OBJECTIVETo investigate the effects of hypoxia induced by cobalt chloride on the expression and the activity of matrix metalloproteinase-2 (MMP-2) in rat hepatic stellate cells (HSC-T6) and to clarify the possible mechanisms.

METHODSHSC-T6 cell line was grown in Dulbecco's modified Eagle medium with 10% fetal calf serum at 37 degrees C and 5% CO2. When reaching confluence, the cells were incubated with serum-free medium in the presence of cobalt chloride (0, 50, 100, 200 micromol/L) for six hours, and then the supernatant and the cells were harvested. The expression of the MMP-2 mRNA and HIF-1alpha protein in HSC-T6 cells was detected using RT-PCR and Western blot respectively. The activity of the MMP-2 in the supernatant was detected by zymography. The binding reaction between HIF-1a protein and MMP-2 gene sequence was investigated by electrophoresis mobility shift assay.

RESULTSWhen the concentration of CoCl2 increased from 0 micromol/L to 200 micromol/L, the expressions of MMP-2 mRNA (the rate of light density) were increased from 0.53+0.12 to 1.57+0.11 and the differences among these four groups were significant (F=34.21). The activity of MMP-2 (the value of light density*band area) decreased gradually from 84.49+5.38 to 53.70+3.42, and the differences among these four groups were also significant (F=29.54). The expressions of HIF-1a were increased gradually with the increase of the CoCl2 concentration. The shift band in the lane of the nuclear protein extraction and the MMP-2 probe containing hypoxia response element showed delays when compared with the lane of the sole probe, and the binding was partially abolished when competing sense oligonucleotides were used.

CONCLUSIONSOur results suggest that chemical hypoxia can up-regulate the expression of MMP-2 mRNA and decrease the activity of the enzyme. HIF-1alpha may play a part in the regulation of MMP-2 transcription under hypoxic conditions.

Animals ; Cell Hypoxia ; Cell Line ; Cobalt ; pharmacology ; Hepatic Stellate Cells ; enzymology ; Hypoxia ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; RNA, Messenger ; genetics ; Rats

Intermittent hypoxia (IH) could induce cognitive impairment through oxidative stress and inflammation. However, the degree of cell damage is closely related to the IH stimulus frequency. IH stimulation with different frequencies also induces opposite results on neuronal cell lines. Therefore, this study was aimed to compare the effects of IH stimulation with three different frequencies on murine hippocampal neuronal HT22 cell activity, and to explore the molecular mechanism of the IH stimulus frequency-related neuron injury. HT22 cells were cultured and divided into control group and three IH stimulation groups with different frequencies. Oxygen concentration in the chamber was circulated between 21% and 1% (IH1 group, 6 cycles/h; IH2 group, 2 cycles/h; IH3 group, 0.6 cycle/h). Cell morphology was observed at 6, 12, 24 and 48 h of IH treatment. Cell viability was determined by the CCK-8 kit, lactate dehydrogenase (LDH) content in cell supernatant was determined by LDH kit, oxidative stress level was detected by the reactive oxygen species (ROS) probe, and protein expression levels of hypoxia inducible factor-1α (Hif-1α) and phosphorylated nuclear factor κB (p-NF-κB) were detected by Western blot. The results showed that, compared with control group, cell number and activity in the three IH groups were decreased, LDH content and ROS levels were increased with the prolongation of IH stimulation time, and the changes were most obvious in the IH1 group among those of the three IH groups. Hif-1α expression and the p-NF-κB/NF-κB ratio were also up-regulated with the prolongation of IH stimulation time, and the changes of IH1 group were the most significant. These results suggest that IH stimulation induces oxidative stress injury in HT22 cells, which is related to increased Hif-1α expression and NF-κB phosphorylation. Moreover, the higher frequency of IH stimulation induces more serious cell injury.
Animals ; Cell Hypoxia ; Cell Survival ; Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Mice ; NF-kappa B/metabolism* ; Oxidative Stress ; Reactive Oxygen Species

OBJECTIVE: To identify and characterize read-through RNAs and read-through circular RNAs (rt-circ-HS) derived from transcriptional read-through hypoxia inducible factor 1α (HIF1α) and small nuclear RNA activating complex polypeptide 1 (SNAPC1) the two adjacent genes located on chromosome 14q23, in renal carcinoma cells and renal carcinoma tissues, and to study the effects of rt-circ-HS on biological behavior of renal carcinoma cells and on regulation of HIF1α. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and Sanger sequencing were used to examine expression of read-through RNAs HIF1α-SNAPC1 and rt-circ-HS in different tumor cells. Tissue microarrays of 437 different types of renal cell carcinoma (RCC) were constructed, and chromogenic in situ hybridization (ISH) was used to investigate expression of rt-circ-HS in different RCC types. Small interference RNA (siRNA) and artificial overexpression plasmids were designed to examine the effects of rt-circ-HS on 786-O and A498 renal carcinoma cell proliferation, migration and invasiveness by cell counting kit 8 (CCK8), EdU incorporation and Transwell cell migration and invasion assays. RT-PCR and Western blot were used to exa-mine expression of HIF1α and SNAPC1 RNA and proteins after interference of rt-circ-HS with siRNA, respectively. The binding of rt-circ-HS with microRNA 539 (miR-539), and miR-539 with HIF1α 3' untranslated region (3' UTR), and the effects of these interactions were investigated by dual luciferase reporter gene assays. RESULTS: We discovered a novel 1 144 nt rt-circ-HS, which was derived from read-through RNA HIF1α-SNAPC1 and consisted of HIF1α exon 2-6 and SNAPC1 exon 2-4. Expression of rt-circ-HS was significantly upregulated in 786-O renal carcinoma cells. ISH showed that the overall positive expression rate of rt-circ-HS in RCC tissue samples was 67.5% (295/437), and the expression was different in different types of RCCs. Mechanistically, rt-circ-HS promoted renal carcinoma cell proliferation, migration and invasiveness by functioning as a competitive endogenous inhibitor of miR-539, which we found to be a potent post-transcriptional suppressor of HIF1α, thus promoting expression of HIF1α. CONCLUSION The novel rt-circ-HS is highly expressed in different types of RCCs and acts as a competitive endogenous inhibitor of miR-539 to promote expression of its parental gene HIF1α and thus the proliferation, migration and invasion of renal cancer cells.
Humans ; Carcinoma, Renal Cell/pathology* ; Cell Proliferation ; Hypoxia ; Kidney Neoplasms ; MicroRNAs/genetics* ; Neoplasm Invasiveness/genetics* ; RNA, Circular/metabolism* ; RNA, Small Interfering ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics*

Phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) is a key factor in pulmonary vascular remodeling. Inhibiting or reversing phenotypic transformation can inhibit pulmonary vascular remodeling and control the progression of hypoxic pulmonary hypertension. Recent studies have shown that hypoxia causes intracellular peroxide metabolism to induce oxidative stress, induces multi-pathway signal transduction, including those related to autophagy, endoplasmic reticulum stress and mitochondrial dysfunction, and also induces non-coding RNA regulation of cell marker protein expression, resulting in PASMCs phenotypic transformation. This article reviews recent research progress on mechanisms of hypoxia-induced phenotypic transformation of PASMCs, which may be helpful for finding targets to inhibit phenotypic transformation and to improve pulmonary vascular remodeling diseases such as hypoxia-induced pulmonary hypertension.
Humans ; Pulmonary Artery ; Hypertension, Pulmonary ; Vascular Remodeling/genetics* ; Hypoxia/genetics* ; Myocytes, Smooth Muscle ; Cell Proliferation/physiology* ; Cells, Cultured ; Cell Hypoxia/genetics*

OBJECTIVETo explore the effects of hypoxia on the proliferation of human leukemia HL-60 cells and the cellular expression of hypoxia inducible factor-1α (HIF-1α).

METHODSHuman acute myeloid leukemia HL-60 cells with exponential growth in routine culture were exposed to 50, 200, 400, 800 µmol/L CoCl(2) to mimic hypoxic conditions. At 24, 48, and 72 h, the cells were collected for morphological observation, MTT assay, and real-time quantitative PCR for HIF-1α mRNA expression.

RESULTSCompared with the cells without CoCl(2) treatment, the cells with CoCl(2) exposure exhibited obvious morphological changes and a significant growth inhibition which increased with CoCl(2)concentration and exposure time. At low concentrations (50-200 µmol/L), CoCl(2) treatment caused a dose- and time-dependent enhancement of HIF-1α expression in HL-60 cells.

CONCLUSIONHypoxia mimicked by CoCl(2) exposure significantly inhibits the proliferation of HL-60 cells, and at the non-toxic doses, CoCl(2) dose- and time-dependently increases the expression of HIF-1α. The mimicked hypoxic conditions do not cause differentiation of HL-60 cells.

Cell Hypoxia ; Cell Proliferation ; Cobalt ; pharmacology ; HL-60 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo investigate the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) on vascular endothelial growth factor (VEGF) expression in Tca8113 cells under hypoxia.

METHODSThe expression of the mRNA of HIF-1 alpha and VEGF in Tca8113 cells was examined by RT-PCR technique at different culture times (1/2 h, 1 h, 3 h, 6 h, 12 h, 24 h) under normoxic and hypoxic conditions.

RESULTSThe expression of HIF-1 alpha under hypoxia showed the trend of increasing first and then decreasing, and was higher than that of the control (normoxic group) at 6h and 12 h (P < 0.05). The expression of VEGF under hypoxia was higher than that of the control group at 1/2 h, 1 h, 3 h, 12 h, 24 h (P < 0.05). The expression of hypoxia-induced VEGF mRNA increased with the increased expression of HIF-1 alpha mRNA in the cell lines tested at the initial stage of hypoxia. But no statistical significant association was observed between HIF-1 alpha and VEGF expression within 24 h under hypoxia (rs = 0.5750, P > .005).

CONCLUSIONSThe increased expression of VEGF in Tca8113 cells might be mediated by multiple factors, including HIF-1 alpha.

Carcinoma, Squamous Cell ; genetics ; metabolism ; Cell Hypoxia ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; RNA, Messenger ; genetics ; Tongue Neoplasms ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

LncRNA H19 encoded by the H19 imprinting gene plays an important regulatory role in the cell. Recently study has found that in hypoxic cells, the expression of H19 gene changes, and the transcription factors and protein involved in the expression change accordingly. Through the involvement of specific protein 1 (SP1), hypoxia-inducible factor-1α (HIF-1α) binds directly to the H19 promoter and induces the up-regulation of H19 expression under hypoxic conditions. The tumor suppressor protein p53 may also mediate the expression of the H19 gene, in part by interfering with HIF-la activity under hypoxia stress. The miR675-5p encoded by exon 1 of H19 promotes hypoxia response by driving the nuclear accumulation of HIF-1α and reducing the expression of VHL gene, which is a physiological HIF-1α inhibitor. In addition, under the condition of hypoxia, the expression of transporter on cell membrane changes, and the transition of the intracellular glucose metabolism pathway from aerobic oxidation to anaerobic glycolysis is also involved in the involvement of H19. Therefore, H19 may be a key gene that maintains intracellular balance under hypoxic conditions and drives adaptive cell survival under conditions of hypoxia stress.
Cell Hypoxia ; genetics ; Genes, Tumor Suppressor ; physiology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; RNA, Long Noncoding ; Up-Regulation ; physiology ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics

OBJECTIVETo study the effects of oxygen and calcium on the expression of eukaryotic vectors harboring wild-type or mutated hypoxia-inducible factor-1alpha (HIF-11alpha) in HEK293 cells.

METHODSHEK293 cells were transiently transfected with pcDNA3.1+/HIF-11alpha, pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha-564Ala-803Ala via lipofectin. Western blotting were used to detect HIF-11alpha protein after normoxic or hypoxic exposure of the transfected HEK293 cells in the presence or absence of Ca(2+). The levels of vascular endothelial growth factor (VEGF) mRNA in the transfected cells in normoxic condition was detected using RT-PCR.

RESULTSThe levels of HIF-11alpha protein and VEGF mRNA increased in HEK293 cells transfected with the vectors harboring mutated HIF-11alpha, but not in the cells transfected with wild-type HIF-11alpha vectors in normoxia. Hypoxia increased the levels of HIF-11alpha protein in the cells transfected with wild-type HIF-11alpha vectors, which was inhibited by the application of Ca(2+). Ca(2+) showed no inhibitory effect on HIF-11alpha levels in HEK293 cells transfected with the vectors containing mutated HIF-11alpha.

CONCLUSIONThe protein products of pcDNA3.1+/HIF-11alpha-564Ala and pcDNA3.1+/HIF-11alpha- 564Ala-803Ala in HEK293 cells enhance the cell tolerance to oxygen and protease.

Calcium ; metabolism ; Cell Hypoxia ; Genetic Vectors ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Oxygen ; metabolism ; RNA, Messenger ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the effects of hypoxia on sumoylation of HIF-1α, as well as transcriptional activity and protein stability of HIF-1α in Jurkat cells, and explore its effect and significance on modulating the transcriptional activity of down stream target gene.

METHODSCoCl(2) was used as a chemical inducer to simulate the hypoxia environment. Real-time fluorescence quantitative PCR and western blot were used to evaluate transcriptional activity and protein stability of HIF-1α, levels of SUMO-1 and SENP1 protein, and gene transcripts of VEGF, mdr1, mdr3, Mcl-1 and survivin respectively.

RESULTSAfter 4 h, 8 h, 16 h, 24 h and 72 h after hypoxia induction, the gene transcripts of HIF-1α were 0.79 ± 0.19, 2.65 ± 2.05, 4.19 ± 4.72, 2.77 ± 3.37, 0.09 ± 0.05 and 0.69 ± 0.55-fold (P > 0.01) of that of 0h in Jurkat cells, respectively, while the protein stability increased first, then decreased (P < 0.01). SENP-1 protein up-regulated first, then down-regulated, and the SUMO-1 protein changed in an opposite trend. Excepting for survivin gene, transcriptional activities of VEGF, mdr1, mdr3, and Mcl-1 were affected by the stability of HIF-1α protein.

CONCLUSIONHypoxia induces changes in SENP-1 expression, which increases the stability of HIF-1α by decreasing the sumoylation of HIF-1α and affects biological process by regulating the transcriptional activities of VEGF, mdr1, mdr3 and Mcl-1 gene.

Cell Hypoxia ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Jurkat Cells ; RNA, Messenger ; genetics ; Sumoylation ; Transcriptional Activation ; Vascular Endothelial Growth Factor A ; metabolism

AIMTo study the effects of ginkgolides (Gin) on the expression of hypoxia inducible factor-1 (HIF-1alpha) in hypoxic/ischemic neurons.

METHODSThe gene expression of HIF-1alpha pretreated with or without Gin (37.5 microg/ml) was observed by RT-PCR on primary cultured cortical neurons in the condition of hypoxia and oxygen-glucose deprivation.

RESULTSSome basic expression of HIF-1alpha mRNA were observed in cultured cortical neurons. The expression of HIF-1alpha mRNA increased after 24 h treatment with Gin. The level of HIF-la mRNA increased also after 1 h hypoxia and further enhanced after the pretreatment with Gin. The expression of HIF-1alpha mRNA decreased with the deprivation of both oxygen and glucose, which reversed after the pretreatment of Gin.

CONCLUSIONGin could increase the expression of HIF-1alpha mRNA in hypoxic/ischemic cortical neurons.

Animals ; Cell Hypoxia ; Gene Expression ; Ginkgolides ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Neurons ; drug effects ; metabolism ; RNA, Messenger ; genetics