Inhibition of Hypoxia Inducible Factor 1α by siRNA‐Induced Apoptosis in Human Retinoblastoma Cells

Hypoxia, which activates the hypoxia inducible factor 1α (HIF‐1α), is an essential feature of retinoblastoma (RB) and contributes to poor prognosis and resistance to conventional therapy. In this study, the effect of HIF‐1α knockdown by small interfering RNA (siRNA) on cell proliferation, apoptosis, and apoptotic pathways of human Y‐79 RB cells was first investigated. Exposure to hypoxia induced the increased expression of HIF‐1α both in mRNA and protein levels. Then, knockdown of HIF‐1α by siRNA_HIF‐1α resulted in inhibition of cell proliferation and induced cell apoptosis in human Y‐79 RB cells under both normoxic and hypoxic conditions, with hypoxic conditions being more sensitive. Furthermore, knockdown of HIF‐1α could enhance hypoxia‐induced slight increase of Bax/Bcl‐2 ratio and activate caspase‐9 and caspase‐3. These results together indicated that suppression of HIF‐1α expression may be a promising strategy for the treatment of human RB in the future.     

Qiliqiangxin attenuates hypoxia‐induced injury in primary rat cardiac microvascular endothelial cells via promoting HIF‐1α‐dependent glycolysis

Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO₂, 1% O₂, 94% N₂) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression.     

Mutual regulation between butyrate and hypoxia‐inducible factor‐1α in epithelial cell promotes expression of tight junction proteins

Inflammatory bowel disease is a kind of multi‐aetiological chronic disease that is driven by multidimensional factors. Hypoxia‐inducible factor‐1α (HIF‐1α) plays an important role in anti‐inflammatory and cellular responses to hypoxia. Previous studies have found that B or T‐cell‐specific HIF‐1α knock out mice exhibit severe colonic inflammation. However, we know very little about other functions of HIF‐1α in intestinal epithelial cells (IECs). In our study, HIF‐1α^ΔIEC mice were used to study the function of HIF‐1α in IECs. HIF‐1α was knocked down in Caco‐2 cells by transfection with a small interfering (si) RNA. Immunohistochemical staining and western blotting were used to detect the expression of zonula occluden‐1 (ZO‐1) and Occludin. The content of colon was harvested for high‐performance liquid chromatography analysis to examine the levels of butyrate in the gut. Our research found that HIF‐1α played a protective role in dextran sulphate sodium‐induced colitis, which was partly due to its regulation of tight junction (TJ) protein expression. Further study revealed that HIF‐1α mediated TJ proteins levels by moderating the content of butyrate. Moreover, we found that butyrate regulated TJ protein expression, which is dependent on HIF‐1α. These results indicated that there is a mutual regulatory mechanism between butyrate and HIF‐1α, which has an important role in the maintenance of barrier function of the gastrointestinal tract.     

Hypoxic stress‐induced changes in adrenergic function: role of HIF1α

Sustaining epinephrine‐elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr‐1 and Sp1 contribute by stimulating the gene encoding the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla‐derived PC12 cells. Hypoxia (5% O₂) also activates hypoxia inducible factor (HIF) 1α, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1α over‐expression also elevate PNMT promoter‐driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1α over‐expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1α inducers CoCl₂ or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1α expression construct show markedly higher levels of Egr‐1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr‐1 and Sp1 binding sites in the proximal ?893 bp of PNMT promoter precludes HIF1α stimulation while a potential hypoxia response element (?282 bp) in the promoter shows weak HIF1α affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1α induction of Egr‐1 and Sp1 rather than by co‐activation by Egr‐1, Sp1 and HIF1α. In addition, the rise in HIF1α protein leading to Egr‐1 and Sp1 stimulation of PNMT appears to include HIF1α gene activation rather than simply prevention of HIF1α proteolytic degradation.     

Sirt1–hypoxia‐inducible factor‐1α interaction is a key mediator of tubulointerstitial damage in the aged kidney

Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia‐inducible factor (HIF)‐1α is largely unknown. In this study, we investigated whether HIF‐1α could be a deacetylation target of Sirt1 and the effect of their interaction on age‐associated renal injury. Five‐week‐old (young) and 24‐month‐old (old) C57Bl/6J mice were assessed for their age‐associated changes. Kidneys from aged mice showed increased infiltration of CD68‐positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF‐1α. The level of Bcl‐2/adenovirus E1B‐interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor‐β1, which are regulated by HIF‐1α, was significantly higher in aged mice suggesting that HIF‐1α activity was increased. In HK‐2 cells, Sirt1 inhibitor sirtinol and siRNA‐mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down‐regulated, which allowed the acetylation and activation of HIF‐1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia‐induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF‐1α activity by Sirt1‐induced deacetylation of HIF‐1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF‐1α‐transfected HK‐2 cells. Finally, we confirmed that chronic activation of HIF‐1α promoted apoptosis and fibrosis, using tubular cell‐specific HIF‐1α transgenic mice. Taken together, our data suggest that Sirt1‐induced deacetylation of HIF‐1α may have protective effects against tubulointerstitial damage in aged kidney.     

HIF‐1α forms regulatory loop with YAP to coordinate hypoxia‐induced adriamycin resistance in acute myeloid leukemia cells

Despite the improvement in acute myeloid leukemia (AML) treatments, most patients had a poor prognosis and suffered from chemoresistance and disease relapse. Therefore, there is an urgent need for elucidation of mechanism(s) underlying drug resistance in AML. In the present study, we found that AML cells showed less susceptibility to adriamycin (ADR) in the presence of hypoxia, while inhibition of hypoxia‐inducible factor 1α (HIF‐1α) by CdCl₂ can make AML cells re‐susceptibile to ADR even under hypoxia. Moreover, HIF‐1α is overexpressed and plays an important role in ADR‐resistance maintenance in resistant AML cells. We further found hypoxia or induction of HIF‐1α can significantly upregulate yes‐associated protein (YAP) expression in AML cells, and resistant cells express a high level of YAP. Finally, we found that YAP may not only enhance HIF‐1α stability but also promote HIF‐1α's activity on the target gene pyruvate kinase M2. In conclusion, our data indicate that HIF‐1α or YAP may represent a therapeutic target for overcoming resistance toward adriamycin‐based chemotherapy in AML.     

HIF1α‐mediated AIMP3 suppression delays stem cell aging via the induction of autophagy

Senescence in stem cells, which occurs as a consequence of chronic responses to the environment, defines the capacity of stem cells for proliferation and differentiation as well as their potential for tissue regeneration and homeostasis maintenance. Although stem cells reside under low oxygen pressure and the availability of oxygen is known to be a crucial determinant in their fate, the key modulators in stem cell aging and the underlying mechanism have yet to be unraveled. Human placenta‐derived mesenchymal stem cells (hpMSCs) were cultured under hypoxia (3% O₂) or normoxia (21% O₂) to investigate the key factors that regulate stem cell senescence under hypoxic conditions. RNA sequencing results suggested that the expression of aminoacyl‐tRNA synthetase‐interacting multifunctional protein 3 (AIMP3, EEF1E1), an aging inducer, in the hpMSCs was dramatically repressed under hypoxia with concurrent suppression of the aging marker p16^INK4a. The hpMSCs that overexpressed AIMP3 under hypoxic conditions displayed significantly decreased proliferation and fewer stem cell characteristics, whereas the downregulation of AIMP3 ameliorated the age‐related senescence of MSCs. Consistent with the results of the hpMSCs, MSCs isolated from the adipose tissue of AIMP3‐overexpressing mice exhibited decreased stem cell functions. Interestingly, AIMP3‐induced senescence is negatively regulated by hypoxia‐inducible factor 1α (HIF1α) and positively regulated by Notch3. Furthermore, we showed that AIMP3 enhanced mitochondrial respiration and suppressed autophagic activity, indicating that the AIMP3‐associated modulation of metabolism and autophagy is a key mechanism in the senescence of stem cells and further suggesting a novel target for interventions against aging.     

Calycosin alleviates allergic contact dermatitis by repairing epithelial tight junctions via down‐regulating HIF‐1α

Calycosin, a bioactive component derived from Astragali Radix (AR; Huang Qi), has been shown to have an effect of anti‐allergic dermatitis with unknown mechanism. This study aims to investigate the mechanism of calycosin related to tight junctions (TJs) and HIF‐1α both in FITC‐induced mice allergic contact dermatitis and in IL‐1β stimulated HaCaT keratinocytes. Th2 cytokines (IL‐4, IL‐5 and IL‐13) were detected by ELISA. The epithelial TJ proteins (occludin, CLDN1 and ZO‐1), initiative key cytokines (TSLP and IL‐33) and HIF‐1α were assessed by Western blot, real‐time PCR, immunohistochemistry or immunofluorescence. Herein, we have demonstrated that allergic inflammation and the Th2 cytokines in ACD mice were reduced significantly by calycosin treatment. Meanwhile, calycosin obviously decreased the expression of HIF‐1α and repaired TJs both in vivo and in vitro. In HaCaT keratinocytes, we noted that IL‐1β induced the deterioration of TJs, as well as the increased levels of TSLP and IL‐33, which could be reversed by silencing HIF‐1α. In addition, administration of 2‐methoxyestradiolin (2‐ME), a HIF‐1α inhibitor,significantly repaired the TJs and alleviated the allergic inflammation in vivo. Furthermore, TJs were destroyed by DMOG or by overexpressing HIF‐1α in HaCaT keratinocytes, and simultaneously, calycosin down‐regulated the expression of HIF‐1α and repaired the TJs in this process. These results revealed that calycosin may act as a potential anti‐allergy and barrier‐repair agent via regulating HIF‐1α in AD and suggested that HIF‐1α and TJs might be possible therapy targets for allergic dermatitis.     

The ROS‐induced cytotoxicity of ascorbate is attenuated by hypoxia and HIF‐1alpha in the NCI60 cancer cell lines

Intravenous application of high‐dose ascorbate is used in complementary palliative medicine to treat cancer patients. Pharmacological doses of ascorbate in the mM range induce cytotoxicity in cancer cells mediated by reactive oxygen species (ROS), namely hydrogen peroxide and ascorbyl radicals. However, little is known about intrinsic or extrinsic factors modulating this ascorbate‐mediated cytotoxicity. Under normoxia and hypoxia, ascorbate IC₅₀ values were determined on the NCI60 cancer cells. The cell cycle, the influence of cobalt chloride‐induced hypoxia‐inducible factor‐1α (HIF‐1α) and the glucose transporter 1 (GLUT‐1) expression (a pro‐survival HIF‐1α‐downstream‐target) were analysed after ascorbate exposure under normoxic and hypoxic conditions. The amount of ascorbyl radicals increased with rising serum concentrations. Hypoxia (0.1% O₂) globally increased the IC₅₀ of ascorbate in the 60 cancer cell lines from 4.5 ± 3.6 mM to 10.1 ± 5.9 mM (2.2‐fold increase, P < 0.001, Mann–Whitney t‐test), thus inducing cellular resistance towards ascorbate. This ascorbate resistance depended on HIF‐1α‐signalling, but did not correlate with cell line‐specific expression of the ascorbate transporter GLUT‐1. However, under normoxic and hypoxic conditions, ascorbate treatment at the individual IC₅₀ reduced the expression of GLUT‐1 in the cancer cells. Our data show a ROS‐induced, HIF‐1α‐ and O₂‐dependent cytotoxicity of ascorbate on 60 different cancer cells. This suggests that for clinical application, cancer patients should additionally be oxygenized to increase the cytotoxic efficacy of ascorbate.     

Effect of HIF1α on Foxp3 expression in CD4+CD25− T lymphocytes

The aim of the present study was to investigate the effect of HIF1α on Foxp3 expression in CD4⁺CD25^? T lymphocytes. CD4⁺CD25^? T lymphocytes were sorted from PBMC using a CD4⁺CD25⁺ regulatory T cell isolation kit. Lentivirus containing lentiviral vector that overexpressed HIF1α (HIF‐lenti) and those containing empty expression vector (control‐lenti) were produced. Meanwhile, lentivirus that contained lentiviral vector that suppressed HIF1α expression (siHIF‐lenti) and those containing control vector (sicontrol‐lenti) were also generated. The sorted CD4⁺CD25^? T lymphocytes were infected with HIF‐lenti, control‐lenti, siHIF‐lenti, and sicontrol‐lenti, respectively. Approximately 72 hr after transduction, real‐time PCR and Western blot were carried out to analyze the RNA and protein expression level of HIF1α and Foxp3. CD4⁺CD25^? T lymphocytes cultured under 21% O₂, 5% CO₂ (normoxia) and 1% O₂, 5% CO₂ (hypoxia) were used as control. Our results showed that overexpression of HIF1α increased both mRNA and protein expression of Foxp3 and, meanwhile, suppression of HIF1α expression by RNAi could reverse high Foxp3 expression in CD4⁺CD25^? T lymphocytes caused by hypoxic culture. These results suggested that hypoxia could stimulate Foxp3 expression by increasing HIF1α expression in CD4⁺ T lymphocytes which may promote CD4⁺ T lymphocytes to convert to Treg.

     

Hypoxia changes chemotaxis behaviour of mesenchymal stem cells via HIF‐1α signalling

Mesenchymal stem cells (MSCs) have drawn great attention because of their therapeutic potential. It has been suggested that intra‐venous infused MSCs could migrate the site of injury to help repair the damaged tissue. However, the mechanism for MSC migration is still not clear so far. In this study, we reported that hypoxia increased chemotaxis migration of MSCs. At 4 and 6 hours after culturing in hypoxic (1% oxygen) conditions, the number of migrated MSCs was significantly increased. Meanwhile, hypoxia also increased the expression of HIF‐1α and SDF‐1. Using small interference RNA, we knocked down the expression of HIF‐1α in MSCs to study the role of HIF‐1α in hypoxia induced migration. Our data indicated that knocking down the expression of HIF‐1α not only abolished the migration of MSCs, but also reduced the expression of SDF‐1. Combining the results of migration assay and expression at RNA and protein level, we demonstrated a novel mechanism that controls the increase of MSCs migration. This mechanism involved HIF‐1α mediated SDF‐1 expression. These findings provide new insight into the role of HIF‐1α in the hypoxia induced MSC migration and can be a benefit for the development of MSC‐based therapeutics for wound healing.     

Plantamajoside inhibits the proliferation and epithelial‐to‐mesenchymal transition in hepatocellular carcinoma cells via modulating hypoxia‐inducible factor‐1α‐dependent gene expression

As a potential antitumor herbal medicine, plantamajoside (PMS) benefits the treatment of many human malignances. However, the role of PMS in the progression of hepatocellular carcinoma (HCC) and the related molecular mechanisms is still unknown. Here, we proved that the cell viabilities of HepG2 cells were gradually decreased with the increasing concentrations of CoCl₂ and/or PMS via cell counting kit‐8 assay. Meanwhile, 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) and western blot assays were used to further confirm that PMS inhibited the CoCl₂‐induced cell proliferation in HepG2 cells via suppressing the Ki67 and proliferating cell nuclear antigen expressions. We also performed wound‐healing and transwell assays and demonstrated that PMS inhibited CoCl₂‐induced migration and invasion in HepG2 cells via suppressing the epithelial–mesenchymal transition (EMT) process. In addition, the use of 3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole further proved that PMS inhibited the malignant biological behaviors of HepG2 cells under hypoxic condition by suppressing the hypoxia‐inducible factor‐1α (HIF‐1α) expression. Besides, we further confirmed that PMS suppressed the growth and metastasis of implanted tumors in vivo. Given that PMS suppressed the proliferation and EMT induced by CoCl₂ in HCC cells via downregulating HIF‐1α signaling pathway, we provided evidence that PMS might be a novel anti‐cancer drug for HCC treatment.     

Long non‐coding RNA MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis

Endometriosis is a common gynecological disease characterized by diminished apoptosis, sustained ectopic survival of dysfunctional endometrial cells. Hypoxia has been implicated as a crucial microenvironmental factor that contributes to endometriosis. It has been reported that long non‐coding RNA MALAT1 (lncRNA‐MALAT1) highly expressed in endometriosis and up‐regulated by hypoxia. Hypoxia may also induce autophagy, which might act as cell protective mechanism. However, the relationship between lncRNA‐MALAT1 and autophagy under hypoxia conditions in endometriosis remains unknown. In the present study, we found that both lncRNA‐MALAT1 and autophagy level were up‐regulated in ectopic endometrium from patients with endometriosis, and its expression level correlates positively with that of hypoxia‐inducible factor‐1α (HIF‐1α). In cultured human endometrial stromal cells, both lncRNA‐MALAT1 and autophagy were induced by hypoxia in a time‐dependent manner and lncRNA‐MALAT1 up‐regulation was dependent on HIF‐1α signalling. Our analyses also show that knockdown of lncRNA‐MALAT1 suppressed hypoxia induced autophagy. Furthermore, inhibiting autophagy with specific inhibitor 3‐Methyladenine (3‐MA) and Beclin1 siRNA enhanced apoptosis of human endometrial stromal cells under hypoxia condition. Collectively, our findings identify that lncRNA‐MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis.