Aldolase A level in malignant pleural effusion and its effects on proliferation,migration and invasion of human lung adenocarcinoma cells

AIM：
To investigate the levels of aldolase A (ALDOA), carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH) in malignant pleural effusion (MPE) from patients with lung cancer and tuberculous pleural effusion (TBPE) from patients with tuberculous pleurisy, and to explore the effects of ALDOA on the proliferation, migration and invasion of human lung adenocarcinoma A549 cells. METHODS：Pleural effusion samples including 65 cases of MPE and 35 cases of TBPE were collected, and the levels of ALDOA, CEA and LDH were detected by ELISA and chemiluminescence assay. After A549 cells were treated with different concentrations of ALDOA, the proliferation, migration and invasion of the cells were investigated by MTT assay, scratch test, Matrigel assay and Transwell invasion assay. RESULTS：The levels of ALDOA, CEA and LDH in MPE were (46.8±21.4) μg/L, (82.2±56.6) μg/L and (755.8±382.5) U/L, respectively, which were significantly higher than those in TBPE ［(23.9±17.2) μg/L, (12.6±9.7) μg/L and (388.4±163.9) U/L, respectively; P<0.01］. The concentration of ALDOA in MPE from adenocarcinoma patients ［(71.7±32.1) μg/L］ was significantly higher than that in MPE from squamous-cell carcinoma patients ［(21.3±14.6) μg/L, P<0.05］. The concentrations of ALDOA in MPE and TBPE were positively correlated with the concentrations of CEA and LDH (P<0.01 or P<0.05). ALDOA enhanced the proliferation, migration and invasion of A549 cells in a concentration-dependent manner. CONCLUSION：The expression level of ALDOA in MPE is significantly higher than that in TBPE, especially in MPE from lung adenocarcinoma patients. There are highly positive correlations between ALDOA and CEA, ALDOA and LDH in pleural effusion. ALDOA concentration-dependently promotes the proliferation, migration and invasion of A549 cells.     

Improving effects of ginsenoside Rb1 on glucose metabolism in cardiomyocytes under hypoxia by hypoxia-inducible factor 1α

AIM: To elucidate the effect of ginsenoside Rb1 (Gs-Rb1) on the glucose metabolism to improve the viability of the cardiomyocytes under hypoxia, and whether hypoxia-inducible factor 1α (HIF-1α) and/or AMPKα are involved in the process.METHODS: The neonatal rat cardiomyocytes were cultured, and randomly divided into control group, hypoxia (1% O₂, 94% N₂ and 5% CO₂) group, Gs-Rb1 (200 μmol/L) group, Ara-A (500 μmol/L) group, Gs-Rb1+Ara-A group, YC-1 (5 μmol/L) group, Gs-Rb1+YC-1 group, Ara-A+YC-1 group and Gs-Rb1+YC-1+Ara-A group. After the intervention for 8 h, the cell viability was analyzed by MTT assay. The protein levels of AMPK, HIF-1α and glucose transporter-4 (GLUT-4) were determined by Western blot. The activities of heterophosphatase (HK), phosphofructokinase (PFK) and lactic dehydrogenase (LDH) were measured by ELISA.RESULTS: Gs-Rb1 significantly improved the viability of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 and Ara-A. In addition, YC-1 and Ara-A had a synergistic effect. Gs-Rb1 increased the protein levels of AMPK and HIF-1α in the hypoxic cardiomyocytes, which was significantly inhibited by Ara-A and YC-1. Gs-Rb1 significantly increased the expression of GLUT-4 on the cytomembrane of hypoxic cardiomyocytes, which was significantly inhibited by YC-1 or Ara-A, especially Ara-A+YC-1. Gs-Rb1 significantly increased the activities of HK, PFK and LDH, all those were significantly inhibited by YC-1 or Ara-A. Besides, YC-1 and Ara-A had a synergistic effect.CONCLUSION: Gs-Rb1 improves the viability of hypoxic cardiomyocytes, which may be related to the regulation of glucose uptake and enhancement of glycolysis by synergy of both HIF-1α and AMPK.     

Effect of AG490 on expression of VEGF and HIF-1α in HEL cells

AIM: To investigate the effect of AG490 on the expression of VEGF and HIF-1α, and the capacity of invasion in human erythroleukemia (HEL) cells. METHODS: The HEL cells were treated with AG490 at different concentrations. The cell viability was detected by CCK-8 assay. The apoptosis was detected by Hoechst staining. The apoptosis and the cell cycle were analyzed by flow cytometry. The capacity of migration was evaluated by Transwell assay. The mRNA expression level of JAK2 was measured by RT-PCR. The protein levels of p-JAK2, VEGF and HIF-1α were determined by Western blot. RESULTS: The HEL cell viabilities were 88%, 75%, 48%, 10% and 0.12% after treated with AG490 at 20, 40, 60, 80 and 100 μmol/L for 48 h, respectively. The results of Hoechst staining showed that brilliant blue cells in 80 μmol/L AG490 group was significantly increased compared with control group for 48 h. The apoptosis rate of 80 μmol/L AG490 group was significantly increased compared with control group at 48 h after AG490 treatment. The number of membrane-permeating HEL cells in 20 μmol/L AG490 group at 24 h after AG490 treatment was significantly lower than that in control group (P<0.05). The mRNA level of JAK2 decreased in a concentration-dependent manner after the HEL cells were treated with different concentrations of AG490 for 48 h. The protein levels of p-JAK2, VEGF and HIF-1α were lower in AG490 treatment groups than those in control group (P<0.05). CONCLUSION: AG490 inhibits the expression of VEGF and HIF-1α in HEL cells by inhibiting JAK2 pathway.     

H IF-1αm ediates effect of interm ittent hypoxia on biological behavior of A 549 cells in vitro

ATM: To investigate whether hypoxia-inducible factor 1α (HIF-1α) mediates the effect of intermittent hypoxia on A549 cell viability, apoptosis and invasive ability METHODS: A549 cells were transfected with HIF-1α-siRNA and cultured under intermittent hypoxia. The expression of HIF-1α and its downstream genes, such as Bcl-2, Bax, P53, P21 and VEGF at mRNA and protein levels was determined by real-time PCR and Western blot. The viability of the A549 cells was measured by MTT assay. The apoptosis and cell cycle distribution of the A549 cells were examined by flow cytometry. The invasive ability of the A549 cells was detected by transwell test. RESULTS: The expression levels of HIF-1α, Bcl-2 and VEGF in non-HIF-1α-siRNA transfected A549 cells cultured in intermittent hypoxia environment[blank controlgroup(IH C),empty vector control group (IH E) and negative control group (IH N)] were higher than those in the A549 cells in normoxia group (RA), but the expression levels of Bax and P21 were lower than those in RA group (P<0.05). The siRNA-mediated HIF-1α gene silencing[intermittent hypoxia silenced group (IHS)] resulted in obvious down-regulation of HIF-1α, Bcl-2 and VEGF, and significant increase in the protein expression of P21 and Bax(P<0.05). The expression level of P53 in intermittent hypoxia groups was significantly higher than that in RA group, and no significant difference of P53 expression in different intermittent hypoxia groups was observed. Compared with normoxia, intermittent hypoxia resulted in significantly enhanced cell viability, decreased apoptosis, and enhanced invasive ability of non-HIF-1α-siRNA transfected A549 cells (P<0.05). The siRNA-mediated HIF-1α gene silencing resulted in significant cell viability inhibition, elevated apoptotic rate and decreased invasive ability under hypoxic condition (P<0.05).CONCLUSION: Intermittent hypoxia promotes the viability and invasion of A549 cells by HIF-1α-mediated downstream gene expression. HIF-1α gene silencing inhibits A549 cell growth and invasion under intermittent hypoxia by inhibition of HIF-1α signal pathways in vitro.     

Effect of hirsutine on hypoxia-induced migration and invasion abilities in human breast cancer MCF-7 cells

AIM: To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS: CCK-8 assay was employed to detect the cytotoxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay, and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS: Hirsutine remarkably reduced the cell viability from 32 μmol/L (P<0.05), and the IC₅₀ value was 62.82 μmol/L. In hypoxia state, MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α, Snail and MMP-9 (P<0.05), lower protein level of E-cadherin (P<0.05), and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05), apart from the mRNA level of HIF-1α. CONCLUSION: Hirsutine inhibits hypoxia-induced migration and invasion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α, Snail and MMP-9, and up-regulation of the protein level of E-cadherin.     

Effects of airway epithelial cells on phenotype and phagocytosis of macrophages and roles of HIF-1α

AIM: To investigate the effects of airway epithelial cells on the phenotype and phagocytosis of macrophages and the roles of hypoxia-inducible factor-1α (HIF-1α).METHODS: Human bronchial epithelial (HBE) cells treated with CoCl₂ (0, 100, 200, 400 and 800 μmol/L) or transfected with HIF-1α siRNA were co-cultured with the macrophages differentiated from human monocyte line THP-1 induced by phorbol 12-myristate 13-acetate (PMA). The mRNA expression of HIF-1α in the HBE cells was detected by RT-qPCR. The expression of macrophage surface markers and the phagocytosis rate of E.coli by macrophages were analyzed by flow cytometry.RESULTS: CoCl₂ upregulated the mRNA expression of HIF-1α in the HBE cells in a concentration-dependent manner and peaked at 8 h. HBE cells treated with CoCl₂ increased the fluorescence intensity ratio of CCL3, CD163, CD206 and CCL18 in co-cultured macrophages, and the strongest effect was seen in the macrophages co-cultured with HBE cells treated with CoCl₂ at 800 μmol/L. The fluorescence intensity ratio of CCL3 in co-cultured macrophages increased most obviously at 8 h and 12 h, while the fluorescence intensity ratio of CD163, CD206 and CCL18 increased more prominently in the macrophages co-cultured for 24 h. The stimulating effects of the HBE cells transfected with HIF-1α-Homo-488 siRNA on CCL3, CD163, CD206 and CCL18 in the macrophages were significantly attenuated. The phagocytosis rate of E.coli by macrophages co-cultured with HBE cells treated with different concentrations of CoCl₂ for 24 h initially increased (up to 60 min), and then it gradually decreased. Compared with normal HBE co-culture group, the phagocytosis rate in 400 and 800 μmol/L stimulation groups decreased at each time point, and that in 800 μmol/L stimulation group was the most.CONCLUSION: In hypoxia environment, airway epithe-lial cells initially transform macrophages predominantly to an M1-phenotype. However, the long-term hypoxia-stimulated airway epithelial cells inhibit the phagocytosis of macrophages and convert them to M2 superiority. HIF-1α may be an important mediator in these processes.     

Effect of digoxin on hypoxia-induced epithelial-mesenchymal transition and invasion in human breast carcinoma MCF-7 cells

AIM: To investigate the effect of digoxin on hypoxia-induced epithelial-mesenchymal transition (EMT), migration and invasion in human breast carcinoma MCF-7 cells. METHODS: MCF-7 cells were treated in vitro with a chemical hypoxia inducer cobalt chloride (CoCl₂) to imitate hypoxia. Cell migration was observed by wound healing assay, and cell invasion was measured by Transwell invasion assay. The protein levels of hypoxia-inducible factor-1α (HIF-1α), Snail, E-cadherin and vimentin in MCF-7 cells were detected by Western blot. RESULTS: Digoxin inhibited CoCl₂-induced EMT and reversed the mesenchymal phenotype. CoCl₂ enhanced the abilities of migration and invasion (P<0.01), significantly decreased the expression of E-cadherin and increased the expression of HIF-1α, Snail and vimentin (P<0.01), but these effects were blocked by digoxin. CONCLUSION: Digoxin inhibits CoCl₂-induced EMT and invasion most likely via HIF1-α-Snail signaling pathway.     

Effect of luteolin on TGF-β1-induced epithelial-mesenchymal transition in lung cancer A549 cells

AIM:To investigate the effects of luteolin on the invasion and epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1) in lung cancer A549 cells. METHODS:The effect of luteolin at 5, 10, 20, 40, 80 and 160 μmol/L on the viability of A549 cells was measured by MTT assay. The invasion ability was analyzed by Transwell method. The morphological changes of the A549 cells were observed under microscope.The protein expression of E-cadherin and vimentin in the A549 cells were determined by Western blot. RESULTS:The viability of the A549 cells was significantly inhibited by luteolin in a dose-time dependent manner (P<0.05). The IC₅₀ of luteolin for the A549 cells (24 h) was 68.79 μmol/L, while that (48 h) was 47.86 μmol/L. TGF-β1 induced morphological alteration of the A549 cells from epithelial to mesenchymal forms. Luteolin significantly inhibited TGF-β1-induced invasion of the A549 cells (P<0.01). The protein expression of E-cadherin was significantly down-regulated and the protein expression of vimentin was significantly up-regulated in the presence of TGF-β1 at 5 μg/L (P<0.01). However, luteolin reversed TGF-β1-induced EMT, up-regulation of E-cadherin and down-regulation of vimentin (P<0.01). CONCLUSION:Lu-teolin reverses TGF-β1-induced EMT in the lung cancer A549 cells.     

Influence of hypoxia-inducible factor-1 alpha on lung cancer cell A549 growth in vitro and in vivo

AIM: To study the role of hypoxia-inducible factor-1alpha(HIF-1α) on lung cancer cells A549 growth in vitro and in vivo. METHODS: To observe the growth rate of A549 cells after HIF-1α transfected, A549 cells (1×10⁶/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group (group A, n=10), the HIF-1α transfected group(group B, n=10). The weights of subcutaneous tumor were detected. The resected specimens were made into paraffin-embedded sections. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemistry(ISH). The expressions of HIF-1α、 apoptosis-related protein survivin and bcl-2 were analyzed by Western blot. RESULTS: The growth rates of the HIF-1α transfected lung cancer cells A549 were significantly increased, and more importantly, the HIF-1α transfected lung cancer cells A549 was able to enhance lung cancer growth in nude mice(P＜0.05). The PCNA were increased significantly in group B, compared with group A. The expressions of HIF-1α, survivin and bcl-2 in group B were increased significantly than that of group A. CONCLUSIONS: HIF-1α increases lung cancer cells A549 growth in vitro and in vivo and its mechanism may be due to promotion of proliferation and inhibition of apoptosis.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

β-estradiol promotes invasion and migration of lung cancer A549 cells via ERβ-mediated ERK1/2 membrane-initiated steroid signaling

AIM: To investigate the regulatory mechanism of β-estradiol in the invasion and migration of lung cancer A549 cells. METHODS: Breast cancer MCF-7 cells and lung cancer A549 cells were cultured in vitro. The MCF-7 cells were used as the estrogen receptor (ER) positive expression cell model. Real-time PCR and immunofluorescence were employed to measure the expression level and the localization of ER in A549 cells. The phosphorylation of ERK1/2 upon β-estradiol stimulation was quantified by Western blot. The invasion and migration abilities of A549 cells upon β-estradiol stimulation with or without ERK1/2 inhibitor PD98059 were measured by Transwell and Cell-IQ assays. RESULTS: ERβ was the dominant ER subtype in the A549 cells and primarily comprised of ERβ2 and ERβ5. Immunofluorescence revealed that ERβ expression was mainly localized in the cytoplasm. β-estradiol induced phosphorylation of ERK1/2 and promoted the invasion and migration of the cells. Inhibition of ERK1/2 signaling reversed β-estradiol-promoted invasion and migration of A549 cells. CONCLUSION: ERβ-mediated membrane-initiated steroid signaling is involved in the process of β-estradiol-promoted invasion and migration of A549 cells, through which ERK1/2 signaling plays a pivotal role.     

Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reperfusion injury by hyperbaric oxygen pretreatment

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway in hyperbaric oxygen (HO) pretreatment in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Healthy male SD rats (n=32) were randomly divided into control group IR group, HO-IR group and HO-IR-HIF-1α inhibitor group (HO-IR-I group). The IR model was established by middle cerebral artery occlusion. The corresponding blood vessels of the rats in control group were only exposed. The rats in HO-IR group and HO-IR-I group were treated with HO for 4 weeks before the animal modeling. The rats in HO-IR-I group received 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazol (YC-1) by intraperitoneal injection at 4 mg/kg before HO preconditioning every day. At 1 d and 7 d after modeling, the neurological assessment was evaluated.At the end of the 7 th day, after observation, the rats were sacrificed by anesthesia to measure the infarct volume of the brain tissue. The protein levels of HIF-1α, VEGF and apoptosis-related proteins Bax and Bcl-2 were determined by Western blot. The number of apoptotic cells was detected by TUNEL. RESULTS: Compared with control group, the neurological function score was decreased, while the cerebral infarction volume ratio, the protein levels of HIF-1α, VEGF, Bcl-2 and Bax, and the apoptotic cells were increased in IR group, HO-IR group and HO-IR-I group (P<0.05). Compared with IR group, the neurological function score, and the protein levels of HIF-1α,VEGF and Bcl-2 were increased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were decreased in HO-IR group and HO-IR-I group (P<0.05). Compared with HO-IR group, the neurological function score, and the protein levels of HIF-1α, VEGF and Bcl-2 were decreased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were increased in HO-IR-I group (P<0.05). CONCLUSION: The mechanism of HO preconditioning attenuating cerebral IR injury may be related to the regulation of apoptosis by inducing HIF-1α/VEGF signaling pathway activation.     

Hypoxia-inducible factor-1α attenuates myocardial inflammatory injury induced by ischemia and reperfusion in rats

AIM:To investigate the role of hypoxia-inducible factor-1α (HIF-1α) stable expression in myocardial inflammatory injury induced by ischemia and reperfusion (I/R) in rats. METHODS:Male Wistar rats were randomly divided into 4 groups:sham operation (sham) group, I/R group, HIF-1α stabilizer dimethyloxalyl glycine (DMOG)+I/R group and HIF-1α inhibitor YC-1+I/R group. The protein expression of myocardial Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was determined by Western blot. The mRNA levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6, TLR4 and NF-κB were detected by real-time PCR. The myeloperoxidase (MPO) activity in the myocardial tissues was measured. HE staining was used to observe the infiltration of inflammatory cells. RESULTS:HIF-1α decreased the infiltration of inflammatory cells, the MPO activity, and the mRNA levels of inflammatory factors IL-1β, IL-6 and TNF-α in the myocardial tissues. HIF-1α also reduced the expression of TLR4 and NF-κB at mRNA and protein levels (P<0.05). CONCLUSION:The stable expression of HIF-1α has an anti-inflammatory effect on the myocardial tissues after I/R injury in rats. The mechanism may be related to the inhibition of TLR4/NF-κB signaling pathway.     

Expression of hypoxia-inducible factor 1α in pulmonary tissues from patients with chronic obstructive pulmonary disease

AIM：To study the expression of hypoxia-inducible factor 1α (HIF-1α) in pulmonary tissues from patients with chronic obstructive pulmonary disease (COPD) μg/L and its effects on the pathogenesis of COPD. METHODS：Pulmonary tissues were obtained from 32 subjects (16 patients with COPD and 16 without COPD as controls) who were undergoing single or bilateral lobectomy or wedge resection for lung cancer. The specimens were obtained as far away from the cancer foci (≥8 cm) as possible. The expression of HIF-1α protein in pulmonary tissues was measured by Western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS：The expression of HIF-1α protein in pulmonary tissues from controls and COPD patients was as follows: (0.96±0.43) μg/L and (0.16±0.07) μg/L (ELISA， P<0.05); 0.71±0.22 and 0.53±0.15 (Western blotting, P<0.05). Furthermore, the level of HIF-1α protein in pulmonary tissues from mild and moderate COPD patients was obviously higher than that from severe COPD patients (P<0.05). CONCLUSION：HIF-1α may play an important role in the progress of COPD.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

An improved method to develop cell culture model of intermittent hypoxia

AIM: To establish and validate a novel model of cultured cells for imitating intermittent hypoxia. METHODS: In a chamber with experiment cabin and simulated air control cabin, the frequency and duration of the intermittent hypoxia model according to the time of hypoxia and reoxygenation were evaluated. The A549 cells were randomly divided into 7 groups, named as control (Con) group, 6 h intermittent hypoxia (6IH) group, 9 h intermittent hypoxia (9IH) group, 6 h simulated air control (6AC) group, 9 h simulated air control (9AC) group, 4 h sustained hypoxia (4SH) group, 6 h sustained hypoxia (6SH) group, respectively. When the model was established, the cellular morphology was observed under inverted microscope. The mRNA expression of hypoxia-inducible factor (HIF)-1α was detected by real-time PCR. The protein expression of HIF-1α was analyzed by immunohistochemistry. RESULTS: The intermittent hypoxia cycle (5% O₂ 60 min-20% O₂ 30 min for 6 cycles) was established. The damaged A549 cells were observed in 6IH group, 9IH group and 6SH group. Compared with 6IH group, the expression of HIF-1α at mRNA and protein levels was significantly increased in 9IH group (P<0.05). The expression of HIF-1α at mRNA and protein levels in 6IH group and 9IH group was higher than that in 4SH group and 6SH group, respectively (P<0.05). No significant difference among the control group, 6AC group and 9AC group was found. CONCLUSION: The model (5% O₂ 60 min-20% O₂ 30 min for 6 cycles) can simulate the pathological process of obstructive sleep apnea hypopnea syndrome. This model is suitable for studying intermittent hypoxia in adherent cells.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.     

Ursolic acid inhibits migration and invasion of human lung cancer A549 cells by targeting miRNA-133a

AIM: To investigate the effects of ursolic acid (UA) on the migration and invasion of human lung cancer cell line A549, and to explore its mechanism. METHODS: The cell viability was detected by MTT assay. The expression of miRNA-133a was detected in the A549 cells treated with UA by real-time PCR. The miRNA-133a mimics and inhibitor were transfected into the A549 cells, and the transfection efficiency was analyzed by real-time PCR. The cell migratory and invasive abilities were determined by wound healing and Transwell methods, respectively. RESULTS: The viability of the human lung cancer A549 cells was significantly inhibited by UA in a dose-dependent manner (P<0.05). IC₅₀ of UA (24 h) for lung cancer A549 cells was 31.04 μmol/L. UA treatment significantly inhibited the migratory and invasive abilities of A549 cells in a concentration-dependent manner, accompanied by significantly elevation of miRNA-133a expression. The mimics and inhibitor of miRNA-133a significantly upregulated and downregulated the expression of miRNA-133a in the transfected A549 cells, respectively. In addition, the viability of the A549 cells was decreased extremely after tansfected with the miRNA-133a mimics (P<0.01), so did the results of the cell migration and invasion test. The A549 cells tansfected with the miRNA-133a inhibitor showed an opposite changes of the cell viability, migration and invasion. CONCLUSION: UA inhibited the viability, migration and invasion of lung cancer A549 cells by elevating the expression of miRNA-133a.