HIF-1α shRNA Research Articles

Metformin suppresses hypoxia-inducible factor-1α expression in cancer-associated fibroblasts to block tumor-stromal cross-talk in breast cancer

To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer. Tumor associated fibroblasts (CAFs) co-cultured with breast cancer cells were treated with metformin, and the changes in expressions of hypoxia-inducible factor-1α (HIF-1α), p-AMPK, stroma-derived factor-1 (SDF-1) and interleukin-8 (IL-8) in the CAFs were detected using ELISA, RT-qPCR or Western blotting; Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1, IL-8 and TGF-β1. The effects of HIF-1α shRNA or overexpression plasmid, AMPK shRNA, and treatment with OG (a proline hydroxylase inhibitor) or 2-OXO (a proline hydroxylase activator) were examined on p-AMPK, HIF-1α, SDF-1 and IL-8 expressions and invasiveness of the CAFs. Metformin treatment significantly increased the expression levels of p-AMPK, SDF-1 and IL-8 (P<0.05) and decreased HIF-1α expression (P<0.05) without affecting AMPK expression level (P>0.05) in the CAFs. The invasion ability of metformintreated breast cancer cells was significantly decreased (P<0.05). Exogenous SDF-1 and IL-8, HIF-1α overexpression, and OGinduced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion (P<0.05) and HIF-1α, SDF-1 and IL-8 expressions in CAFs (P<0.05). Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells (P<0.05). P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells (P<0.05). Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells (P<0.05). Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.

Regulation of Long Noncoding RNA NEAT1/miR-320a/HIF-1α Competitive Endogenous RNA Regulatory Network in Diabetic Retinopathy.

To determine the mechanism that long noncoding RNA NEAT1 (lncNEAT1)/miR-320a competitive endogenous RNA (ceRNA) network regulates hypoxia-inducible factor-1α (HIF-1α) in ARPE-19 cells and its potential role in diabetic retinopathy (DR). ARPE-19 cells were cultured in a normal or high-glucose (HG) medium, and cell migration, invasion, and permeability were detected by scratch, transwell, and FITC-dextran staining assays. LncNEAT1, HIF-1α, ZO-1, occludin, N-cadherin, and vimentin levels were tested. The binding of lncNEAT1 to miR-320a was verified by dual-luciferase reporter assay, and the binding of miR-320a to HIF-1α by RIP assay. ARPE-19 cells were treated with lncNEAT1 or HIF-1α shRNA or miR-320a agomir to determine the activation of ANGPTL4/p-STAT3 pathway. The effect of lncNEAT1 in DR and its regulations on miR-320a and HIF-1α were determined in a rat model of DR. HG treatment promoted the migration, invasion, and permeability of ARPE-19 cells. After lncNEAT1 silencing, HIF-1α, N-cadherin, and vimentin levels were downregulated, ZO-1 and occludin levels were upregulated, and the migration, permeability, and invasion of HG-treated ARPE-19 cells were inhibited. However, HIF-1α overexpression increased N-cadherin and vimentin expression, reduced ZO-1 and occludin expression, and promoted the migration, permeability, and invasion of ARPE-19 cells. The binding of miR-320a with both lncNEAT1 and HIF-1α was predicted and confirmed. In a diabetic rat model, silencing lncNEAT1 inhibited HIF-1α/ANGPTL4/p-STAT3 pathway activation and alleviated retinopathy. The lncNETA1/miR-320a/HIF-1α ceRNA network activates the ANGPTL4/p-STAT3 pathway and promotes HG-induced ARPE-19 cell invasion and migration.

Advanced platelet-rich fibrin promotes the paracrine function and proliferation of adipose-derived stem cells and contributes to micro-autologous fat transplantation by modulating HIF-1α and VEGF

BackgroundThe micro-autologous fat transplantation (MAFT) technique has demonstrated its feasibility in multiple medical fields, such as facial rejuvenation. Advanced platelet-rich fibrin (APRF), an autologous platelet concentrated on a fibrin membrane without added external factors, has shown significant potential for tissue restoration. However, the role of APRF in the modulation of MAFT remains unclear. Here, we aimed to explore the effect of APRF on MAFT.MethodsAdipose-derived stem cells (ASCs) were isolated from human gastric subcutaneous fat and treated with APRF. ELISA assays measured cytokines. The proliferation of ASCs was analyzed by CCK-8 assays. The levels of hypoxia-inducible factor-1α (HIF-1α), heat shock protein 70 (HSP70), insulin like growth factor 2 (IGF-2), interleukin-6 (IL-6), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) were measured by ELISA assays, quantitative reverse transcription-PCR (qRT-PCR), and Western blot analysis. The effect of APRF/HIF-1α/VEGF on MAFT in vivo was analyzed in Balb/c nude mice. The BALB/c mice were subcutaneously co-transplanted with fat, APRF, and control shRNA, HIF-1α shRNA, or VEGF shRNA into the dorsal area. The serum and protein levels of the above cytokines were analyzed by ELISA assays and Western blot analysis. Lipid accumulation was measured by Oil Red O staining. The expression of CD34 was assessed by immunohistochemical staining.ResultsAPRF continuously secreted multiple cytokines, including epidermal growth factor (EGF), FGF-2, insulin like growth factor 1 (IGF-1), interleukin-1beta (IL-1β), interleukin-4 (IL-4), platelet-derived growth factor alpha polypeptide b (PDGF-AB), platelet-derived growth factor beta polypeptide b (PDGF-BB), transforming growth factor-beta (TGF-β), and VEGF. APRF was able to promote the proliferation of ASCs. APRF dose-dependently activated the expression of HIF-1α, HSP70, IGF-2, IL-6, IL-8, and VEGF in ASCs. APRF regulated the paracrine function of ASCs by modulating HIF-1α and VEGF. APRF increased the survival of MAFT by modulating HIF-1α and VEGF in vivo.ConclusionsAPRF promotes the paracrine function and proliferation of ASCs and contributes to MAFT by modulating HIF-1α and VEGF. Our findings provide new insights into the mechanism by which APRF regulates MAFT.

HIF1 and ID1 Interplay Confers Adaptive Survival to HIF1α-Inhibition.

Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.

Hypoxia Inducible Factor (HIF)‐1α is a Transcriptional Mediator for Thiamine Insufficiency Induced Neurotoxicity

Age-dependent decline in vitamin B1 (thiamine) levels has a devastating impact on the brain parenchyma. The neurotoxicity promoted by chronic thiamine insufficiency (TI) is a well-established comorbidity of Alzheimer's Disease (AD), the most prevalent age-related metabolic neurodegenerative disease. Inadequate levels of blood thiamine and derivates highly correlate with senile dementia prognosis as predictive peripheral AD biomarker. Impaired activity of thiamine-dependent enzymes due to TI, dramatically decreased cerebral oxidative metabolism promoting oxidative stress, mitochondrial damage, inflammation, and eventually neuronal loss. Furthermore, age-related decline in TI potentiates the AD neuropathology triggering the amyloid plaque formation and hyperphosphorylation of tau. We previously showed that TI stabilizes Hypoxia Inducible Factor -1α (HIF-1α), the main transcription factor involved in hypoxic stress, leading to HIF-1α induced pro-apoptotic and pro-inflammatory response. Among the transcriptionally activated HIF-1α target genes, TI triggered the expression of the pro-apoptotic protein BCL2/adenovirus E1B 19 kDa protein-interacting protein-3 (BNIP-3). Induction of BNIP-3 leads to its mitochondria migration where eventually triggers cell death via loss of membrane potential and increase in reactive oxygen species. To date, despite the well-established correlation between TI and neuropathogenesis, the molecular mechanism underlying the TI mediated neurotoxicity is still unknown. Therefore, we hypothesize that HIF-1α may be a critical upstream mediator between thiamine insufficiency and neurotoxicity. In this study, hippocampal murine cells (HT22) treated in TI conditions exhibited a significant increase in protein and mRNA levels of HIF-1α. Increase in BNIP-3 and other HIF-1α target genes (LDHA, VEGF, BACE-1, MCP-1) implicated in pro-inflammatory and pro-apoptotic response was also detected. Treatment with 50 μM and 100 μM of the thiamine antagonist Pyrithiamine (PT) up to 5 days significantly decrease HT22 viability to 30% and 20%, respectively. HIF-1α silencing via shRNA attenuated the neurotoxic response improving viability up to 60% with the same PT doses. In order to better characterize the HIF-1α response, BNIP-3 was also silenced via shRNA. In congruency with HIF-1α shRNA result, we found a 2-fold decrease in PT-induced toxicity in BNIP-3 shRNA transduced cells. Localization of BNIP-3 in mitochondria and loss of mitochondrial potential after PT treatment further highlighted the involvement of the HIF-1α - BNIP-3 response in TI induced neuronal death. Overall, these findings suggest a central role for HIF-1α as upstream regulator in TI mediated neurotoxicity.

Treatment for Liver Tumor Using Combined Transarterial Embolization and Interaarterial Transfecting HIF-1α shRNA in a Rabbit VX2 Model.

BackgroundHypoxia-inducible factor-1α (HIF-1α) has been selected as therapeutic gene in gene therapy. The aim of this study was to explore the treatment effect of combined transarterial embolization using microsphere treatment (MD) and intraarterial transfecting HIF-1α shRNA on hepatocellular carcinoma (HCC).Materials and MethodsRabbit skin fibroblast was transfected with HIF-1α shRNA to evaluate the knocking down efficiency. Sixteen rabbit VX2 liver tumor models were randomly divided into four groups: the control group without any treatment, the MD group, the shRNA group (HIF-1α shRNA transfection by transcatheter intraarterial infusion), and the shRNA+MD group. The necrotic score, mitotic count and expression of HIF-1α, vascular endothelial growth factor (VEGF), CD34 and periodic acid-Schiff (PAS) stain were evaluated at the 14th and 28th day after treatment. The expression of HIF-1α and VEGF of VX2 tumors was also evaluated by real-time polymerase chain reaction on the 28th day.ResultsThe expression of HIF-1α-mRNA was lower in HIF-1α shRNA group than the control (p < 0.01). The tumor size was smaller in the shRNA + MD group than the shRNA group and the MD group (p < 0.05) on the 28th day. The growth rate of tumors in the shRNA + MD group was also lower than in other groups. The gene and protein expressions of both HIF-1α and VEGF in the shRNA + MD group were lower than the MD group, shRNA group and control group on the 28th day (p < 0.05). The necrotic score was higher in the shRNA + MD group than the MD group and control group (p < 0.05). The mitotic count and PAS-positive cells in shRNA + MD group were lower and CD34 was higher than the other three groups (p < 0.05).ConclusionCompared to therapy with MD or HIF-1α shRNA with transcatheter intraarterial transfection alone, the combined treatment has a better effect on HCC.

Inhibiting miR-155 protects against myocardial ischemia/reperfusion injury via targeted regulation of HIF-1α in rats.

Objective(s):The aim of this study was to identify the role of miR-155 in the myocardial ischemia/reperfusion (I/R) injury through targeting hypoxia-inducible factor 1-alpha (HIF-1α). Materials and Methods:We constructed rat models with myocardial I/R injury and H9C2 cell models with hypoxia/reoxygenation (H/R) damage. Anti-miR-155 and HIF-1α short hairpin RNA (shRNA) were used to treat rats and H9C2 cells to measure infarct area (IA) by TTC staining, determine creatine kinase (CK) and lactate dehydrogenase (LDH) activities by automatic biochemical analyzer, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) levels by ELISA, and detect apoptosis-related proteins by Western blotting. TUNEL staining and flowcytometry were employed to evaluate the apoptosis, JC-1 staining to detect mitochondrial membrane potential (MMP), and MTT assay to determine H9C2 cell viability.Results:After I/R and H/R, significant elevations were observed in IA, apoptosis, CK, LDH, cTnT, cTnI, and miR-155 levels with reduced HIF-1α. Besides, H/R-induced H9C2 cells presented decreases in MMP and Bcl-2/Bax, but increases in cytosolic/mitochondrial ratio of cytochrome C (Cyt-C) and expressions of cleaved caspase-3 and cleaved caspase-9. However, both rats and H9C2 cells showed an opposite tendency concerning the above after anti-miR-155 treatment. Nevertheless, HIF-1α shRNA effectively reversed protective effects of anti-miR-155 on alleviating I/R- and H/R- induced injury.Conclusion:Inhibiting miR-155 could reduce myocardial infarct size, suppress I/R-induced cardiomyocyte apoptosis, and maintain the MMP to alleviate I/R-induced injury via specific regulation of HIF-1α.

Chrysophanol Suppresses Hypoxia-Induced Epithelial-Mesenchymal Transition in Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common human malignancy, accounting for 600,000 death cases annually worldwide. Chrysophanol is a naturally occurring anthraquinone compound and exhibits anti-neoplastic activities. This study aims to explore the biological effects of chrysophanol on CRC metastasis and the relevant underlying mechanism. Cell proliferation assay, wound scratch assay, and Transwell invasion assay were used to examine the effect of chrysophanol on proliferation, migration, and invasion of CRC cells. Hypoxia-inducible factor-1α (HIF-1α) shRNA was utilized to transfect CRC cells to examine the role of HIF-1α in chrysophanol suppression of hypoxia-induced epithelial to mesenchymal transition (EMT). The suppression effect of chrysophanol on hypoxia-induced EMT in vivo was also validated in xenograft tumor models. In the present study, our findings indicated that chrysophanol has the capability to suppress hypoxia-induced EMT in CRC in vitro and in vivo, and the possible mechanism involved is the inhibition of HIF-1α via modulating PI3k/Akt signaling pathway. Collectively, the results indicated that chrysophanol can be used as an EMT and cancer metastasis inhibitor in the treatment of CRC. Anat Rec, 302:1561-1570, 2019. © 2019 American Association for Anatomy.

A Combination of UTMD-Mediated HIF-1α shRNA Transfection and TAE in the Treatment of Hepatic Cancer.

To explore the antitumor effect of hypoxia-inducible factor-1α short hairpin RNA (HIF-1α shRNA) delivered by ultrasound targeted microbubble destruction (UTMD) and transcatheter arterial embolization (TAE) on rats with hepatic cancer. After the models of transplantation hepatoma were established, Wistar rats were randomly divided into 4 groups: Control group, UTMD group, TAE group, and UTMD+TAE group. Contrast-enhanced ultrasound (CEUS) was used to monitor tumor size on day 14 after four different treatments. Western blotting and immunohistochemistry were applied to measure the protein level of HIF-1α and VEGF in the hepatic cancer tissue. In comparison with UTMD+TAE group (21.25±10.68 days), the mean survival time was noticeably shorter in the Control group and TAE group (13.02±4.30 days and 15.03±7.32 days) (p<0.05, respectively). There was no statistical difference between UTMD+TAE group and UTMD group of the mean survival time (p>0.05). In addition, our results proved that the tumor sizes in UTMD+TAE group were obviously smaller than those in other groups (p<0.05, respectively). By CEUS, we clearly found that the tumor size was the smallest on day 14 in the UTMD+TAE group. The western blotting and immunohistochemistry results proved that the protein levels of HIF-1α and VEGF in UTMD+TAE group were obviously lower than those in TAE group and Control group on days 7 and 14 (p<0.05, respectively). However, there was no statistical difference between UTMD+TAE group and UTMD group (p>0.05). In this study we tried to explore the antitumor effect through a combination of UTMD-mediated HIF-1α shRNA transfection and TAE on rats with hepatic cancer. Our results showed that UTMD-mediated HIF-1α shRNA transfection and TAE can obviously silence HIF-1α and VEGF expression, thereby successfully inhibiting the growth of the tumor.

Abstract TP332: Hypoxia-Inducible Factor 1-Alpha (HIF1α)-Mediated Phosphoenolpyruvate (PEP) Carboxykinase-1 (PCK1) Upregulation &amp; Metabolic Injury in Stroke

Introduction: In acute ischemic stroke (AIS), neurons upregulate glycolysis in an attempt to meet their energy demands, which causes lactic acidosis and reactive oxygen species (ROS) formation. These metabolic changes injure brain tissue by decreasing pH and disrupting local redox balance. There is emerging evidence that gluconeogenesis occurs in the brain. In AIS, the energy-requiring gluconeogenic pathway may be incomplete due to lack of ATP. In hepatocytes, HIF1α upregulates gluconeogenesis at the PCK1 promoter, which catalyzes oxaloacetate (OAA) decarboxylation to CO 2 and PEP. In AIS, HIF1α may act similarly, but its precise molecular mechanism in neuronal gluconeogenesis is unknown. It is hypothesized that gluconeogenesis is stunted through HIF1α upregulation of PCK1 in ischemia, which contributes to increased acidotic &amp; oxidative injury and neuron damage. Methods: Four cell culture groups (SH-SY5Y neurons) were cultivated: (1) control, (2) control cells transfected with HIF1α shRNA, (3) cells with oxygen-glucose deprivation (OGD) for 2 h and reoxygenation for 24 h, and (4) OGD cells transfected with HIF1α knockdown shRNA. The amount of lactate and ROS were assayed. Cell damage was determined by the WST-1 viability assay and apoptotic protein expression. The mRNA and protein levels were measured through qRT-PCR and Western blotting. Results: Cell viability decreased amidst increased lactate and ROS levels after OGD, and there was decreased Bcl2 (anti-apoptotic) and increased Bax (pro-apoptotic) protein expression. HIF1α and PCK1 mRNA and protein levels increased after OGD along with decreased glucose and OAA. The lactate and ROS were reduced (p&lt;0.05) and glucose and OAA were elevated (p&lt;0.05) with HIF1α shRNA treatment. Cell viability was enhanced by HIF1α shRNA after OGD, as expression of Bax decreased and Bcl2 increased. HIF1α shRNA decreased HIF1α and PCK1 mRNA and protein levels. Conclusion: Decreased acidotic, apoptotic, and oxidative damage by HIF1α knockdown aligned with enhanced cell survival through decreased PCK1 and decreased gluconeogenic activity. HIF1α-upregulated PCK1 stunted gluconeogenesis, leading to neuronal damage through lactate and ROS toxicity.

Effects of short hairpin RNA-mediated hypoxia inducible factor-1α gene silence on the proliferation of osteosarcoma cells under hypoxic condition

Objective To explore the effect of hypoxic condition on the proliferation of primary osteosarcoma cells, and the proliferation change of primary osteosarcoma cells under hypoxia after hypoxia inducible factor (HIF)-1α gene silence with short hairpin RNA (shRNA). Methods Primary osteosarcoma cells were isolated from osteosarcoma tumor and cultured under hypoxic (37℃, 1% O2, 5% CO2) or normoxic (37℃, 21% O2, 5% CO2) condition separately. Firstly, the cell proliferation, cell cycle and the HIF-1α protein expression of the cells were detected by methyl thiazol tetrazolium (MTT) assay, flow cytometry and Western blotting separately. Secondly, the primary osteosarcoma cells were transfected by lentiviral vectors carrying HIF-1α or scramble (SCR) shRNA, which was respectively denoted as HIF-1α/shRNA group or SCR/shRNA group. HIF-1α mRNA expression in both groups was quantified using quantitative polymerase chain reaction (qPCR) and the HIF-1α protein expression was detected by Western blotting. Finally, the transfected cells were cultured under hypoxic condition, and subsequently MTT assay was carried out for the evaluation of their proliferation. Results Hypoxia could inhibit the proliferation of primary osteosarcoma cells within 24 h, and this inhibition attenuated with the continually hypoxic condition. The expression of HIF-1α increased under continually hypoxic condition, indicating that the osteosarcoma cell proliferation might be related to the HIF-1α expression, and the cell proliferation rate of hypoxia cells decreased by 30.5% in 12 h and 25.0% in 24 h. As compared with SCR/shRNA group, the mRNA and protein expression levels of HIF-1α in HIF-1α/shRNA group significantly decreased (P=0.000), suggesting that the specific gene silence of HIF-1α could inhibit the osteosarcoma cell proliferation. Conclusion shRNA-mediated HIF-1α gene silence can effectively reverse the promoting effect of HIF-1α on osteosarcoma cell proliferation under hypoxic condition, which may further interfere with the osteosarcoma development and growth. Key words: Osteosarcoma; Hypoxia-inducible factor-1α; Gene silence; Short hairpin RNA

Hypoxia inducible factor-1\u03b1 mediates the profibrotic effect of albumin in renal tubular cells

Proteinuria is closely associated with the progression of chronic kidney diseases (CKD) by producing renal tubulointerstitial fibrosis. Over-activation of hypoxia inducible factor (HIF)-1α has been implicated in the progression of CKD. The present study tested the hypothesis that HIF-1α mediates albumin-induced profibrotic effect in cultured renal proximal tubular cells. Incubation of the cells with albumin (40 μg/ml) for 72 hrs significantly increased the protein levels of HIF-1α, tissue inhibitor of metalloproteinase (TIMP)-1 and collagen-I, which were blocked by HIF-1α shRNA. Albumin also stimulated an epithelial-mesenchymal transition (EMT) as indicated by the decrease in epithelial marker E-cadherin, and the increase in mesenchymal markers α-smooth muscle actin and fibroblast-specific protein 1. HIF-1α shRNA blocked albumin-induced changes in these EMT markers as well. Furthermore, albumin reduced the level of hydroxylated HIF-1α, indicating an inhibition of the activity of prolyl-hydroxylases, enzymes promoting the degradation of HIF-1α. An anti-oxidant ascorbate reversed albumin-induced inhibition of prolyl-hydroxylase activity. Overexpression of prolyl-hydroxylase 2 (PHD2) transgene, a predominant isoform of PHDs in renal tubules, to reduce HIF-1α level significantly attenuated albumin-induced increases in TIMP-1 and collagen-I levels. These results suggest that albumin-induced oxidative stress inhibits PHD activity to accumulate HIF-1α, which mediates albumin-induced profibrotic effects in renal tubular cells.

Hypoxia-Inducible Factor 1α Signaling Promotes Repair of the Alveolar Epithelium after Acute Lung Injury

During the acute respiratory distress syndrome, epithelial cells, primarily alveolar type (AT) I cells, die and slough off, resulting in enhanced permeability. ATII cells proliferate and spread onto the denuded basement membrane to reseal the barrier. Repair of the alveolar epithelium is critical for clinical recovery; however, mechanisms underlying ATII cell proliferation and spreading are not well understood. We hypothesized that hypoxia-inducible factor (HIF)1α promotes proliferation and spreading of ATII cells during repair after lung injury. Mice were treated with lipopolysaccharide or hydrochloric acid. HIF activation in ATII cells after injury was demonstrated by increased luciferase activity in oxygen degradation domain-Luc (HIF reporter) mice and expression of the HIF1α target gene GLUT1. ATII cell proliferation during repair was attenuated in ATII cell-specific HIF1α knockout (SftpcCreERT2+/-;HIF1αf/f) mice. The HIF target vascular endothelial growth factor promoted ATII cell proliferation invitro and after lung injury invivo. In the scratch wound assay of cell spreading, HIF stabilization accelerated, whereas HIF1α shRNA delayed wound closure. SDF1 and its receptor, CXCR4, were found to be HIF1α-regulated genes in ATII cells and were up-regulated during lung injury. Stromal cell-derived factor 1/CXCR4 inhibition impaired cell spreading and delayed the resolution of permeability after lung injury. We conclude that HIF1α is activated in ATII cells after lung injury and promotes proliferation and spreading during repair.

Cell type-dependent HIF1 α-mediated effects of hypoxia on proliferation, migration and metastatic potential of human tumor cells.

Tumor hypoxia promotes neoangiogenesis and contributes to the radio- and chemotherapy resistant and aggressive phenotype of cancer cells. However, the migratory response of tumor cells and the role of small GTPases regulating the organization of cytoskeleton under hypoxic conditions have yet to be established. Accordingly, we measured the proliferation, migration, RhoA activation, the mRNA and protein levels of hypoxia inducible factor-1alpha (HIF-1α) and three small G-proteins, Rac1, cdc42 and RhoA in a panel of five human tumor cell lines under normoxic and hypoxic conditions. Importantly, HT168-M1 human melanoma cells with high baseline migration capacity showed increased HIF-1α and small GTPases expression, RhoA activation and migration under hypoxia. These activities were blocked by anti- HIF-1α shRNA. Moreover, the in vivo metastatic potential was promoted by hypoxia mimicking CoCl2 treatment and reduced upon inhibition of HIF-1α in a spleen to liver colonization experiment. In contrast, HT29 human colon cancer cells with low migration capacity showed limited response to in vitro hypoxia. The expression of the small G-proteins decreased both at mRNA and protein levels and the RhoA activation was reduced. Nevertheless, the number of lung or liver metastatic colonies disseminating from orthotopic HT29 grafts did not change upon CoCl2 or chetomin treatment. Our data demonstrates that the hypoxic environment induces cell-type dependent changes in the levels and activation of small GTPases and results in varying migratory and metastasis promoting responses in different human tumor cell lines.

Cold Inducible RNA Binding Protein Is Involved in Chronic Hypoxia Induced Neuron Apoptosis by Down-Regulating HIF-1α Expression and Regulated By microRNA-23a.

Background: Neuron apoptosis mediated by hypoxia inducible factor 1α (HIF-1α) in hippocampus is one of the most important factors accounting for the chronic hypobaric hypoxia induced cognitive impairment. As a neuroprotective molecule that is up-regulated in response to various environmental stress, CIRBP was reported to crosstalk with HIF-1α under cellular stress. However, its function under chronic hypobaric hypoxia remains unknown.Objective: In this study, we tried to identify the role of CIRBP in HIF-1α mediated neuron apoptosis under chronic hypobaric hypoxia and find a possible method to maintain its potential neuroprotective in long-term high altitude environmental exposure.Methods: We established a chronic hypobaric hypoxia rat model as well as a tissue culture model where SH-SY5Y cells were exposed to 1% hypoxia. Based on these models, we measured the expressions of HIF-1α and CIRBP under hypoxia exposure and examined the apoptosis of neurons by TUNEL immunofluorescence staining and western blot analysis of apoptosis related proteins. In addition, by establishing HIF-1α shRNA and pEGFP-CIRBP plasmid transfected cells, we confirmed the role of HIF-1α in chronic hypoxia induced neuron apoptosis and identified the influence of CIRBP over-expression upon HIF-1α and neuron apoptosis in the process of exposure. Furthermore, we measured the expression of the reported hypoxia related miRNAs in both models and the influence of miRNAs' over-expression/knock-down upon CIRBP in the process of HIF-1α mediated neuron apoptosis.Results: HIF-1α expression as well as neuron apoptosis was significantly elevated by chronic hypobaric hypoxia both in vivo and in vitro. CIRBP was induced in the early stage of exposure (3d/7d); however as the exposure was prolonged (21d), CIRBP level of the hypoxia group became significantly lower than that of control. In addition, HIF-1α knockdown significantly decreased neuron apoptosis under hypoxia, suggesting HIF-1α may be pro-apoptotic in the process of exposure. CIRBP over-expression significantly suppressed HIF-1α up-regulation in hypoxia and inhibited HIF-1α mediated neuron apoptosis. Interestingly, miR-23a was also induced by hypoxia exposure and showed the same changing tendency with CIRBP (increasing in 3d/7d, decreasing in 21d). In addition, over-expressing miR-23a up-regulated CIRBP, down-regulated HIF-1α and attenuated neuron apoptosis.Conclusion: Cold inducible RNA binding protein is involved in chronic hypoxia induced neuron apoptosis by down-regulating HIF-1α expression, and MiR-23a may be an important tool to maintain CIRBP level and function.

Suppression of the expression of hypoxia-inducible factor-1α by RNA interference alleviates hypoxia-induced pulmonary hypertension in adult rats

Hypoxia-inducible factor-1α (HIF-1α) has been implicated in the pathogenesis of hypoxic pulmonary hypertension (PH). However, the potential clinical value of HIF-1α as a therapeutic target in the treatment of PH has not yet been evaluated. In this study, an animal model of hypoxia-induced PH was established by exposing adult rats to 10% O2 for 3 weeks, and the effects of the lentivirus-mediated delivery of HIF-1α short hairpin RNA (shRNA) by intratracheal instillation prior to exposure to hypoxia on the manifestations of hypoxia-induced PH were assessed. The successful delivery of HIF-1α shRNA into the pulmonary arteries effectively suppressed the hypoxia-induced upregulation of HIF-1α, accompanied by the prominent attenuation the symptoms associated with hypoxia-induced PH, including the elevation of pulmonary arterial pressure, hypertrophy and hyperplasia of pulmonary artery smooth muscle cells (PASMCs), as well as the muscularization of pulmonary arterioles. In addition, the knockdown of HIF-1α in cultured rat primary PASMCs significantly inhibited the hypoxia-induced acceleration of the cell cycle and the proliferation of the PASMCs, suggesting that HIF-1α may be a direct mediator of PASMC hyperplasia in hypoxia-induced PH. In conclusion, this study demonstrates the potent suppressive effects of HIF-1α shRNA on hypoxia-induced PH and PASMC hyperplasia, providing evidence for the potential application of HIF-1α shRNA in the treatment of hypoxic PH.

Silencing of hypoxia inducible factor-1α gene attenuated angiotensin Ⅱ-induced abdominal aortic aneurysm in apolipoprotein E-deficient mice

Background and aimsWe aimed to determine the effect of HIF-1α, the main regulatory subunit of the hypoxia inducible factor 1 (HIF-1), on the development of the abdominal aortic aneurysm (AAA). MethodsAAA was induced in ApoE−/− mice by angiotensinⅡ (AngⅡ) infusion. In vivo silencing of HIF-1α was achieved by transfection of lentivirus expressing HIF-1α shRNA. ResultsTime course analysis of the AngⅡ infusion model revealed that HIF-1α was persistently upregulated during a 28-day period of AAA development. Silencing of the HIF-1α gene reduced the aneurysm size (2.84 ± 1.96 mm vs. 1.41 ± 0.85 mm respectively at day 28, p = 0.0002). Silencing of HIF-1α also alleviated infiltration of macrophages (38.8 ± 14.7 vs. 11.4 ± 4.4 macrophages/0.1 mm2, p = 0.0006) and neovascularity (5.56 ± 2.14 vs. 1.27 ± 1.05 microvessels/0.1 mm2, p = 0.0008) in the AngⅡ infusion model, at day 28. The activity of MMP-2 and MMP-9 was also decreased by knockdown of HIF-1α. The early increased expression of pro-inflammatory factors, angiogenic factors, and MMPs during AAA induction was alleviated by HIF-1α silencing. ConclusionsActivation of HIF-1 signaling pathway participates in the Ang Ⅱ-induced AAA formation in mice.

Downregulation of HIF-1a sensitizes U251 glioma cells to the temozolomide (TMZ) treatment

PurposeThe aim of this study was to investigate the effect of downregulation of HIF-1α gene on human U251 glioma cells and examine the consequent changes of TMZ induced effects and explore the molecular mechanisms. MethodsU251 cell line stably expressing HIF-1α shRNA was acquired via lentiviral vector transfection. The mRNA and protein expression alterations of genes involved in our study were determined respectively by qRT-PCR and Western blot. Cell proliferation was measured by MTT assay and colony formation assay, cell invasion/migration capacity was determined by transwell invasion assay/wound healing assay, and cell apoptosis was detected by flow cytometry. ResultsWe successfully established a U251 cell line with highly efficient HIF-1α knockdown. HIF-1a downregulation sensitized U251 cells to TMZ treatment and enhanced the proliferation-inhibiting, invasion/migration-suppressing, apoptosis-inducing and differentiation-promoting effects exerted by TMZ. The related molecular mechanisms demonstrated that expression of O6-methylguanine DNA methyltransferase gene (MGMT) and genes of Notch1 pathway were significantly upregulated by TMZ treatment. However, this upregulation was abrogated by HIF-1α knockdown. We further confirmed important regulatory roles of HIF-1α in the expression of MGMT and activation of Notch1 pathways. ConclusionHIF-1α downregulation sensitizes U251 glioma cells to the temozolomide treatment via inhibiting MGMT expression and Notch1 pathway activation.

Targeting HIF-1α and VEGF by lentivirus-mediated RNA interference reduces liver tumor cells migration and invasion under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) is a key transcription factor to initiate the expressions of distinct pro-angiogenic growth genes, particularly the expression of vascular endothelial growth factor (VEGF).CoCl2 was used in rat liver tumor cell line McA RH-7777 to stimulate hypoxia to mimic the hypoxic conditions induced by transcatheter arterial chemoembolization (TACE). CCK8 assays were performed to examine the effect of hypoxia on cell viability. Real-time qRT-PCR, western blot and ELISA assays were used to measure the expression of HIF-1α and VEGF in McA RH-7777 cells under hypoxic conditions, respectively. Lentivirus-mediated HIF-1α and/or VEGF-specific shRNA was used to establish single or HIF-1α and VEGF double knocking-down McA RH-7777 cells. Transwell assays were performed to examine the effect of HIF-1α and VEGF knocking-down on McA RH-7777 cells migration and invasion.The mRNA and protein expression level of HIF-1α and VEGF were remarkably up-regulated in McA RH-7777 cells under hypoxic conditions, respectively. The knockdown of HIF-1α or VEGF significantly reduced the expression of the secreted VEGF. More importantly, knockdown of both HIF-1α and VEGF resulted in the best effective inhibitory effect in VEGF expression, and in turn remarkably reduced the cell migration and invasion activity.Our findings showed that HIF-1α play an important role in the stimulation of the secreted VEGF expression under hypoxic conditions, suggesting that targeting both HIF-1α and VEGF could represent a potential therapeutic strategy in combination with TACE in the treatment of liver tumors.

Abstract 5080: HIF-1 is responsible for the induction of cancer-associated fibroblasts in hypoxic tumor microenvironment

Abstract In tumor tissues, tumor cells and tumor stromal cells interact with each other, generating the complex tumor microenvironment (TME). Whereas cancer-associated fibroblasts (CAFs) are dominant cells among tumor stromal cell types, the induction of CAFs in TME still remains unclear. Given that TME turns to be hypoxic while tumor is growing, we hypothesized that hypoxia in TME would contribute to induction of CAFs. In this study, we focused on a role of hypoxia-inducible factor-1 (HIF-1), a key transcription factor in hypoxic responses, and examined the implication of HIF-1 in the induction of CAFs in TME. The mouse lymphoma cell line, E.G7 cells, which had been transfected with HIF-1α shRNA (E.G7- HIF-1α shRNA) were cultured under hypoxia with an O2 concentration of 1.0%. As controls, E.G7 cells or those which had been transfected with control shRNA (E.G7-control shRNA) were also cultured under the same hypoxic condition as in case of E.G7- HIF-1α shRNA. Three days later, the culture medium was harvested for following experiments. To evaluate a role of HIF-1α in migration of CAFs progenitor cells into TME, mouse bone marrow cells as CAFs progenitor cells were put in the upper compartment, and hypoxic culture medium was added in the lower compartment of a transwell chamber. We observed that the number of α-smooth muscle actin (α-SMA)-positive cells that had migrated was decreased when hypoxic culture medium from E.G7- HIF-1α shRNA was added in the lower compartment. Transforming growth factor-beta (TGF-β) has been reported to be one of soluble factors which are responsible for induction of CAFs. Thus, we examined the level of TGF-β in hypoxic culture medium by ELISA, demonstrating that it was decreased in the medium from E.G7- HIF-1α shRNA as compared with E.G7-control shRNA or E.G7 cells. Next, we examined the association between HIF-1α inhibition and CAFs induction in a tumor-bearing mouse model. C57BL/6 mice were subcutaneously administered E.G7-HIF-1α shRNA, E.G7-control shRNA, or E.G7 cells at the flank. Three weeks later, tumor tissues were harvested from the mice, and α-SMA-positive cells in tumor tissues were examined by immunohistochemistry. The data showed that α-SMA-positive CAFs were decreased in E.G7-HIF-1α shRNA tumor tissues as compared with E.G7-control shRNA, or E.G7 tumor tissues. In addition, tumor growth was significantly suppressed in mice bearing E.G7-HIF-1α shRNA, while inhibition of HIF-1α did not exert a suppressive effect on the proliferation of E.G7 cells in vitro. These data indicated that HIF-1α would play a critical role in the induction of CAFs via TGF-β in hypoxic TME. Inhibition of HIF-1α could have contributed to reduction of CAFs in tumor tissues, leading to suppression of tumor growth. These results provide a novel rationale with TME-targeted strategy against cancer. Citation Format: Koji Teramoto, Yoko Kataoka, Tomoyuki Igarashi, Yasuhiko Ohshio, Jun Hanaoka, Yataro Daigo. HIF-1 is responsible for the induction of cancer-associated fibroblasts in hypoxic tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5080. doi:10.1158/1538-7445.AM2015-5080