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.     

Hypoxia-induced caveolin-1 up-regulation is involved in migration and invasion of lung adenocarcinoma A549 cells

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride (CoCl2) on caveolin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcinoma A549 cells. METHODS:The concentrations of Cav-1 and hypoxia-inducible factor (HIF)-1α in pleural effusion of the patients with lung cancer (MPE) or tuberculous pleurisy (TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1α inhibitor YC-1. The concentrations of Cav-1 and HIF-1α in the cell supernatants were measured by ELISA. The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respectively. RESULTS:The levels of Cav-1 and HIF-1α in MPE were significantly higher than those in TBPE. There was a highly positive correlation between Cav-1 and HIF-1α levels in the pleural effusion. CoCl2 induced the generation of Cav-1 and HIF-1α in A549 cells in a concentration- and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration- or time-dependent inhibition was observed. HIF-1α inhibitor YC-1 concentration-dependently inhibited the generation of HIF-1α and Cav-1 induced by CoCl2 in A549 cells. CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1. CONCLUSION: The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells. A possible role for HIF-1α is indicated in Cav-1 generation.     

Effect of siRNA for survivin gene on growth and apoptosis in A549 cells

AIM:To observe the influence of lentiviral vectors expressing siRNA for survivin gene knockdown in A549 cells, sequentially as tools to explore the molecule pathogenesis and new gene therapy of lung adenocarcinoma. METHODS:The lentiviral vectors, which express survivin siRNA, were constructed and transfected into A549 cell strain. The titers of the lentiviruses were determined by 293T cells. The expressions of survivin and caspase-3 were detected by Western blotting and RT-PCR. The cell cycle and cell growth of A549 cells were examined by MTT and FCM．RESULTS:The expression of survivin was suppressed effectively by siRNA targeting survivin. The expression of survivin mRNA decreased by 97%. The expression of survivin protein decreased by 94%. The rate of cell growth was decreased. The G1 phase cells were increased, whereas S phase cells were decreased. CONCLUSION:The lentivirus vectors expressing siRNA for survivin can significantly inhibit gene expression and the cell growth, and markedly induce the apoptosis. It is hopeful to be a new gene therapy of lung adenocarcinoma.     

Effects of NF-κB decoy oligonucleotides on apoptosis in lung cancer cell A549

AIM: To investigate the effects of NF-κB decoy oligodeoxynucleotides (ODNs) on apoptosis in lung cancer cell A549. METHODS: The treatments of lung cancer cells (A549) were divided into three groups: group A (control group); group B (decoy ODN group) and group C (scramble decoy ODN group). FITC-labeled NF-κB decoy ODNs was transfected into A549 with LipofectAMINE^TM2000. The activation was observed by electrophoretic mobility shift assays (EMSA). The proliferation was observed by growth curve. The apoptosis of cells were observed by flow cytometry and TdT mediated dUTP-biotin Nick End Labeling (TUNEL). The expression of Bcl-2 and Fas were observed by Western blot. RESULTS: After FITC-labeled decoy ODNs was transfected for 1 hour, the decoy ODNs was detected in the nuclei of A549 cells. EMSA performed the depression of the NF-κB binding to the nucleus. The growth curve showed the inhibition of the A549 cell growth and the percentage of apoptosis was increased compare with control group by flow cytometry and TUNEL. The amount of apoptosis inhibitor (Bcl-2) in group A and group C were 2.0 times and 2.1 times more than that in group B, respectively. The level of apoptosis accelerator (Fas) in group B were 2.6 times and 2.3 times more than that in group A and group C, respectively via Western blot. CONCLUSION: The NF-κB decoy ODNs accelerate the apoptosis of lung cancer cell A549 and the mechanism may be due to its inhibiting the expression of Bcl-2 and increasing the level of Fas.     

Expression of miRNA-181a in human lung adenocarcinoma cells and its effect on cell function

AIM: To study the expression of microRNA (miRNA)-181a in different human lung adenocarcinoma cell lines, and to investigate the effect of miRNA-181a on cell function and its mechanism in human lung adenocarcinoma drug resistant cell A549/DDP. METHODS: Real-time PCR was used to detect the expression of miRNA-181a in BEAS-2B cells, A549 cells and A549/DDP cells. The A549/DDP cells were transfected with pGenesil-miRNA-181a eukaryotic expression plasmid. At the same time, the untransfection group and negative transfection group were also set up. The expression of miRNA-181a, cell viability, cell growth inhibition and apoptosis rate during cis-diamminedichloroplatinum (DDP) treatment, cell cycle, cell invasion, the protein expression of miRNA-181a target genes bcl-2 and p53 in the A549/DDP cells were determined by real-time fluorescence quantitative PCR, MTT assay, flow cytometry, Transwell method and Western blot, respectivly. RESULTS: The expression of miRNA-181a in A549 cells and A549/DDP cells was significantly lower than that in BEAS-2B cells, and the lowest expression level was observed in A549/DDP cells (P<0.05). The expression of miRNA-181a in A549/DDP cells was significantly increased after transfection with pGenesil-miRNA-181a (P<0.05). The cell viability, cell cycle and invasion ability of the A549/DDP cells were inhibited after miRNA-181a transfection (P<0.05). The cell growth inhibition rate and apoptotic rate of the A549/DDP cells were increased (P<0.05). The expression of Bcl-2 was reduced, but the expression of P53 was increased after transfection with miRNA-181a in A549/DDP cells (P<0.05). CONCLUSION: miRNA-181a may be correlated with the development of human lung adenocarcinoma. miRNA-181a can serve as a new target for treatment of lung cancer.     

PGRN gene silencing on proliferation and migration abilities of human non-small-cell lung cancer A549 cells

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated progranulin (PGRN) gene silencing on the proliferation and migration abilities of human non-small-cell lung cancer A549 cells and its mechanism. METHODS: The mRNA and protein expression levels of PGRN in the A549 cells and human bronchial epithelial (HBE) cells were detected by qPCR and Western blot. A549 cells were transfected with PGRN-siRNA by liposome method. The expression of PGRN at mRNA and protein levels in the A549 cells transfected with PGRN-siRNA was detected by qPCR and Western blot, respectively. The cell viability was measured by MTT assay. The cell proliferation ability was measured by living cells counting and crystal violet staining assays. The cell migration ability was measured by wound-healing and Transwell assays. The protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1, Bcl-2 and Bax were determined by Western blot. The protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated protein kinase B (p-Akt) were also determined by Western blot. RESULTS: The expression of PGRN at mRNA and protein levels was higher in the A549 cells than that in the HBE cells (P<0.05). The expression of PGRN at mRNA and protein levels in the A549 cells transfected with PGRN-siRNA was significantly decreased, and the cell proliferation and migration abilities were significantly decreased. The protein expression levels of PCNA, cyclin D1 and Bcl-2 were significantly reduced and the protein expression level of Bax was significantly increased (P<0.05). Meanwhile, the protein levels of p-ERK1/2 and p-Akt were down-regulated (P<0.05). CONCLUSION: PGRN gene silencing obviously inhibits the proliferation and migration abilities of human non-small-cell lung cancer A549 cells. The PI3K/Akt and MAPK/ERK signaling pathways may play an important role in these processes.     

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.     

Effect of RNA interference on HIF-2 in the renal cancer cell line 786-0

AIM: To study the effect of RNA interference on hypoxia-inducible factor-2 (HIF-2) in the renal cell cancer in vitro and in vivo. METHODS: HIF-2 RNAi was synthesized and inserted into RNA interference eukaryotic expression vector which was confirmed by sequencing. The vector was transfected into the renal cancer cell 786-0 and positive clone was selected by using G418. The HIF-2 expression was detected by RT-PCR and Western blotting method. The growth of cells was measured by MTT method. Nude mouse xenograft assays were also done. RESULTS: Compared with empty vector group and control group, the amounts of HIF-2 mRNA and protein expression were lower in the HIF-2 RNAi group, the difference was significant (P<0.01). No significant difference between empty vector group and control group was observed. The cell growth in the HIF-2 RNAi group become slower. Compared with control group, the growth of tumor was slower in the RNAi group in the nude mice (P<0.01). CONCLUSION: HIF-2 RNAi inhibits the expression of 786-0 and cell growth, and slows the growth of tumor in the nude mice. The result provides new application for biological therapy in the renal cell cancer.     

Tripchlorolide activates p-ERK and induces autophagy in lung cancer A549 cells

AIM: To investigate the effects of tripchlorolide (TP) on proliferation and autophagy of human lung cancer A549 cells, and explore its mechanism. METHODS: MTT assay was performed to analyze the effect of TP on the viability of human lung cancer A549 cells. The A549 cells were treated with TP, and their autophagy was observed under the fluorescence microscope through acridine orange staining. Green fluorescence spots were observed by fluorescence microscopy through GFP-LC3 plasmid transfection experiment. The levels of LC3 and p-ERK in the A549 cells after TP treatment were determined by Western blot. RESULTS: The viability of human lung cancer A549 cells was significantly inhibited by TP in a dose-time dependent manner (P<0.05). The number of the intracellular acidic follicles dyed with bright red fluorescence was significantly increased after TP treatment in A549 cells. The number of green dot-like congregate autophagosomes in cell cytoplasm was significantly increased after TP treatment in the A549 cells transfected with GFP-LC3 plasmid, while the normal treatment only induced a few cells with autophagosome formation. At the same time, we did not observe the dot-like congregate autophagosomes after TP treatment in the A549 cells transfected with GFP-control plasmid. Compared with control group, the expression of LC3-Ⅱ protein was up-regulated in A549 cells after TP treatment (P<0.01). Furthermore, treatment with TP in A549 cells for 48 h also led to a significant upregulation of phosphorylated form of ERK (P<0.01). In contrast, no significant change in the levels of total ERK protein was observed. Compared with 100 nmol/L TP group, TP+3-MA group down-regulated the protein levels of LC3-Ⅱ (P<0.01) and p-ERK (P<0.01) in the A549 cells. CONCLUSION: TP significantly inhibits the growth of A549 lung cancer cells and induces the autophagy, which may be correlated with upregulation of p-ERK protein.     

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.     

Effect of antisense RNA targeting Polo-like kinase 1 on cell growth in A549 lung cancer cells

AIM: To investigate the effect of Polo-like kinase-1 (Plk1) depletion on cell cycle progression and cell growth in lung cancer cells.METHODS: A recombinant plasmid containing antisense RNA targeting Plk1 (pcDNA3-Plk1) was transfected into A549 cells by lipofectine. RT-PCR and Western blotting were used to examine Plk1 gene expression. Cell proliferation was evaluated by cell counting and BrdU labeling. Cell cycle distribution and apoptosis were examined by flow cytometry. Inhibition rate (IR) of vinorebline (NVB) was determined by MTT assay. RESULTS: After transfected with pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. Abnormal morphological changes of cells and growth inhibition were observed in pcDNA3-Plk1 transfected cells. The BrdU labeling index was significantly lower than that in control group (P<0.05). Cells showed a strong G₂/M arrest and apoptosis 72 h post transfection. IR of vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than that in other groups. CONCLUSION: Antisense RNA targeting Plk1 is capable of suppressing Plk1 expression, and therefore, significantly inhibits cellular proliferation, induces cell cycle arrest and apoptosis. Moreover, the sensitivity of lung cancer cells to chemotherapy is increased.     

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.     

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.     

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.     

miR-134 regulates cisplatin resistance of human lung adenocarcinoma cells

AIM: To investigate the roles of microRNA-134 (miR-134) in the cisplatin resistance of lung adenocarcinoma cells. METHODS: miRNA microarray was applied to compare the miRNA expression profile between A549/CDDP and A549 cells. Real-time PCR was used to confirm the expression of miR-134. miR-134 mimics and inhibitors were transfected into A549/CDDP and A549 cells, respectively. MTT assay was used to detect the sensitivity of lung cancer cells to cisplatin. Western blot was applied to test whether miR-134 regulated forkhead box protein M1 (FOXM1) and multidrug-associated protein 1 (MRP1) expression. RESULTS: Based on the data of miRNA microarray, 13 miRNAs were found to be differentially expressed in A549/CDDP cells compared with A549 cells, among which miR-134 was the most significantly down-regulated one. Compared with control group, A549/CDDP cells transfected with miR-134 mimics showed greatly enhanced sensitivity to cisplatin as indicated by IC₅₀ values (P<0.01). In contrast, suppression of the miR-134 level in the A549 cells resulted in a decreased sensitivity to cisplatin (P<0.01). FOXM1 siRNA down-regulated the protein levels of FOXM1. A549/CDDP cells transfected with si-FOXM1 showed enhanced sensitivity to cisplatin (P<0.01). In addition, the result of Western blot showed that miR-134 repressed MRP1 protein expression. CONCLUSION: miR-134 effectively increases the sensitivity of lung adenocarcinoma cells to cisplatin, and this effect of miR-134 may be partly due to its regulation of FOXM1 and MRP1 expression.     

Let-7a-3p inhibits activity of cancer stem cells in human lung cancer A549 cells by targeting IGF1R

AIM:To study the effect of let-7a-3p on the activity of cancer stem cells in human lung cancer A549 cells and its molecular biological mechanism. METHODS:The exepression levels of let-7a-3p in lung cancer cell lines A549, NCI-H1299, SPC-A1, H1650 and HCC-827, and human normal bronchial epithilial cell line BEAS-2B were compared by RT-qPCR. The lung cancer A549 cells were transfected with let-7a-3p mimic and negative control mimic, as let-7a-3p group and negative control group, respectively, and non-transfected control group was also set up. The content of let-7a-3p in each group was detected by RT-qPCR. Tumor sphere formation assay was used to detect the tumor sphere formation ability in 3 groups of the cancer stem cells. The proportion of cancer stem cells was detected by flow cytometry. The protein levels of NANOG, OCT4 and insulin-like growth factor 1 receptor (IGF1R) were determined by Western blot. The target gene of let-7a-3p was predicted by the bioinformatic method. The relationship between let-7a-3p and IGF1R was analyzed by double luciferase assay. Western blot was used to detect whether IGF1R over-expression antagonized the inhibitory effect of let-7a-3p on the activity of cancer stem cells. A subcutaneous transplantation tumor model was also established and the effect of let-7a-3p in vivo was observed. RESULTS:The expression level of let-7a-3p in the lung cancer cell lines was significantly lower than that in the normal bronchial epithelial cell line (P<0.01). The expression level of let-7a-3p in the A549 cells of let-7a-3p group was significantly up-regulated compared with non-transfected group (P<0.01). The number of tumor spheres in let-7a-3p group was significantly lower than that in non-transfected group. The percentage of CD133⁺ cells in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). The protein expression of NANOG and OCT4 in let-7a-3p group was significantly lower than that in non-transfected group (P<0.01). Bioinformatic prediction showed that let-7a-3p complementarily matched the 3'-UTR of IGF1R, and IGF1R might be the target gene of let-7a-3p. Luciferase assay confirmed that IGF1R was the direct downstream target gene of let-7a-3p. The protein expression of IGF1R in let-7a-3p group was significantly decreased (P<0.01). Subcutaneously transplantated tumor in let-7a-3p group was significantly smaller than that in non-transfected group. CONCLUSION:Let-7a-3p may affect the expression of lung cancer stem cell-related proteins and inhibit the potential of lung cancer stem cells by down-regulating its downstream target gene IGF1R.     

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.     

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.