HIF-1α调控β-catenin对肺腺癌细胞放射敏感性的影响
发布时间：2018-12-31 22:06
【摘要】：目的:构建肺腺癌细胞放射抗拒株模型;慢病毒介导sh RNA靶向沉默抗拒株细胞中HIF-1α的表达,探讨其对β-catenin的调控及对肺腺癌细胞放射敏感性的影响。方法:采用亚致死剂量法诱导人肺腺癌H1299及A549放射抗拒细胞H1299R及A549R,克隆形成实验验证放射抗性,CCK-8实验检测增细胞殖能力变化,q RT-PCR及Western Blot检测HIF-1α、β-catenin、Cyclin D1、Survivin的基因及蛋白表达差异;通过RNAi技术,设计靶向沉默HIF-1α的sh RNA,构建慢病毒表达载体;慢病毒转染H1299R、A549R细胞,通过克隆形成实验验证放射抗拒性的变化,Western Blot检测下调组及对照组细胞HIF-1α、β-catenin、Cyclin D1、Survivin的蛋白表达变化。结果:克隆形成实验结果显示:抗拒株细胞比亲本株细胞具有更高的克隆形成率(P0.05),H1299R细胞SF2值是H1299细胞的1.56倍,A549R细胞SF2值是A549细胞的1.55倍,抗拒株细胞放疗抗性增强;CCK-8结果提示:在接受4Gy射线干预后亲本株细胞增殖能力显著抑制(P0.05);q RT-PCR及Western Blot结果显示:抗拒株细胞比亲本株细胞中HIF-1α、β-catenin、Cyclin D1、Survivin的基因及蛋白表达水平明显升高(P0.05);通过慢病毒介导sh RNA下调抗拒株细胞HIF-1α后,下调组细胞β-catenin、Cyclin D1、Survivin蛋白表达明显下降(P0.05);在不同剂量射线干预后,随照射剂量增加,下调组细胞HIF-1α、β-catenin、Cyclin D1、Survivin的蛋白表达明显低于对照组(P0.05);克隆形成实验结果显示:下调组比对照组细胞克隆形成率明显降低(P0.05),通过放射敏感性参数分析H1299R对照组细胞SF2值是下调组细胞的3.56倍,A549R对照组细胞SF2值是下调组细胞的2.45倍,下调组细胞放射敏感性显著提高;CCK-8结果显示:在细胞增殖能力方面,下调组比对照组细胞明显降低(P0.05);在接受6Gy射线干预后,下调组细胞增殖能力显著抑制(P0.05)。结论:HIF-1α可能调控β-catenin影响肺腺癌细胞放射敏感性,其机制可能与影响下游Cyclin D1、Survivin等蛋白的表达有关。
[Abstract]:Aim: to establish a lung adenocarcinoma cell line model of radioresistance and to investigate the effect of lentivirus-mediated HIF-1 伪 expression on 尾-catenin and radiosensitivity of lung adenocarcinoma cells. Methods: human lung adenocarcinoma cell lines H1299R and A549R were induced by sublethal dose method. The radioresistance of H1299R and A549R was verified by clone formation assay. The colonization ability was detected by CCK-8 assay. HIF-1 伪 and 尾-catenin, were detected by Q RT-PCR and Western Blot. The expression of survivin gene and protein in Cyclin D1 was different. The expression vector of lentivirus was constructed by designing sh RNA, targeting silencing HIF-1 伪 by RNAi technique. Lentivirus was transfected into H1299RtA549R cells. The change of radioresistance was verified by clone formation assay. The expression of HIF-1 伪, 尾-catenin,Cyclin D1 survivin was detected by, Western Blot in down-regulation group and control group. Results: the clone forming rate of resistant cell was higher than that of parent cell (P0.05). The SF2 value of H1299R cell was 1.56 times of that of H1299 cell, and the SF2 value of A549R cell was 1.55 times of that of A549 cell. The cell resistance to radiotherapy of the resistant strain was enhanced. The results of CCK-8 showed that the cell proliferation ability of parental lines was significantly inhibited after 4Gy irradiation (P0.05). The results of Q RT-PCR and Western Blot showed that the expression of HIF-1 伪, 尾-catenin,Cyclin D1 survivin gene and protein in resistant cell were significantly higher than those in parent cell line (P0.05). After down-regulation of HIF-1 伪 by sh RNA mediated by lentivirus, the expression of 尾-catenin,Cyclin D1 survivin protein decreased significantly (P0.05). The protein expression of HIF-1 伪, 尾-catenin,Cyclin D 1 and survivin was significantly lower than that in control group with the increase of irradiation dose after different doses of radiation (P0.05). The results of clone formation test showed that the colony formation rate of down-regulation group was significantly lower than that of control group (P0.05). The SF2 value of H1299R control group was 3.56 times of that of down-regulation group by radiosensitivity parameter analysis. The SF2 value of A549R control group was 2.45 times of that of down-regulated cells, and the radiosensitivity of down-regulated cells was significantly increased. CCK-8 results showed that the cell proliferation ability of down-regulation group was significantly lower than that of control group (P0.05), and that of down-regulation group was significantly inhibited after 6Gy irradiation (P0.05). Conclusion: HIF-1 伪 may regulate 尾-catenin to affect radiosensitivity of lung adenocarcinoma cells, and its mechanism may be related to the expression of downstream Cyclin D1 survivin and other proteins.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
[Abstract]:Aim: to establish a lung adenocarcinoma cell line model of radioresistance and to investigate the effect of lentivirus-mediated HIF-1 伪 expression on 尾-catenin and radiosensitivity of lung adenocarcinoma cells. Methods: human lung adenocarcinoma cell lines H1299R and A549R were induced by sublethal dose method. The radioresistance of H1299R and A549R was verified by clone formation assay. The colonization ability was detected by CCK-8 assay. HIF-1 伪 and 尾-catenin, were detected by Q RT-PCR and Western Blot. The expression of survivin gene and protein in Cyclin D1 was different. The expression vector of lentivirus was constructed by designing sh RNA, targeting silencing HIF-1 伪 by RNAi technique. Lentivirus was transfected into H1299RtA549R cells. The change of radioresistance was verified by clone formation assay. The expression of HIF-1 伪, 尾-catenin,Cyclin D1 survivin was detected by, Western Blot in down-regulation group and control group. Results: the clone forming rate of resistant cell was higher than that of parent cell (P0.05). The SF2 value of H1299R cell was 1.56 times of that of H1299 cell, and the SF2 value of A549R cell was 1.55 times of that of A549 cell. The cell resistance to radiotherapy of the resistant strain was enhanced. The results of CCK-8 showed that the cell proliferation ability of parental lines was significantly inhibited after 4Gy irradiation (P0.05). The results of Q RT-PCR and Western Blot showed that the expression of HIF-1 伪, 尾-catenin,Cyclin D1 survivin gene and protein in resistant cell were significantly higher than those in parent cell line (P0.05). After down-regulation of HIF-1 伪 by sh RNA mediated by lentivirus, the expression of 尾-catenin,Cyclin D1 survivin protein decreased significantly (P0.05). The protein expression of HIF-1 伪, 尾-catenin,Cyclin D 1 and survivin was significantly lower than that in control group with the increase of irradiation dose after different doses of radiation (P0.05). The results of clone formation test showed that the colony formation rate of down-regulation group was significantly lower than that of control group (P0.05). The SF2 value of H1299R control group was 3.56 times of that of down-regulation group by radiosensitivity parameter analysis. The SF2 value of A549R control group was 2.45 times of that of down-regulated cells, and the radiosensitivity of down-regulated cells was significantly increased. CCK-8 results showed that the cell proliferation ability of down-regulation group was significantly lower than that of control group (P0.05), and that of down-regulation group was significantly inhibited after 6Gy irradiation (P0.05). Conclusion: HIF-1 伪 may regulate 尾-catenin to affect radiosensitivity of lung adenocarcinoma cells, and its mechanism may be related to the expression of downstream Cyclin D1 survivin and other proteins.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2
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