Effects of HIF-1α silencing on expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919

AIM: To study the effect of hypoxia-inducible factor 1α(HIF-1α) silencing on the expression of p27 and Ki67 in rat hepatoma cell line CBRH-7919 under hypoxia. METHODS: Hypoxic condition was induced by CoCl₂ and the expression of HIF-1α was silenced by small interference RNA. HIF-1α-specific RNAi lentiviral vector was constructed. Real-time RT-PCR and Western blotting were used to detect the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells under hypoxia. The expression of p27 and Ki67 was observed by Western blotting after HIF-1α silencing was performed. The cell cycle of hepatoma cells was detected by flow cytometry. RESULTS: Under hypoxic condition, the expression of HIF-1α at mRNA and protein levels in CBRH-7919 cells increased significantly (P<0.05). HIF-1α silencing significantly reduced the expression of Ki67 but increased the expression of p27 (P<0.05) in CBRH-7919 cells. In transfected cells, the number of cells in G₀/G₁ phase was much higher and that in S phase was much lower than those in the control cells. CONCLUSION: Hypoxia induces the expression of HIF-1α. HIF-1α silencing can regulate the proliferation of hepatoma cells through reducing the expression of Ki67 and increasing the expression of p27.     

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.     

Effects of tanshinone IIA on proliferation,apoptosis and expression of HIF-1α, VEGF and wild-type P53 in human hepatoma HepG2 cells under hypoxia

AIM:To investigate the effects of tanshinone IIA (Tan IIA) on proliferation, apoptosis and its molecular mechanism in human hepatoma HepG2 cells under hypoxic condition. METHODS:Hypoxia model was established by treatment with cobalt chloride (CoCl2). The cells were divided into normoxia control group, hypoxia control group and hypoxia combined at different concentrations of Tan IIA groups. After HepG2 cells were incubated with different concentrations of Tan IIA (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) for 24 h, 48 h and 72 h under hypoxic condition, the cell proliferation was determined by MTT assay. After Tan IIA was added to the media at different concentrations for 24 h and 48 h, the apoptotic cells were observed by Hoechst 33258 staining. The protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and wild-type P53 were detected by Western blotting after cultured with different concentrations of Tan IIA for 48 h. RESULTS:Tan IIA inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner. Tan IIA induced the typical morphology of apoptotic cells and increased the apoptotic rate in a dose- and time-dependent manner after treatment with 1.0 mg/L~5.0 mg/L for 24 h and 48 h under hypoxic condition. The protein levels of HIF-1α and VEGF were weakly expressed in HepG2 cells under normoxia but up-regulated after incubated under hypoxia for 48 h. The protein expression of HIF-1α and VEGF were decreased with the increase in the concentration of Tan IIA under hypoxia. The protein expression of wild-type P53 was increased with the increase in the concentrations of Tan IIA under hypoxia. CONCLUSION: Tan IIA significantly inhibits the proliferation and induces the apoptosis of human hepatoma HepG2 cells under hypoxia, which may be related to the down-regulation of HIF-1α and VEGF and up-regulation of wild-type P53.     

Effects of G6PD silencing by siRNA on expression of HIF-1α in human colon cancer cells

AIM: To study the effects of glucose-6-phosphate dehydrogenase (G6PD) silencing by small interference RNA(siRNA) on the levels of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and hypoxia-inducible factor 1α(HIF-1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells. The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD. LoVo cells were cultured in vitro under hypoxic condition. NADPH levels were determined.The mRNA and protein levels of HIF-1α were analyzed by RT-PCR and Western blotting,respectively. RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells. Compared with untransfected cells,the mRNA expression of G6PD in transfected cells was decreased by 43% and G6PD activity was decreased by 63.5%. Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased (41% vs 100%, P<0.05).HIF-1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells. CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF-1α stability in cancer cells under hypoxic condition. By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.     

Silencing of TKTL1 gene by siRNA down-regulates HIF-1α expression and activity of glycolytic key enzymes in human cervical cancer cells

AIM：To explore the relationship between the expression of transketolase-like gene 1 (TKTL1) and glycolysis metabolism in human cervical cells. METHODS：The changes of hypoxia-inducible factor 1α (HIF-1α) expression and the activity of glycolytic key enzymes, hexokinase Ⅱ (HK-Ⅱ) and lactate dehydrogenase (LDH), under hypoxia in human cervical cell line HeLa were observed after TKTL1 was knockdown by siRNA. The specific siRNA expression vector targeting TKTL1 gene was constructed, and the recombinant plasmid was transfected into HeLa cells. The effects of TKTL1 silencing were evaluated by detecting transketolase (TKT) activity and TKTL1 mRNA expression using RT-PCR. The changes of HIF-1α expression and HK-Ⅱ expression, and HK-Ⅱ and LDH activity were also observed in transfected HeLa cells. RESULTS：The mRNA expression of TKTL1 and the activity of TKT decreased significantly (P<0.01) after TKTL1 silencing. Meanwhile, all HIF-1α expression, HK-Ⅱexpression, and HK-Ⅱ and LDH activity decreased significantly compared with the untransfected cells (P<0.01). CONCLUSION：Silencing of TKTL1 gene in human cervical cancer cells by siRNA down-regulates HIF-1α expression and the activity of glycolytic key enzymes, thus changing the malignant phenotype of carcinoma 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.     

Angiogenic effect of HIF-1α on extranodal nasal-type NK/T-cell lymphoma

AIM: To study the angiogenic effect of hypoxia inducible factor 1α(HIF-1α) and its significance on human extranodal nasal-type NK/T-cell lymphoma. METHODS: The protein levels of HIF-1α, vascular endothelial growth factor(VEGF) and VEGF receptor 2(VEGFR2) in human extranodal nasal-type NK/T-cell lymphoma were detected by immunohistochemistry. Microvessel density (MVD) of the tumor tissues was determined by labeling of microvessel endothelium with CD34 antibody. The correlation between the expression of HIF-1α, VEGF and VEGFR2 and MVD was analyzed with SPSS 13.0 statistical software. RESULTS: The positive expression of HIF-1α was observed in 39 cases (39/50, 78%) and the positive expression of VEGFR2 was 27 cases (27/50, 54%) of human extranodal nasal-type NK/T-cell lymphoma. A statistical difference of HIF-1α and VEGFR2 expression between tumor tissues and normal lymphocytes in lymph node was observed (P<0.05). In the tumor tissues, the co-expression of VEGF or VEGFR2 with HIF-1α was 72% and 64%, respectively, significantly higher than that without HIF-1α co-expression (P<0.05). The expression of HIF-1α, VEGFR and VEGFR2 was positively correlated with MVD of the tumor tissues (P<0.01). HIF-1α was expressed in all 15 cases of extranodal nasal-type NK/T-cell lymphoma with angiocentric infiltration.CONCLUSION: HIF-1α may promote angiogenesis of extranodal nasal-type NK/T-cell lymphoma through VEGF/VEGFR2 signaling pathway.     

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.     

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.     

Expression of hypoxia-inducible factor 1α in human gingival tissues with chronic periodontitis

AIM：To observe the expression of hypoxia-inducible factor 1α (HIF-1α) in human gingival tissues with chronic periodontitis. METHODS：A total of 55 volunteers, including 15 healthy controls, 20 cases of moderate chronic periodontitis and 20 cases of severe chronic periodontitis, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining, and the expression of HIF-1α in gingival tissues was detected by immunohistochemical staining. RESULTS： The proportion of HIF-1α positive cells in gingival tissues was significantly higher in chronic periodontitis groups than that in healthy control group (P<0.01), and that in severe chronic periodontitis group was significantly higher than that in moderate chronic periodontitis group (P<0.05). There was a significantly positive correlation between the severity of chronic periodontitis and the proportion of HIF-1α positive cells in gingival tissues. CONCLUSION：The expression of HIF-1α in human gingival tissues is increased with the severity of chronic periodontitis, suggesting that hypoxia may play an important role in chronic periodontitis.     

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.     

Effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells

AIM:To investigate the effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells. METHODS:Transient transfection of NANOG targeting siRNA into HepG2 cells was performed. The expression of NANOG and cyclin D1 at mRNA and protein levels was detected by real-time PCR and Western blotting. Cell proliferation was examined by CCK-8 assay and colony formation assay, and cell cycle was tested by flow cytometry. RESULTS:After transfection with NANOG-targeting siRNA, the inhibition of NANOG expression was observed. Compared with mock group, the mRNA and protein expression levels of NANOG and cyclin D1 were decreased (P<005). In addition, knockdown of NANOG expression inhibited the cell proliferation and increased the proportion of G 0/G 1-phase cells (P<0.05). CONCLUSION:Silencing of NANOG expression in HepG2 cells causes down-regulation of cyclin D1 expression and decreases the cell proliferation ability.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.     

Expression of volume-activated chloride channel in rat PASMCs treated with hypoxia and hypercapnia and its relationship with MAPK pathway

AIM：To investigate the expression of volume-activated chloride channel (CLC3) in rat pulmonary artery smooth muscle cells (PASMCs) treated with hypoxia and hypercapnia and its relationship with MAPK pathway. METHODS：The method of enzyme digestion was used to isolate the PASMCs in male SD rat for cell primary culture. The cells were identified by immunofluorescence cytochemical method with mouse anti-rat α-smooth muscle actin antibody. The rat model of hypoxia and hypercapnia was established. The protein expression of CLC3 was detected by Western blotting. The mRNA expression of CLC3 was determined by RT-PCR. RESULTS：Compared with control group, the mRNA and protein expression of CLC3 in PASMCs was significantly raised in hypoxia and hypercapnia group. Compared with hypoxic and hypercapnic group, the expression of CLC3 was significantly reduced in ERK inhibitor U0126+ hypoxia and hypercapnia group, and was up-regulated in p38 inhibitor SB203580+ hypoxia and hypercapnia group. p38 activator anisomycin significantly decreased the expression of CLC3 at mRNA and protein levels in hypoxia and hypercapnia group. CONCLUSION：The expression of CLC3 at mRNA and protein levels in PASMCs increases under hypoxia and hypercapnia conditions. The ERK1/2 pathway mediates CLC3 expression in PASMCs induced by hypoxia and hypercapnia. Activation of p38 MAPK pathway down-regulates the expression of CLC3 at mRNA and protein levels induced by hypoxia and hypercapnia.     

Relationship between peritubular capillary injury and renal interstitial fibrosis in mice with unilateral ureteral obstruction

AIM: To observe the injury of peritubular capillary (PTC), hypoxia and interstitial fibrosis after unilateral ureteral obstruction (UUO), and to explore the effects of PTC injury and hypoxia on interstitial fibrosis in mouse model of UUO. METHODS: Forty-eight male KM mice were randomly divided into control group and UUO group. On the 1st, 3rd, 7th and 14th days, 6 mice in each group were sacrificed. The changes of pathomorphism in the kidney were observed by HE and Masson staining. The expression levels of thrombospondin-1 (TSP-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), and PTC density were detected by immunohistochemistry. The protein expression of VEGF was also determined by Western blotting. RESULTS: No histological abnormalities of the kidneys were observed in sham-operated mice. The expression of TSP-1 was increased 1 day after UUO, and significantly increased on the 3rd, 7th and 14th days(P<0.05). The expression of VEGF was obviously decreased(P<0.05). PTC density was gradually decreased. The expression of HIF-1α was gradually increased, and renal interstitial area was gradually expanded. PTC density was negatively correlated with the expression of TSP-1 and HIF-1α (r=-0.874 and r=-0.930, respectively). VEGF expression was positively correlated with PTC density (r=0.745). PTC density was negatively correlated with the area of renal fibrosis (r=-0.787). HIF-1α expression was positively correlated with the area of renal fibrosis (r=0.835, P<0.05). CONCLUSION: In mouse UUO model, the expression of TSP-1 is increased. The expression of VEGF is reduced. The peritubular capillary injury and tissue hypoxia are aggravated, and renal interstitial fibrosis area is expanded. Ischemia and hypoxia may play an important role in the progression of UUO.     

Protective effect of delayed ischemic preconditioning on renal ischemia/reperfusion injury via induction of hypoxia inducible factor

AIM: To investigate the effects of delayed ischemic preconditioning on renal ischemia-reperfusion injury in mice and to study the role of hypoxia inducible factor 1α(HIF-1α). METHODS: Male C57/BL6N mice were randomly divided into 3 groups: sham operation group(sham), ischemia/reperfusion group(IR) and ischemic preconditioning group(IPC). Thirty-minute ischemia was induced by clamping renal bilateral pedicles followed by reperfusion in IR group. Fifteen-minute pre-ischemia was performed 4 days before IR in IPC group. Serum creatinine(Scr), blood urea nitrogen(BUN), kidney morphology and apoptosis were observed at different time points following reperfusion. The expression of HIF-1α in the renal tissues was evaluated by the methods of immunohistochemistry and Western blotting analysis. The mRNA expression of vascular endothelial growth factor(VEGF) and glucose transporter-1(Glut-1) was detected by real-time quantitative RT-PCR.RESULTS: Compared with IR group at 24 h following reperfusion, acute tubulointerstitial injury was significantly relieved in IPC group. The levels of Scr and BUN, and apoptosis of tubular epithelial cells were also decreased in IPC group. Nuclear expression of HIF-1α was higher in IPC group than that in IR group. The mRNA expression of VEGF and Glut-1, the target genes of HIF-1, was also increased significantly in IPC group. CONCLUSION: Delayed ischemic preconditioning attenuates both morphologic and functional injuries induced by renal ischemia/reperfusion. This protective effect may be related to the increased expression of hypoxia inducible factor.     

Effect of extracellular HSP70/HSP70-PCs on epithelial-mesenchymal transition of human hepatocellular carcinoma HepG2 cells

AIM：To investigate the effect of extracellular heat-shock protein 70 (HSP70)/HSP70-peptide complexes (HSP70-PCs) on epithelial-mesenchymal transition (EMT) of human hepatocellular carcinoma HepG2 cells and its probable mechanism. METHODS：HepG2 cells were divided into 3 groups: control group, HSP70/HSP70-PCs (2 mg/L) group and LY294002+HSP70/HSP70-PCs group. The mRNA and protein expression of epithelial cell surface marker E-cadherin, mesenchymal cell surface marker α-smooth muscle actin (α-SMA), phosphatidylinositol 3-kinase (PI3K) and hypoxia-inducible factor 1α (HIF-1α) was examined by real-time RT-PCR and Western blotting. RESULTS：Extracellular HSP70/HSP70-PCs promoted the initiation of EMT of HepG2 cells. The expression of HIF-1α and PI3K significantly increased in the process of EMT of HepG2 cells. After PI3K was blocked by LY294002, EMT did not occur and HIF-1α was not up-regulated in HepG2 cells. CONCLUSION：Extracellular HSP70/HSP70-PCs may promote EMT of hepatocellular carcinoma cells via PI3K/HIF-1α signaling pathway.     

Effects of fucoidan on angiogenesis of human multiple myeloma RPMI 8226 cells

AIM: To investigate the effects of fucoidan on the angiogenesis of multiple myeloma cells in vitro, and its related mechanisms. METHODS: The human multiple myeloma RPMI 8226 cells and human endothelial cells were cultured in vitro. The growth inhibition rate of RPMI 8226 cells was examined by MTT assay. The cell cycle and apoptosis rate were measured by flow cytometry. RPMI 8226 cells were treated with fucoidan for 72 h, and the cell culture supernatant was collected. The VEGF concentration was examined by ELISA, and the tube formation assay was applied to assess the angiogenic activity. After treatment with fucoidan for 72 h at different concentrations, the protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 were detected by Western blot. RESULTS: Fucoidan inhibited the growth of RPMI 8226 cells in a dose- and time-dependent manner. After treatment with fucoidan for 72 h, the cell cycle was arrested at G₁ phase, and the apoptotic rate of RPMI 8226 cells was increased with the increasing concentration of fucoidan, which was much higher than that in control group (P<0.05). The VEGF concentration was significantly decreased with the increa-sing concentration of fucoidan. The numbers and areas of the capillary-like structures decreased while the concentration of fucoidan increased, and those at 100 mg/L were less than those in the control (P<0.05). The protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 in fucoidan group were significantly lower than those in control group (P<0.05). CONCLUSION: Fucoidan inhibits the secretion of VEGF in multiple myeloma cells, and reduces angiogenesis induced by multiple myeloma cells. It inhibits the protein expression of HIF-1α and VEGF, which may be related to inhibiting the phosphorylation of AKT and ERK1/2.     

Effects of RNA interference targeting CDC25agene on proliferation of human liver cancer HepG2 cells

AIM: To investigate the effect of silencing cell division cycle 25a (CDC25a) gene on the proliferation of human hepatoma HepG2 cells. METHODS: CDC25agene in human hepatoma HepG2 cells was silenced by RNA interference. Real-time PCR was applied to detect the expression of CDC25a, cyclin E and CDK2 at mRNA levels in the HepG2 cells. Western blotting was applied to detect the expression of CDC25a at protein level. In addition, MTT assay, Giemsa staining and flow cytometry were used to measure the proliferation of human hepatoma HepG2 cells. RESULTS: The expression of CDC25a at mRNA and protein levels in RNA silence group was lower than those in negative control group and normal control group (P＜0.05). The mRNA expression of cyclin E and CDK2 in silence group was lower than that in negative control group and normal control group (P＜0.05). The cell proliferation in silence group was lower than that in negative control group and normal control group (P＜0.05). The results of flow cytometry revealed that the cells in silence group were blocked in G1 phase. CONCLUSION: Infection of LV-CDC25a-RNAi recombinant to the HepG2 cells effectively inhibits the CDC25agene expression and the proliferation of human hepatoma cells, and arrests the cells in G1 phase, suggesting that CDC25agene may be a key target for the treatment of liver cancer.