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Effects of hypoxia-inducible factor-1α silencing on the proliferation of cbrh-7919 hepatoma cells

机译:缺氧诱导因子-1α沉默对cbrh-7919肝癌​​细胞增殖的影响

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AIM: To study the effects of hypoxia-inducible factor-1α (HIF-1α) silencing on the proliferation of hypoxic CBRH-7919 rat hepatoma cells. METHODS: The CBRH-7919 rat hepatoma cell line was used in this study and the hypoxic model was constructed using CoCl2. The HIF-1α-specifc RNAi sequences were designed according to the gene coding sequence of rat HIF-1α obtained from GeneBank. The secondary structure of the HIF-1α gene sequence was analyzed using RNA draw software. The small interfering RNA (siRNA) transfection mixture was produced by mixing the siRNA and Lipofectamine2000?, and transfected into the hypoxic hepatoma cells. Real time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assay were used to detect the expression levels of mRNA and protein. HIF-1α and vascular endothelial growth factor (VEGF) mRNA was determined using real time RT-PCR; the protein expression levels of AKT, p-AKT, p21 and cyclinD1 were determined using Western blotting. The proliferation of hepatoma cells was observed using the methyl thiazolyl tetrazolium (MTT) assay and the bromodeoxyuridine (BrdU) incorporation cell proliferation assay. RESULTS: Under induced hypoxia, the viability of the hepatoma cells reached a minimum at 800 μmol/L CoCl2; the viability of the cells was relatively high at CoCl2 concentrations between 100 μmol/L and 200 μmol/L. Under hypoxia, the mRNA and protein expression levels of HIF-1α and VEGF were significantly higher than that of hepatoma cells that were cultured in normaxia. HIF-1α-specifc RNAi sequences were successfully transfected into hepatoma cells. The transfection of specific siRNAs significantly inhibited the mRNA and protein expression levels of HIF-1α and VEGF, along with the protein expression levels of p-AKT and cyclinD1; the protein expression of p21 was significantly increased, and there was no significant difference in the expression of AKT. The MTT assay showed that the amount of hepatoma cells in S phase in the siRNA transfection group was obviously smaller than that in the control group; in the siRNA transfection group, the amount of hepatoma cells in G1 phase was more than that in the control group. The BrdU incorporation assay showed that the number of BrdU positive hepatoma cells in the siRNA transfection group was less than that in the control group. The data of the MTT assay and BrdU incorporation assay suggested that HIF-1α silencing using siRNAs significantly inhibited the proliferation of hepatoma cells. CONCLUSION: Hypoxia increases the expression of HIF-1α, and HIF-1α silencing significantly inhibits the proliferation of hypoxic CBRH-7919 rat hepatoma cells.
机译:目的:研究缺氧诱导因子-1α(HIF-1α)沉默对低氧CBRH-7919大鼠肝癌细胞增殖的影响。方法:使用CBRH-7919大鼠肝癌细胞系,并用CoCl2建立低氧模型。根据从GeneBank获得的大鼠HIF-1α的基因编码序列设计HIF-1α特异性RNAi序列。使用RNA绘图软件分析了HIF-1α基因序列的二级结构。通过将siRNA和Lipofectamine2000?混合产生小的干扰RNA(siRNA)转染混合物,并将其转染到低氧肝癌细胞中。实时逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法用于检测mRNA和蛋白质的表达水平。使用实时RT-PCR测定HIF-1α和血管内皮生长因子(VEGF)的mRNA。用蛋白质印迹法测定AKT,p-AKT,p21和cyclinD1的蛋白表达水平。使用甲基噻唑基四唑(MTT)测定法和溴脱氧尿苷(BrdU)掺入细胞增殖测定法观察到肝癌细胞的增殖。结果:在诱导的缺氧条件下,肝癌细胞的活力在800μmol/ L CoCl2时达到最小值。当CoCl2浓度在100μmol/ L和200μmol/ L之间时,细胞的活力相对较高。在缺氧条件下,HIF-1α和VEGF的mRNA和蛋白表达水平显着高于常氧培养的肝癌细胞。 HIF-1α特异的RNAi序列已成功转染到肝癌细胞中。特异性siRNA的转染显着抑制了HIF-1α和VEGF的mRNA和蛋白表达水平,以及p-AKT和cyclinD1的蛋白表达水平。 p21的蛋白表达明显增加,而AKT的表达无明显差异。 MTT法检测发现,siRNA转染组S期肝癌细胞数量明显少于对照组。在siRNA转染组中,G1期肝癌细胞的数量要多于对照组。 BrdU掺入法显示,siRNA转染组中BrdU阳性肝癌细胞的数量少于对照组。 MTT试验和BrdU掺入试验的数据表明,使用siRNA沉默HIF-1α可显着抑制肝癌细胞的增殖。结论:低氧可增加HIF-1α的表达,而HIF-1α沉默可明显抑制低氧CBRH-7919大鼠肝癌细胞的增殖。

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