RESUMO
Objective To observe the skin ultrastructure change of electric shock death rats and to test the expression changes of hypoxia-inducible factor-2α （HIF-2α） and heart type-fatty acid-binding protein （H-FABP） of myocardial cells, in order to provide basis for forensic identification of electric shock death. Methods The electric shock model of rats was established. The 72 rats were randomly divided into control group, electric shock death group and postmortem electric shock group. Each group was divided into three subgroups, immediate （0 min）, 30 min and 60 min after death. The skin changes of rats were observed by HE staining, the changes of skin ultrastructure were observed by scanning electron microscopy, and the expression of HIF-2α and H-FABP in rats myocardium was tested by immunohistochemical staining. Results The skin in the electric shock death group and postmortem electric shock group had no significant difference through the naked eye or by HE staining. Under the scanning electron microscope, a large number of cellular debris, cells with unclear boundaries, withered cracks, circular or elliptical holes scattered on the cell surface and irregular edges were observed. A large number of spherical foreign body particles were observed. Compared with the control group, the expression of HIF-2α in all electric shock death subgroups increased, reaching the peak immediately after death. In the postmortem electric shock group, HIF-2α expression only increased immediately after death, but was lower than that of electric shock death group （P<0.05）. Compared with the control group, the expression of H-FABP in all subgroups of electric shock death group and postmortem electric shock group significantly decreased. The expression of H-FABP in all subgroups of electric shock death group was lower than that of the postmortem electric shock group （P<0.05）. Conclusion Electric shock can increase HIF-2α expression and decrease H-FABP expression in the myocardium, which may be of forensic significance for the determination of electric shock death and identification of antemortem and postmortem electric shock.
Assuntos
Animais , Ratos , Autopsia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína 3 Ligante de Ácido Graxo/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Pele/ultraestruturaRESUMO
Cardiotrophin-1 (CT-1) is an important regulator of organ protection and glucose and lipid metabolism. Aims: To explore the role of hypoxia-inducible factors (HIFs) in regulating CT-1 expression in cholestatic disease. Methods: Twenty-three pediatric biliary atresia patients and 7 healthy liver donors were enrolled from Jun. 2016 to Jun. 2017 at Renji Hospital, School of Medicine, Shanghai Jiao Tong University. Cellular localization and expressions of HIF-1α, HIF-2α and CT-1 in liver tissue were detected by immunohistochemistry and Western blotting. In in vitro study, human umbilical vein endothelial cells (HUVECs) were treated with H2O2, a strong oxidizer, with or without silencing the expression of HIF-2α with RNA interference, and the expressions of HIF-2α and CT-1 were determined by Western blotting and real-time PCR. Results: In pediatric cholestatic liver tissue, HIF-2α and CT-1 were co-distributed in cholangiocytes and stromal cells such as vascular endothelial cells and inflammatory cells, while HIF-1α was only expressed in some inflammatory cells occasionally. Western blotting showed elevated expressions of HIF-2α and CT-1 in pediatric cholestatic liver tissue when compared with that of healthy controls. H2O2-induced oxidative stress could significantly increase the expressions of HIF-2α and CT-1 in HUVECs, while silencing of HIF-2α could significantly decrease the transcription of CT-1 induced by H2O2. Conclusions: HIF-2α but not HIF-1α regulates the expression of CT-1 in pediatric cholestatic disease. In cholestasis, oxidative stress may induce CT-1 expression via HIF-2α-dependent pathway.
RESUMO
Purpose To identify the binding of hypoxia in-ducible factor-2a (HIF-2 a) to the hypoxia-response element(HRE) of the matrixmetallo proteinases-2 (MMP-2) gene promoter region and clear the binding site. Methods Electro-phoretic mobility shift assay (EMSA) was used to identify the binding of HIF-2 a to HRE of the MMP-2 gene promoter region in vitro. At the same time, the chromatin immunoprecipitation assay (CHIP) was used to further determine the binding site. Results Successful prediction of two potential HIF-2a binding sites of MMP-2 the promoter region, which were-217～-204 and-1 029 ～-1 007, respectively. Probe test shows that the marked efficiency of sense chain and antisense chain was above 50％, and they could be used for EMSA-electrophoretic mobility shift assay. The results of EMSA showed that there was a binding site of HIF-2 a sense chain and antisense chain moter region int-217～-204. The results of chromatin immuno-precipitation showed that in the experimental group and control group an about 250 bp fragment in MMP-2 promoter containing HRE region was amplified, suggesting that the protein of HIF-2a binded to the HRE in MMP-2 promoter region in vivo. Conclusion HIF-2 a in MMP-2 promoter regionne promoter region in vitro and in vivo.
RESUMO
Objective To explore the expressions of hypoxia inducible factor ( HIF), vascular endothelial growth factor receptor 2 ( VEGFR 2), and microvessel density ( MVD) in adrenocortical adenoma ( ACA) and adrenocortical carcinoma ( ACC), in order to discuss their potential role in the development of adrenal tumours. Methods Fifty-five adrenal tumour specimens resected in the hospital with complete clinical data (including 30 ACA cases and 25 ACC cases) were examined by immunohistochemistry for the expressions of HIF-2α, HIF-1α, VEGFR 2, and MVD. Results VEGFR 2 and MVD up-regulated were found in the ACC group (P＜0.05). The expression of HIF-2α and HIF-1α correlated with VEGFR 2 (P＜0.05). The expressions of VEGFR 2 and MVD were related to some clinicopathological features ( P＜0. 05 ). Additionally, tumour size, expression of VEGFR 2 and MVD were independently associated with ACC (P＜0.05). Conclusions The high expression of HIF-2α, VEGFR 2, and MVD in adrenal tumours suggested their roles in tumour angiogenesis, which indicated that anti-angiogenesis therapies deserve intensive studies for malignant adrenocortical tumours.
RESUMO
There is increasing evidence that microcirculation hypoxia plays an important role in pathogenesis of inflammatory bowel disease (IBD).Hypoxia-inducible factors (HIFs)are transcriptional factors that serve as master regulators in ischemic and hypoxia injuries.Aims:To investigate the effect of HIF-2αon dextran sulfate sodium (DSS)-induced colitis in mice and its possible mechanism.Methods:Mx-Cre/LoxP recombination system was utilized to establish a conditional HIF-2αgene knockout (HIF-2α-/-)mouse model.C57BL/6,HIF-2α+/+and HIF-2α-/-mice were randomly allocated into DSS colitis group and water drinking group,respectively.Experimental colitis was induced by treatment with 4% DSS in drinking water for 7 days,and the disease activity index (DAI)was assessed daily.Mice in each group were sacrificed on day 1,3,5 and 7 in batch;the histopathological changes of colonic tissue were observed, and mRNA expressions of HIF-2αand tumor necrosis factor-α(TNF-α)were measured by real-time PCR.Results:During model establishment,expression of HIF-2αmRNA in colonic tissue was elevated in C57BL/6 and HIF-2α+/+DSS colitis groups,and the DAI and colonic inflammatory score were significantly higher than those in C57BL/6 water drinking group (P<0.05 on day 5 and day 7).Compared with HIF-2α+/+DSS colitis group,HIF-2α-/-DSS colitis group had more severe colonic inflammatory injury and the DAI and inflammatory score were further increased (P all<0.05,except the inflammatory score on day 7);expression of TNF-αmRNA in colonic tissue was also increased significantly in HIF-2α-/-DSS colitis group (P<0.05 on day 5 and day 7).Conclusions:HIF-2αmay ameliorate colonic inflammatory injury in mice with DSS colitis via inhibition of TNF-αexpression.
RESUMO
Purpose To investigate the effects of hypoxia inducible factor-2α (HIF-2ot) siRNA on proliferation and chemotherapy sensitivity of breast carcinoma MCF-7.Methods RNA interference was used to silence the expression of HIF-2α in MCF-7 cells.The changes of HIF-2α gene expression were detected by immunocytochemistry and RT-PCR.Under hypoxia environment simulated by CoCl2,MTT assay and flow cytometry (FCM) were used to measure cell growth inhibition rate and cell apoptosis of MCF-7 cells under different dosages of chemotherapeutic agents (5-fluorouracil,adriamycin,and paclitaxel).Results Expression of HIF-2α in MCF-7 were down-regulated by HIF-2α siRNA (P ＜ 0.05).The proliferation inhibition and apoptosis rates were evidently increased after transfection with HIF-2α siRNA (P ＜ 0.05),chemotherapy drug sensitivity was enhanced.Conclusion HIF-2α siRNA can induce the apoptosis and inhibit the proliferation and enhance the sensitivity of breast carcinoma MCF-7 cell line to chemotherapeutic agents.Blocking HIF-2α maybe a very promising strategy for breast carcinoma gene therapy in combination with chemotherapy.
RESUMO
Objective@#To investigate the effect of hypoxia inducible factor 2α (HIF-2α) on regulating CUB domain-containing protein 1 (CDCP1) and its role in hepatocellular carcinoma metastasis.@*Methods@#HIF-2α-knocked down and HIF-2α-stably overexpressing cells (MHCC97H) were prepared by small interfering RNA (siRNA) and lentivirus transfection, respectively. The expression of CDCP1 protein and mRNA in the above cells was detected by western blot and real-time PCR. The effect of HIF-2α on cell invasion ability was determined by Transwell assay. Furthermore, immunohistochemical staining was performed to detect the expression of CDCP1 in human HCC tissue samples.@*Results@#Both HIF-2α and CDCP1 were induced under hypoxic conditions. The activation of CDCP1 under hypoxic conditions was dependent on the expression of HIF-2α.When HIF-2α was overexpressed, the mRNA level of CDCP1 was greatly upregulated (5.92±0.28, P<0.05). When HIF-2α was knocked down by siRNA for 48 h and 72 h, the expression of CDCP1 was significantly downregulated (48 h: 0.25±0.04; 72 h: 0.18±0.02, all P<0.05). Moreover, analysis of human HCC samples showed that CDCP1 expression was correlated with tumor-free survival (P<0.05).@*Conclusions@#The results of this study indicate that the expression of CDCP1 is regulated by HIF-2α and is correlated with the progression of HCC. Inhibition of HIF-2α/CDCP1 may play certain inhibitory role in the metastasis of HCC.
RESUMO
Objective To study the expression of HIF-2α and VEGF in colorectal cancer and to investigate the relationship between them and clinicopathologic parameter.Methods Immunohistochemistry staining was conducted to detect the expression of HIF-2α and VEGF protein in 67 samples of colorectal tumor tissues and 67 samples of normal adjacent tissue.Results The expression of HIF-2α and VEGF in colorectal cancer tissues was significantly higher than that in adjacent tissues.The expression of HIF-2α and VEGF increased with the clinical stage and lymph node metastasis.The expression of HIF-2α increased with the tumor volume.The expression of HIF-2α and VEGF was not related to the age,sex,tumor location and tumor differentiation of the patients.HIF-2α was positively correlated with VEGF expression.Kaplan-Meier survival analysis showed that HIF-2α expression was associated with survival,that is,the higher expression of HIF-2α the worse of prognosis was obtained.Conclusion HIF-2α is involved in the process of growth,invasion and metastasis of colorectal cancer.This process may be related to the regulation of VEGF expression.
RESUMO
Objective:To investigate the expression of hypoxia-inducible factor-2α (HIF-2α)in non-small cell lung cancer (NSCLC)tissue,and to analyze its relationships with angiogenesis,cell proliferation and chemotherapy resistance. Methods: Total 112 cases of NSCLC and 20 cases of normal lung tissues were selected, immunohistochemical method was used to detect the expressions of HIF-2α,CD31,Ki67 and GST-π in 112 cases of cancer tissue and 20 cases of normal lung tissue,and the correlations of HIF-2α expression with microvessel density (MVD),Ki67, and GST-π were analyzed.Results:The positive expression rate of HIF-2α in NSCLC tissue was significantly higher than that in normal lung tissue (P < 0.05 ), the expression rate of HIF-2α in 112 cases of NSCLC was 47.3% (53/112).The MVD in HIF-2α protein high expression NSCLC group (31.1 ± 14.7)was higher than that in HIF-2αprotein low expression NSCLC group (24.3±15.8)(P <0.05).The cases of high expression of Ki67 in HIF-2αhigh expression group occupied 54.7% (29/53),and it was higher than that in HIF-2αlow expression group (16/59,27.1%);there was significant difference (r = 0.281,P = 0.003).The high expression of HIF-2α had no obvious correlation with the expression of GST-π (r = 0.122,P = 0.202). Conclusion:HIF-2αmay play an important role in the carcinogenesis and development of NSCLC by promoting the angiogenesis and enhancing the cell proliferation of NSCLC,but it may have no correlation with chemotherapy resistance.
RESUMO
Objective To investigate the mechanisms of hypoxia inducible factor-2 alpha (HIF-2a) regulating human umbilical vein endothelial cells (HUVECs) under hypoxic conditions.Methods The experimental study was adopted.(1) HUVECs in logarithmic growth phase were taken:HUVECs without any disposals as control group,HUVECs with shRNA transfection control as shRNA control group,HUVECs with HIF-2α shRNA transfection as HIF-2α shRNA group and HUVECs with HIF-2α shRNA transfection then added rhAng-2 as HIF-2α ± rh-Ang-2 group.(2) Western blot testing:the expressions of Ang-2 and HIF-2α proteins in HUVECs were cultured under hypoxia conditions at 0,2,4,8,12,16,20 hours,and the levels of which were detected in the control group,shRNA control group and HIF-2α shRNA group.(3) Enzyme-linked immunosorbent assay(ELISA):the level of Ang-2 protein in supernatant of HUVECs was detected in the control group,shRNA control group and HIF-2α shRNA group.(4)The amounts of endothelial cell tubes in HUVECs among the 4 groups were detected by tube formation experimental testing.(5) Transwell method was performed to detect the amounts of cells migration in HUVECs and hepatoma cells SMMC-7721 migration intervened by supernatant of HUVECs among the 4 groups.Measurement data with normal distribution were presented as x ± s,repeated measurement data were analyzed by the repeated measures ANOVA,comparison among groups and pairwise comparison were conducted respectively by the one-way ANOVA and Dunnett's test.Results (1) Western blot test:the expression levels of Ang-2 and HIF-2α proteins in HUVECs under hypoxia conditions at 0,2,4,8,12,16,20 hours were 0.110 ±0.011,0.120 ±0.020,0.210 ±0.070,0.410 ±0.100,0.520 ± 0.090,0.790±0.130 1.010 ±0.220 and 0.180 ±0.090,0.410 ±0.070,0.470 ±0.110,0.470 ±0.070,0.580 ± 0.120,0.690 ± 0.140,0.920 ± 0.130,respectively,and which were increased after culturing under hypoxia conditions and had an ascending tendency as the hypoxia time extended,with statistically significant differences (F =403.550,3 265.587,P ＜ 0.05).The expression levels of Ang-2 and HIF-2α proteins in the control group,shRNA control group and HIF-2α shRNA group were 1.030 ±0.180,1.070 ±0.120,0.210 ± 0.070,and 0.940 ± 0.110,0.930 ± 0.190,0.170 ± 0.021,respectively,showing statistically significant differences (F =290.242,26.688,P ＜ 0.05).(2) The results of ELISA:the expression levels of Ang-2 in the control group,shRNA control group and HIF-2α shRNA group were (433.2 ±9.7)ng/L,(438.3 ± 2.6)ng/L,(114.6 ± 4.2) ng/L,with a statistically significant difference (F =2 642.180,P ＜ 0.05).(3) The results of tube formation experiments:the number of endothelial cell tubes in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α ± rh-Ang-2 group were 48.3 ± 2.5,47.4 ± 3.1,19.7 ± 1.5 and 38.3 ± 2.1,respectively,with a statistically significant difference (F =148.196,P ＜ 0.05).(4) The results of Transwell method:① the number of HUVECs migration in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α + rh-Ang-2 group were 140.3-± 3.5,142.7 ± 2.1,42.7 ± 3.1 and 78.1 ± 4.2,respectively,showing a statistically significant differences (F =212.205,P ＜ 0.05).②The results of Transwell method:the number of SMMC-7721 cells migration after intervening using four different supernatant in the control group,shRNA control group,HIF-2α shRNA group and HIF-2α ± rh-Ang-2 group were 106.7 ± 5.5,102.7 ± 6.6,63.0 ± 3.3 and 96.7 ± 2.1,respectively,showing a statistically significant difference (F =55.122,P ＜ 0.05).Conclusion HIF-2a could not only affect HUVECs formation but also promote SMMC-7721 cells migration via regulating Ang-2 expression.
RESUMO
Objective To investigate the role of hypoxia?inducible factor?2α(HIF?2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia condition. Methods The expressions of the HIF?2α and tight junction proteins such as occludin and ZO?1 of rGENCs were examined after exposed to 5%oxygen at different treatment time periods (0 h, 12 h, 24 h and 48 h). Then lentiviral transfection was used to knock down HIF?2α expression in rGENCs. The cells were split into four groups, including i) control group where rGENCs were cultured under normal oxygen conditions, ii) hypoxia group, iii) negative control group where rGENCs were infected with a negative vector, iv) HIF?2α lentivirus transfection group. Group ii, iii and iv were kept in hypoxic chamber (5% O2, 5% CO2 and 90% N2) for 24 h. The expressions of occludin, ZO?1 and HIF?2α were assessed by Western blotting. The permeability of rGENCs was measured using trans?epithelium electrical resistant (TEER) by Millicell? ERS voltohmmeter. Results With the elongation of hypoxia time, the expression of HIF?2α was increased gradually, while the occludin expression was decreased, there was statistically significance difference in each group (all P0.05). And a dramatic decrease in TEER of hypoxia cells was detected as compare with control cells (P0.05). Conclusion Hypoxia may promote HIF?2α expression, which could increase the permeability of rGENCs by reducing the expression of occludin and ZO?1.
RESUMO
Objective To investigate the effect of HIF-2a silencing by transfection of siRNA into MG-63 cells un-der hypoxia. Methods HIF-2αexpression level in MG-63 cells under hypoxia was determined by Western Blot. Small in-terfering RNA (siRNA) was used to construct MG-63/siHIF-2α(siHIF-2α)cells and control MG-63/scramble (NC) cells. The expression levels of HIF-2α, Vascular endothelial growth factor (VEGF), p-Erk/ErK and Mcl-1 in MG-63, NC and si-HIF-2αcells was determined by Western Blot. NC and siHIF-2αcells were cultured under hypoxia. Cell viability was as-sessed by MTT assay. Migration was identified by scratch migration assay. Tumor formation was identified by clone formation assay. Nude mouse subcutaneous xenograft model was used to investigate tumor development in vivo. Results Hypoxia im-proved HIF-2αexpression in MG-63 cells in a time-dependent manner (F=2 037.412,P<0.001). HIF-2αexpression un-der hypoxia in siHIF-2αcells was lower than that in NC cells (P<0.01). Cell viability of siHIF-2αcells under hypoxia for 12 h and 24 h were lower than that in NC cells (P<0.05 or P<0.01). The relative width of scratch in siHIF-2αgroup under hypoxia for 12 h and 24 h were larger than that in NC group (P<0.01 or P<0.01). When cell counts reach 1 000-5 000, the clone formation rates of siHIF-2αcells were lower than that in NC cells (P<0.05 or P<0.01). The expression of VEGF, p-Erk/Erk and Mcl-1 protein under hypoxia in siHIF-2αcells was lower than that in NC cells(P<0.01). Tumor sizes, weights and density of siHIF-2α group in nude mice were suppressed compared with those in NC group (P<0.01). Conclusion Blocking HIF-2αsignal pathway warrants its investigation as a potential strategy in osteosarcoma treatment.
RESUMO
Objective To investigate the location and expression of hypoxia inducible factor (HIF) subunits in the remnant kidney of 5/6 nephrectomy rats. Methods Remnant kidneys were produced in adult male SD rats by 5/6 nephrectomy. The renal function and histopathological changes were evaluated at week 1, 2, 4, 6, 8 and 12 after operation. Tissues of remnant kidneys were collected to detect the location and expression of HIF-1α and HIF-2α by immunohistochemistry staining and Western blotting. The mRNA levels of HIF targeted genes vascular endothelial growth factor (VEGF) and heme oxygenase-1 (HO-1) were determined by RTPCR. Results (1) 5/6 nephrectomy rats underwent one week of acute renal failure at first[Scr (122.8±22.1) μmol/L] and then developed compensative chronic renal failure [(66.0±3.7)-(66.4±8.4) μmol/L], but the level of Scr increased quickly after week 6 [(66.4±8.4)-(127.8±22.7) μmol/L],concomitantly with progressive tubulointerstitial fibrosis in remnant kidney cortex. (2) In cortex, HIF-1α was expressed only in the atrophic and dilated tubular cells while HIF-2α was located in endothelial, interstitial fibroblasts, and vascular smooth muscle cells. The semiquantitative results of imunohistochemistry and Western blotting revealed that HIF-1α and HIF-2α were both gradually up-regulated during the early stage of remnant kidney, peaked at week 4 and 6, and then gradually down-regulated. (3) The mRNA levels of HIF targeted genes VEGF and HO-1 transiently peeked at week 4 and 6, and then decreased gradually. Conclusions The increased stabilization of HIF-αprotein and transcription of HIF targeted genes at the early stage of this model is a compensation reaction towards hypoxia. The mechanism of decreased expression of HIF-α at the end stage of chronic kidney disease deserves further investigation.
RESUMO
Objective To investigate the effects of hypoxia inducible factor-2?(HIF-2?) on the proliferation and invasion of hepatocellular carcinoma cell line SMMC-7721 under the anoxia condition.Methods Lipofectamine-mediated HIF-2? oligodeoxynucleotide(ODN) was transfected into SMMC-7721 cells and the expressions of HIF-2? mRNA and protein were detected to evaluate the transfection.The proliferative and adhesive activity was determined by methyl thiazolyl tetrazolium(MTT) method and the invasive activity was determined by a transwell cell culture chamber method.Results The expressions of HIF-2? mRNA and protein were inhibited significantly by HIF-2? antisense oligodeoxyribonucleotide(ASODN)(P0.05).Conclusion The blockage of HIF-2? expression inhibits the adhesive and invasive activity of SMMC-7721cells,and has no evident impact on their proliferation.
RESUMO
AIM: To study the effect of RNA interference on hypoxia-inducible factor-2(HIF-2) in the renal cell cancer in vitro and in vivo.METHODS: HIF-2 RNAi was synthesized and inserted into RNA interference eukaryotic expression vector which was confirmed by sequencing.The vector was transfected into the renal cancer cell 786-0 and positive clone was selected by using G418.The HIF-2 expression was detected by RT-PCR and Western blotting method.The growth of cells was measured by MTT method.Nude mouse xenograft assays were also done.RESULTS: Compared with empty vector group and control group,the amounts of HIF-2 mRNA and protein expression were lower in the HIF-2 RNAi group,the difference was significant(P