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.     

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 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.     

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.     

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.     

Effects of TKTL1 silencing by siRNA on growth microenvironment of human hepatocellular carcinoma HepG2 cells

AIM:To explore the relationship between the expression of transketolase-like 1 protein(TKTL1) and the metastasis of hepatocellular carcinoma by investigating the change of some indicators in growth microenvironment including lactate production, reduced glutathione(GSH) level and ratio of NADPH/NADP⁺ in hepatocellular carcinoma cell line HepG2 after silencing of TKTL1 expression by siRNA. METHODS:Specific siRNA expression vector targeting TKTL1 gene was constructed and transfected into HepG2 cell line. The effect of TKTL1 silencing was evaluated by detecting the mRNA level and the activity of transketolase(TKTL1). The changes of lactate production, GSH level, the ratio of NADPH/NADP⁺ and glucose-6-phosphate dehydrogenase(G-6-PD) activity were observed in transfected HepG2 cells compared with the untransfected control cells. RESULTS:Compared with the untransfected control cells, the mRNA expression of TKTL1 and the TKT activity decreased significantly(P<0.01). Meanwhile, the lactate production, GSH level and ratio of NADPH/NADP⁺ also decreased significantly(P<0.01). However, no change of the G-6-PD activity was observed. CONCLUSION:Carcinoma cells switch the glucose metabolism by overexpression of TKTL1 to modify the lactate production and the levels of reactive oxygen species, thus changing the growth microenvironment in favor of tumor metastasis.     

Effects of Nrf2 overexpression on proliferation,activation and collagen type I synthesis in cultured hepatic stellate cells stimulated by ethanol

AIM：To investigate the effects of nuclear factor E2-related factor 2 (Nrf2) overexpression on the proliferation, cell cycle distribution, collagen type I (Col I) synthesis and alpha-smooth muscle actin (α-SMA) expression in cultured hepatic stellate cell line HSC-T6 stimulated by ethanol. METHODS：Cultured HSC-T6 cells were transfected with pEGFP-Nrf2 or pEGFP-N1 (empty vector) plasmid by liposome transient transfection. The cells were divided into control group, ethanol group, ethanol+pEGFP-Nrf2 group and ethanol+pEGFP-N1 group. The mRNA expression of Nrf2, α-SMA and Col I was determined by RT-PCR, and their protein expression was detected by Western blotting. Cell proliferation was assessed by MTT assay, and cell cycle was detected by flow cytometry. RESULTS：The pEGFP-Nrf2 plasmid was successfully transfected into HSC-T6 cells, and the mRNA and protein expression of Nrf2 was higher than other three groups 48 h after transfection (P<0.05). Compared with control group, the cell proliferation and the mRNA and protein expression of α-SMA and Col I in ethanol group and ethanol+pEGFP-N1 group were significantly increased (P<0.05), and the numbers of HSC-T6 cells were decreased in G1 phase and increased in S phase (P<0.05), without significant differences between the two groups (P>0.05). Meanwhile, the cells in ethanol+pEGFP-Nrf2 group showed significantly decreased proliferation level, down-regulated mRNA and protein expression of α-SMA and Col I, higher numbers in G1 phase and lower numbers in S phase compared with ethanol group and ethanol+pEGFP-N1 group (P<0.05). CONCLUSION：Nrf2 overexpression could significantly down-regulate the expression of α-SMA and Col I and cause G1/S phase arrest in HSC-T6 cells cultured with ethanol, thus inhibiting the proliferation and activation of the cells.     

Inhibitory effects of septin 2 gene silencing induced by siRNA on migration of human colorectal cancer cell line LoVo

AIM:To investigate the expression of septin 2 in human colorectal cancer cell lines and the effect of septin 2 on the capacity of migration of human colorectal cancer cell line LoVo. METHODS:Real-time fluorescence quantitative RT-PCR and Western blotting analysis were used to determine the mRNA and protein levels of septin 2 in different metastatic potential cell lines, respectively. The expression of septin 2 in LoVo cells was silenced by siRNA. The efficacy of siRNA was confirmed by real-time fluorescence quantitative RT-PCR. Septin 2 and its co-localization with F-actin were measured by immunofluorescence method. The migration of transfected cells was analyzed by scratch test. RESULTS:The expression of septin 2 in LoVo cells was significantly higher than that in low metastatic potential cell lines such as SW480, HCT-116 and HT-29 at both mRNA and protein levels. The mRNA expression of septin 2was successfully silenced in LoVo cells by siRNA. Cell wound closure rate was also decreased in septin 2 siRNA group as compared with control group(both P<0.05). The co-expression of septin 2 and F-actin formed the typical filamentous-granular organization, and down-regulation of septin 2 resulted in cell skeleton disturbance with less or shorter pseudopodium and decreased stress fibers. CONCLUSION:Septin 2 is highly expressed in LoVo cells. Partial deletion of septin 2 represses the capability of tumor cell migration.     

Effect of specific hTERT RNA interference on biological characteristics of colon carcinoma in vivo and in vitro

AIM: To investigate the effect of specific hTERT RNA interference on biological characteristics of colon carcinoma in vivo and in vitro. METHODS: A small interference RNA (siRNA) targeting to hTERT mRNA (pU6-hTERT-siRNA) was constructed. The siRNA was transfected into LoVo colon cancer cells in vivo and in vitro with Lipofectamine^TM2000. The groups of non-specific siRNA (pU6-hTERT) and non-treatment were designed as negative control and blank control,respectively. The cell growth in vitro was detected by MTT method. The effect of pU6-hTERT-siRNA on xenografts in nude mice was observed by determining the tumor size. The mRNA expression of hTERT in vitro and in vivo was detected by FQ-PCR quantitatively. The protein level of hTERT was determined by Western blotting. RESULTS: The inhibition rate of cell growth in vitro 72 h after transfection with recombinant plasmids containing hTERT-target sequences was 42.1%, significantly higher than that in control group (3.2%, P<0.01). The size of xenografts in pU6-hTERT-siRNA group was (85.9±18.7)mm³, significantly smaller than that in control group and blank group , P<0.01. The mRNA expression and the protein level of hTERT were both specifically inhibited by pU6-hTERT-siRNAs in LoVo colon cancer cells and xenografts (P<0.01). No difference between control group and blank group was observed (P>0.05).CONCLUSION: hTERT expression in LoVo colon cancer cells is inhibited significantly in vivo and in vitro by using plasmid-based siRNA. Down-regulation of hTERT expression distinctly inhibits the growth of LoVo colon cancer cells in vitro or subcutaneously transplanted in athymic mice.     

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.     

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.     

Construction and identification of eukaryotic expression vector of bcl-2 siRNA

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.     

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.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

Effect of DEC1 gene silencing on viability,invasion and migration of human breast cancer MDA-MB-231 cells under hypoxia

AIM: To investigate the effect of differentiated embryonic chondrocyte gene 1 (DEC1) expression silencing on viability, invasion and migration of human breast cancer MDA-MB-231 cells and its possible mechanism under hypoxia. METHODS: The expression of DEC1 was detected by RT-qPCR and Western blot in breast cancer MDA-MB-231 cells under normoxia and hypoxia. MDA-MB-231 cells were transfected with the siRNA targeting DEC1 and the protein levels of DEC1, Smad3 and phosphorylated Smad3 (p-Smad3) were examined under hypoxia. Subsequently, the changes in the viability, invasion and migration abilities of MDA-MB-231 cells were analyzed by CCK-8 assay, Transwell experiment and Scratch test, respectively. RESULTS: The expression of DEC1 in MDA-MB-231 cells under hypoxia was higher than that in the MDA-MB-231 cells under normoxia condition at both mRNA and protein levels (P<0.05). The viability, invasion and migration abilities of MDA-MB-231 cells in siRNA-DEC1 group were decreased significantly as compared with control group (P<0.01). Besides, the protein level of p-Smad3 in the MDA-MB-231 cells in siRNA-DEC1 group was lower than that in negative control group under hypoxia condition (P<0.05). CONCLUSION: Down-regulated DEC1 expression significantly decreases the viability, invasion and migration abilities of breast cancer MDA-MB-231 cells by blocking the TGF-β/Smad3 signaling pathway under hypoxia condition.     

Effect of Wip1 gene silencing on chemotherapy sensitivity of human colon cancer cells

AIM: To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells. METHODS: Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene. The mRNA expression of Wip1 was measured by real-time PCR. The protein level of Wip1 was detected by Western blotting. The viability of RKO colon cancer cells was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels. The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression. The viability of RKO colon cancer cells was decreased from (89.4±6.6)% to (74.7±3.9)% after treated with 5-fluorouracil (P<0.05) and decreased from (77.9±2.4)% to (66.7±2.9)% after treated with oxaliplatin (P<0.05). The cell apoptotic rate was increased from (7.7±0.5)% to (12.3±3.2)% and from (14.7±2.1)% to (34.0±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05). CONCLUSION: Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.     

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.     

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.     

Inhibition of plasminogen activator inhibitor-1 by small interfering RNA attenuates pulmonary fibrosis in rats

AIM: To examine the effects of silencing of plasminogen activator inhibitor-1 (PAI-1) expression by small interfering RNA (siRNA) on bleomycin (BLM)-induced rat pulmonary fibrosis. METHODS: Total 72 Wistar rats were divided into 4 groups: control, BLM, BLM+non-specific siRNA (BLM+N), and BLM+ PAI-1 siRNA (BLM+P). Pulmonary fibrosis was induced by intratracheal injection of BLM (5 mg/kg), whereas equal volume of normal saline was used in control group. After the administration of BLM or normal saline, the rats were treated with tracheal injection of PAI-1-siRNA (7.5 nmol/0.2 mL per rat) in BLM+P group, non-specific siRNA (7.5 nmol/0.2 mL per rat) in BLM+N group, and 0.2 mL normal saline in BLM group and control group, twice a week, 8 times in 28 d. On day 7, 14, and 28, the rats (n=6 at each time point) were sacrificed. The bronchoalveolar lavage fluid (BALF) from the left lung was harvested to examine the activity of PAI-1. The mRNA expression of collagen type Ⅲ, α-smooth muscle actin (α-SMA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the middle lobe of the right lung was detected by RT-PCR. RESULTS: PAI-1 activity and the expression of collagen type Ⅲ, α-SMA and TIMP-1 were increased in BLM group on day 7, 14 and 28. Intratracheal injection of PAI-1 siRNA twice a week continuously reduced PAI-1 activity in the BALF (P<0.05),and decreased the expression of collagen type Ⅲ, α-SMA and TIMP-1 in the fibrotic lung tissues on day 7, 14 and 28. Statistical differences in the expression of collagen type Ⅲ, α-SMA and TIMP-1 between BLM+P group and BLM group at the same time point were observed. CONCLUSION: Intratracheal injection of PAI-1 siRNA twice a week continuously inhibits the expression of PAI-1. PAI-1 siRNA ameliorates BLM-induced pulmonary fibrosis by down-regulation of TIMP-1 expression.