (TRAP). Cell number and neural sphere diameter were measured under a microscope. The activity of lactate dehydrogenase (LDH) was examined by chemiluminescence. Cell proliferation rate and apoptosis were detected by flow cytometry. RESULTS: After OGD insults, obvious injury of neural stem cells was observed, including less cell number, smaller neural sphere, more dead cells, lower proliferation rate and decreased survival rate. In CAG-treated groups, there were higher TERT expression level and telomerase activity compared with the control group (P<0.05). In comparison with the OGD group, CAG treatment attenuated cell loss (P<0.05) and neural sphere diameter decrease (P<0.05), promoted cell proliferation (P<0.05), and increased cell survival rate (P<0.05). Low and high concentrations of CAG had similar effects on proliferation and survival of neural stem cells (P>0.05). In the normal cultural condition, CAG treatment also enhanced TERT expression (P<0.05) and increased cell numbers (P<0.05) and neural sphere diameter (P<0.05) compared with the control group. CONCLUSIONS: Telomerase activation can promote the proliferation and improve survival of neural stem cells under the state of hypoxic-ischemic insults, suggesting telomerase activators might be potential agents for the therapy of hypoxic-ischemic brain injury.

[Protective effect of histone acetylation against cortical injury in neonatal rats].

OBJECTIVE: To investigate the protective effect of histone acetylation against hypoxic-ischemic cortical injury in neonatal rats. METHODS: A total of 90 neonatal rats aged 3 days were divided into three groups: sham-operation, cortical injury model, and sodium butyrate (a histone deacetylase inhibitor) treatment. The rats in the model and the sodium butyrate treatment groups were intraperitoneally injected with lipopolysaccharide (0.05 mg/kg), and then right common carotid artery ligation was performed 2 hours later and the rats were put in a hypoxic chamber (oxygen concentration 6.5%) for 90 minutes. The rats in the sham-operation group were intraperitoneally injected with normal saline and the right common carotid artery was only separated and exposed without ligation or hypoxic treatment. The rats in the sodium butyrate treatment group were intraperitoneally injected with sodium butyrate (300 mg/kg) immediately after establishment of the cortical injury model once a day for 7 days. Those in the sham-operation and the model groups were injected with the same volume of normal saline. At 7 days after establishment of the model, Western blot was used to measure the protein expression of histone H3 (HH3), acetylated histone H3 (AH3), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (BAX), cleaved caspase-3 (CC3), and brain-derived neurotrophic factor (BDNF). Immunofluorescence assay was used to measure the expression of 5-bromo-2'-deoxyuridine (BrdU) as the cortex cell proliferation index. RESULTS: The sodium butyrate treatment group had a significantly lower HH3/AH3 ratio than the model group (P<0.05), which suggested that the sodium butyrate treatment group had increased acetylation of HH3. Compared with the model group, the sodium butyrate treatment group had a significant increase in Bcl-2/Bax ratio, a significant reduction in CC3 expression, and a significant increase in BDNF expression (P<0.05). The sodium butyrate treatment group had a significant increase in the number of BrdU-positive cells in the cortex compared with the model group (P<0.05), and BrdU was mainly expressed in the neurons. CONCLUSIONS: Increased histone acetylation may protect neonatal rats against cortical injury by reducing apoptosis and promoting regeneration of neurons. The mechanism may be associated with increased expression of BDNF.

[The neuroprotective role of exogenous TERT gene in neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: To study the effect of telomerase reverse transcriptase (TERT) on cell apoptosis in neonatal rat brains after hypoxic-ischemic brain injury (HIBD). METHODS: A total of 72 neonatal rats were divided into sham, vehicle, HIBD and TERT groups. HIBD was induced by Rice method in the later three groups. The neonatal rats in the vehicle and TERT groups were injected with plasmids containing mock or full length TERT by an intracerebroventricular injection 30 minutes after hypoxic-ischemic (HI) injury. Pathological changes of brain tissue were observed by hematoxylin and eosin (HE) staining. Western blot was used to detect the protein expression of TERT, apoptosis-inducing factor (AIF) and cleaved caspase 3 (CC3). Apoptotic cells were detected by TUNEL staining. RESULTS: Western blot showed that TERT protein was dramatically increased in the vehicle, HIBD and TERT groups compared with the sham group. Compared with the vehicle and HIBD groups, TERT protein in the TERT group was significantly upregulated. Compared with the sham group, there was a significant increase in apoptotic index and expression of AIF and CC3 proteins in the vehicle and HIBD groups (p<0.01). The TERT group showed decreased expression of AIF and CC3 proteins and apoptotic index compared with the vehicle and HIBD groups (p<0.01). CONCLUSIONS: TERT can inhibit cell apoptosis induced by HI and might have a neuroprotective role in developing brain with HIBD.

[Long non-coding RNAs and hypoxic-ischemic brain damage].

Long non-coding RNAs (lncRNAs) are transcripts with a complex structure and a length of >200 nt which are unable to encode proteins. The lncRNAs interact with DNA, mRNA, and proteins and regulate gene expression through various mechanisms, thus participating in the regulation of various biological processes. Studies have shown that lncRNAs play important roles in neural development and the pathogenesis of diseases. This article reviews the roles of lncRNAs in hypoxic-ischemic brain damage.

[Effects of PINK1 gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage].

OBJECTIVE: To study the effect of PINK1 (phosphatase and tensin homolog deleted on chromosome ten induced putative kinase 1) gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage (HIBD). METHODS: Seventy-two wild-type C57BL/6 mice and 72 PINK1 gene knockout neonatal C57BL/6 mice were randomly divided into four groups: sham-operated wild-type (SWT), HIBD model wild-type (MWT), sham-operated knockout (SKO) and HIBD model knockout (MKO). HIBD model was prepared by low oxygen exposure for 2.5 hours after right carotid artery ligation. After 24 hours of hypoxia-ischemia treatment, TTC (2,3,5-triphenyl four azole nitrogen chloride) staining was used to measure brain infarct volume. The immunohistochemical staining was used to measure the expression of cell apoptosis protein cleaved-caspase-3 (CC3) in brain tissues. The TUNEL method was used to measure cell apoptosis. The immunofluorescence staining and Western blot were used to measure the expression of cell autophagy protein LC3. RESULTS: Compared with the MWT group, the infarct volume of brain tissues was markedly reduced in the MKO group (P<0.05), the number of apoptotic cells and the cell apoptosis index were markedly decreased in the MKO group (P<0.05), the expression of apoptosis protein CC3 was significantly reduced in the MKO group (P<0.05), the expression of cell autophagy protein LC3 was significantly decreased in the MKO group, and the autophagy indicator LC3II/LC3I was also markedly reduced in the MKO group (P<0.05). CONCLUSIONS: PINK1 gene knockout can protect neonatal mice from HIBD.

[Role of STAT3 signaling pathway in hypoxic-ischemic brain damage of neonatal rats].

OBJECTIVE: To study the role and mechanisms of STAT3 signaling pathway in hypoxic-ischemic brain damage (HIBD) of neonatal rats. METHODS: Eighty 7-day-old Sprague-Dawley rats were randomly divided into two groups: HI and sham-operated (n=40 each). The rats in the HI group were subjected to right carotid artery ligation and subsequent hypoxia exposure (8% O2) for 2.5 hours, and the rats in the sham-operated group underwent the right carotid artery dissection without subsequent ligation or hypoxia treatment. Brain tissue samples were collected at 4, 6, 8, 12 and 24 hours after operation and hypoxic exposure. Immunohistochemistry and Western blot were used to detect the expression of STAT3, phosphorylated STAT3 (p-STAT3) and vascular endothelial growth factor (VEGF) proteins. TUNEL staining was used to detect apoptotic cells. RESULTS: No significant difference in STAT3 expression was observed at all time points between the HI and sham-operated groups (P>0.05). Compared with the sham-operated group, the expression of p-STAT3 protein in the HI group was significantly upregulated at 4, 6, 8, 12 hours after operation and hypoxic exposure, and peaked at 6 hours (P<0.01). The VEGF expression in the HI group was higher than that in the sham-operated group at all time points, which peaked at 8 hours (P<0.05). TUNEL staining showed that the apoptotic cells increased significantly in a time-dependent manner compared with the sham-operated group (P<0.01). CONCLUSIONS: HI may lead to phosphorylation of STAT3 which probably induces the VEGF expression in the brain of neonatal rats. The activated STAT3 signaling pathway may be involved in the apoptosis regulation of nerve cells, and related to apoptosis inhibition of nerve cells.

[Role of long non-coding RNA BC088414 in hypoxic-ischemic injury of neural cells].

OBJECTIVE: To investigate the role of long non-coding RNA (lncRNA) BC088414 in hypoxic-ischemic injury of neural cells. METHODS: Rat adrenal pheochromocytoma (PC12) cells were divided into four groups: normoxic, oxygen glucose deprivation (OGD), siRNA-normoxic (siRNA group) and siRNA-OGD (n=3 each). Cells were incubated in glucose-free and serum-free DMEM medium under the conditions of 37℃ and 1% O2+99% N2/CO2 for 6 hours to establish an in vitro hypoxic-ischemic model. Quantitative real-time PCR was used to measure mRNA expression of lncRNA BC088414, ß2-adrenoceptor (Adrb2), and caspase-6 (CASP6). siRNAs were used to inhibit BC088414 expression in PC12 cells. The TUNEL method was used to measure cell apoptosis. RESULTS: The OGD group had a significantly higher cell apoptotic index than the normoxic group (P<0.01). After inhibition of BC088414 expression, the OGD group had a significantly reduced apoptotic index (P<0.05). The OGD group had significantly higher mRNA expression levels of lncRNA BC088414, Adrb2, and CASP6 compared with the normoxic group (P<0.05). The siRNA -normoxic group had significantly lower mRNA expression levels of Adrb2 and CASP6 than the normoxic group (P<0.05), and the siRNA-OGD group also had significantly lower mRNA expression levels of Adrb2 and CASP6 than the OGD group (P<0.05). CONCLUSIONS: LncRNA BC088414 may promote apoptosis through Adrb2 and CASP6 and aggravate neural cell injury induced by hypoxia-ischemia.

[Effects of dimethyloxalyl glycine on hypoxic-ischemic brain damage in newborn rats].

OBJECTIVE: To investigate the effects of dimethyloxalyl glycine on hypoxic-ischemic brain damage in newborn rats. METHODS: Forty eight postnatal day 10 SD rats were divided into 3 groups, including sham surgery group, hypoxic-ischemic group and DMOG treated group. The brain tissues were collected at 4, 8, 24 and 72 hours after the hypoxic-ischemic treatment. The expressions of hypoxia inducible factor-1alpha (HIF-1alfa) protein and anti apoptoticprotein cleaved caspase 3 (CC3) were detected by immunohistochemistry. The apoptotic cells were detected by TUNEL staining. RESULTS: The expression level of HIF-1alpha was significantly higher in DMOG treated group than in hypoxic-ischemic group. While the expression level of CC3 was lower and the number of tunel positive cells was fewer in DMOG treated group than that in hypoxic-ischemic group. CONCLUSION: Dimethyloxalyl glycine may play a neuro-protective role in hypoxic-ischemic brain damage in newborn rats by stabilizing HIF-1alpha.

[The studies of GLUT1 expression and neuron apoptosis in neonatal hypoxic-ischemia brain damage rat model].

OBJECTIVE: To investigate the relationship between the expression of glucose transporter protein 1 and the apoptosis of neuron during hypoxic-ischemia brain damage in neonatal rats. METHODS: Total 120 10-day old SD rats were divided into normal group, sham control group and hypoxic-ischemia (HI) group. In HI group, hypoxic-ischemia brain damage (HIBD) were generated according to Rice-Vannucci method, brain tissues were harvested at 2, 8, 24, 48 and 72 h after HI. The brain samples were also collected at the same time points in normal group and sham control group. The pathological changes was observed by hematoxylin-eosin (HE) staining, the mRNA expression of glucose transporter 1 (GLUT1) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR), the protein expressions of GLUT1 and cleaved caspase-3 (CC3) were detected by immunohistochemistry, the apoptosis of neuron was measured by TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining. RESULTS: HE staining showed that the degree of brain cell damage increased with time after HI, the loss of neuronal cells peaked at 48 h, while the cells in control group apperanted in an orderly and normal morphology. The mRNA and protein expressions of GLUT1 were increased after HI, which began to increase at 2 h, and reach the peak at 24 h. and the expression levels at each time points were statistically higher (P< 0.01) than those in control group. CC3 protein expression also began to increase at 2 h, peaked at 48 h after HI, which was higher than that of control group (P<0.01). The number of positive cells was significantly increased after HI,with a peak at 48 h. CONCLUSION: The mRNA and protein expression of GLUT1 in brain tissue increased significantly after hypoxic-ischemia, and the peak time was earlier than that of CC3 protein and cellular apoptosis. This suggests that GLUT1 expression upregulation may be a certain degree of inhibition on neuronal apoptosis.

[Preparation and application of biotinylated anti-AIB1 moniclonal antibody].

OBJECTIVE: To cross-link the McAb 1A2E1 to BNHS reagents and apply this biotinylated McAb to detect AIB1 antigen of target cells. METHODS: The McAb 1A2E1 was mixed with BNHS reagents to generate Biotin-1A2E1. The competitive inhibition ELISA and immunocytochemical method were applied to detect the biologic activity of antibody. RESULTS: The antibody kept high biological activities (with a titer of 1 : 3200) and sensitivities in detecting the AIB1 protein of breast cancer cell. CONCLUSION: The method of preparing biotinylated McAb is successful, and the prepared biotinylated McAb can be applied to detect target cells which express AIB1 antigen. This McAb provide useful tool for tumor auxiliary diagnosis.

[Hypoxia-inducible factor-1alpha: a promising target for tumor therapy].

Hypoxia-inducible factor-1alpha (HIF-1alpha), a nuclear transcriptional factor, is constitutively expressed in mammalian cells under hypoxia, which contributes a lot to the regulation of internal O2 homeostasis. Micro-environmental hypoxia is a common feature of solid tumors. Under the stress of hypoxia, HIF-1alpha is accumulated and activated, which leads to activation of a vast array of downstream genes that contribute to tumor O2 homeostasis and energy metabolic equilibrium. HIF-1alpha weighs heavily in favor of tumor genesis and progression. So far, HIF-1alpha has became an attracting tumor research topic, which improves understanding on how HIF-1alpha functions in tumor progression and key signaling pathways that regulate HIF-1alpha, therefore, provides new scientific supports and ideas to look for novel target for tumor therapy.

[Effect of dominant negative HIF-1alpha (dn HIF-1alpha) on biological characteristics of uterine cervix cancer cells].

OBJECTIVE: To explore the effect of dominant negative HIF-1alpha (dn HIF-1alpha) on biological characteristics of uterine cervix cancer cell SiHa and elucidate the related mechanism. METHODS: pcDNA3. 1-dn HIF-1alpha was transfected into SiHa cells. The expression of HIF-1alpha and VEGF protein were detected by immunocytochemical method and Western Blotting. The growth proliferation of cells was surveyed by the MTT assay and cell apoptosis was detected through TUNEL after treated with CoCl2, meanwhile the results were compared with the group transfected with mock plasmid and untransfected group. RESULTS: After successfully transfected with relevant plasmid, there's no obvious difference of expression of HIF-1alpha among dn HIF-1alpha group, pcDNA3. 1 group, and untransfected group, however the expression of VEGF of dn HIF-1alpha group was significantly lower than that of the others (P < 0. 05). The proliferation ability of dn HIF-1alpha group was obviously lower than that of the other two (P < 0.05), whether it was under normoxia or chemical hypoxia induced by CoCl2. The characteristic apoptotic morphology was most significantly apparent in dn HIF-1alpha group among these three (P < 0.05). CONCLUSION: Domain negative HIF-1alpha can inhibit the proliferation of uterine cervix cancer cell and accelerate its apoptosis under hypoxia induced by CoCl2, as well as decrease the expression of VEGF protein. The implications of all this were that the domain negative HIF-1alpha may play an important role in the therapy of uterine cervix cancer.

[Effect of HIF-1alpha on the proliferation and apoptosis of uterine cervix cancer SiHa cells].

OBJECTIVE: To explore the effect of HIF-1alpha on the proliferation and apoptosis of SiHa cells in hypoxia, as well as the role of HIF-1alpha in the development of uterine cervix cancer. METHODS: The eukaryotic expression vector containing full length HIF-1alpha (pcDNA3. 1-full length HIF-1alpha) was transfected into SiHa cell, the mock plasmid (pcDNA3. 1) was also transfected into SiHa cell as the negative control. The expression of HIF-1alpha and VEGF were detected by Western Blotting and immunocytochemistry. Cell proliferation was detected by MTT colorimetric assay after the treatment of varied concentration of CoCl2, and cell apoptosis was detected by TUNEL. RESULTS: The level of HIF-1alpha and VEGF expression in the cells transfected with full length HIF-1alpha was higher than that in the untransfected group and pcDNA3. 1 group (P < 0.05), the survival ability of the cells in HIF-1alpha group was also higher than that of other two groups no matter in anoxia or in hypoxia (P < 0.05), the ratio of apoptosis was less than untransfected group and pcDNA3. 1 group (P < 0.05). CONCLUSION: HIF-1alpha inhibited cell apoptosis in CoCl2 induced hypoxia and stimulated cell proliferation. These results suggest that HIF-1alpha may play an important role in the development of uterine cervix cancer.

[Effect of aurora A on carcinogenesis of human prostate cancer].

OBJECTIVE: To investigate whether Aurora A is involved in prostate cancer carcinogenesis. METHODS: The expressions of Aurora A mRNA and protein in prostate cancer tissue and cell line were measured using RT-PCR, immunohistochemistry and Western blot analyses. Meanwhile, A DNAzyme targeting at Aurora A mRNA was performed to detect the inhibition effect of Aurora A in regulating the growth of prostate cancer cell line PC3. RESULTS: Aurora A expression was up-regulated in 91% prostate cancer tissue as well as in prostate cancer cell lines such as PC3, LNCaP and Du145. Furthermore, Aurora A expression suppressed by cell transduction of a DNAzyme targeting at Aurora A mRNA made the PC3 cells both arrest the cell cycle in G2/M phase and increase the cell apoptosis. CONCLUSIONS: These findings suggest that Aurora A up-regulation may be involved in carcinogenesis of prostate cancer, and that aurora A gene may be a valuable target for treatment of prostate cancer.

[Effect of Aurora A on biological phenotypes of prostate cancer cells].

OBJECTIVE: To examine the effect of Aurora A on the biological phenotypes of prostate cancer cells and the role of Aurora A in the carcinogenesis of prostate cancer. METHODS: Full length Aurora A gene was cloned and transfected into the prostate cancer cell line LNCaP to construct a positive cell clone with high expression of Aurora A. The growth and proliferation of the cells was detected by MTT. The mobility of the cells was examined by transwell cell layer infiltration assay. The growth and motility of LNCaP-Aurora A cells were compared with those of the LNCaP-pcDNA3. 1/LNCaP cells. RESULTS: Compared to the LNCaP-pcDNA3. 1/ LNCaP cells, the cells with high expression of Aurora A had faster proliferation and stronger invasive ability. Three days after cultivation, more LNCaP-Aurora A cells had grown than the LNCaP-pcDNA3. 1/LNCaP cells (P < 0.05). CONCLUSION: The increased expression of Aurora A is closely associated with the malignant phenotypes of prostate cancer. Aurora A may play a very important role in the carcinogenesis of prostate cancer.