自噬及PARP-1对鼻咽癌CNE-2细胞放射敏感性影响的实验研究
发布时间：2018-08-09 13:53
【摘要】：第一部分沉默自噬相关基因ATG5对鼻咽癌CNE-2细胞放射敏感性的影响 [目的] 本部分通过慢病毒介导的RNA干扰技术抑制自噬相关基因ATG5的表达,观察ATG5沉默后对鼻咽癌CNE-2细胞增殖、凋亡及放射敏感性的影响,以探讨自噬与鼻咽癌CNE-2细胞放射敏感性的关系,为鼻咽癌放射增敏及基因治疗提供新的靶点。 [方法] 针对自噬相关基因ATG5设计并合成3条干扰靶点,筛选最佳沉默片段进行慢病毒包装,感染鼻咽癌CNE-2细胞并经流式分选获得稳定沉默的细胞株。应用RT-PCR及Western-blot实验验证干扰效果。采用CCK-8法、流式细胞技术和克隆形成实验检测自噬相关基因ATG5沉默后鼻咽癌CNE-2细胞放射敏感性的变化。同时,进行裸鼠成瘤体内试验,观察ATG5基因沉默后,射线照射对裸鼠移植瘤生长的影响,进一步验证自噬与鼻咽癌放射敏感性的关系。 [结果] CCK-8实验结果显示,与未转染的Control组和阴性对照NC组相比,各剂点ATG5沉默组的细胞存活率均显著降低(P0.05),绘制细胞生存曲线可见下调ATG5的表达后可以增加CNE-2细胞的放射敏感性。流式细胞术结果显示,经6Gy射线照射后,与NC组及Control组相比,ATG5沉默组细胞的凋亡指数明显升高(F=394.876,P0.01)。克隆形成实验结果显示,在各照射剂量点,ATG5组的细胞存活分数较Control及NC组均有所下降(P0.05),二次线性模型拟合剂量存活曲线示沉默ATG5基因可增加鼻咽癌CNE-2细胞的放射敏感性。裸鼠成瘤体内实验结果显示,经10Gy射线照射后,与Control及NC组相比,ATG5组移植瘤的生长明显减缓,ATG5组瘤体重量明显小于其他两组(F=4.035,P0.05)。 [结论] 沉默自噬相关基因ATG5可以增加鼻咽癌CNE-2细胞的放射敏感性,初步判断自噬是鼻咽癌CNE-2细胞放射过程中的一种保护性机制。 第二部分沉默PARP-1基因对鼻咽癌CNE-2细胞放射敏感性的影响 [目的] 本部分采用慢病毒介导的RNA干扰技术抑制基因PARP-1的表达,观察其沉默后对射线所致的自噬现象及鼻咽癌CNE-2细胞放射敏感性的影响,进一步探讨PARP-1与自噬及鼻咽癌放射敏感性的关系,为寻找鼻咽癌放射增敏途径提供理论基础。 [方法] 针对基因PARP-1设计并合成的3条干扰靶点,进行慢病毒包装,应用RT-PCR及Western-blot实验筛选出沉默效果最佳的靶点,感染目的细胞CNE-2并流式分选获得稳定沉默PARP-1基因的细胞株。Western-blot实验分别检测PARP-1及ATG5基因沉默的CNE-2细胞射线照射前后LC3-Ⅱ、ATG5及PARP-1蛋白的变化情况,研究PARP-1与自噬的关系。另外应用CCK-8法、流式细胞术及克隆形成实验检测]PARP-1基因沉默后鼻咽癌CNE-2细胞放射敏感性的变化。[结果] Western-blot实验结果显示,经10Gy射线照射,PARP-1基因沉默组LC3-Ⅱ的相对表达量较单纯照射组明显降低(F=34.856,P0.01),这一现象在ATG5沉默组中也有相似表现。同时,沉默PARP-1基因后,PARP-1蛋白的相对表达量较单纯照射组降低(F=14.853,P0.01),ATG5的蛋白表达量也相应降低(F=10.863,P0.01),而沉默自噬相关基因ATG5后,ATG5蛋白的表达量有所降低,PARP-1的表达却未受影响。CCK-8实验结果显示,降低PARP-1的表达后各实验组细胞的存活率均有所降低(P0.05),绘制细胞生存曲线可见下调PARP-1的表达后可增加CNE-2细胞的放射敏感性。流式细胞术结果显示,经6Gy射线照射后,与NC组及Control组相比,PARP-1沉默组细胞的凋亡率明显升高(F=501.048,P0.01)。克隆形成实验结果显示,各剂量点PARP-1沉默组细胞的存活分数均有所降低(P0.05),二次线性模型拟合剂量存活曲线示下调PARP-1的表达后可以增加鼻咽癌CNE-2细胞的放射敏感性。 [结论] 射线可以诱导PARP-1的激活。PARP-1参与了射线所致的鼻咽癌CNE-2细胞的自噬现象,且可能处于相应信号通路的上游。PARP-1基因表达量的降低增加了CNE-2细胞对射线的敏感性,即PARP-1在射线所致鼻咽癌CNE-2细胞死亡过程中起保护性作用。
[Abstract]:Part I Effect of silencing autophagy-related gene ATG5 on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
In this part, the expression of autophagy related gene ATG5 was suppressed by lentivirus mediated RNA interference technique, and the effects of ATG5 silence on the proliferation, apoptosis and radiosensitivity of nasopharyngeal carcinoma CNE-2 cells were observed in order to explore the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma CNE-2 cells, and to provide new targets for the radiation sensitization and gene therapy of nasopharyngeal carcinoma.
[method]
3 interference targets were designed and synthesized for autophagy related gene ATG5, and the best silent fragment was screened for lentivirus package, CNE-2 cells of nasopharyngeal carcinoma were infected and the cell lines stable and silent by flow sorting. The interference effect was verified by RT-PCR and Western-blot experiments. The CCK-8 method, flow cytometry and clone formation test were used to detect autophagy. The changes in the radiosensitivity of the nasopharyngeal carcinoma CNE-2 cells after the ATG5 gene was silenced. At the same time, the nude mice were tested in vivo to observe the effect of radiation on the growth of xenografts in nude mice after the ATG5 gene silencing, and to further verify the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma.
[results]
The results of CCK-8 experiment showed that compared with the untransfected Control group and the negative control NC group, the cell survival rate of each point ATG5 silencing group decreased significantly (P0.05). The cell survival curve could be shown to increase the radiosensitivity of the CNE-2 cells after the expression of the down-regulation of ATG5. The results of flow cytometry showed that after the 6Gy ray irradiation, it was associated with the NC group and Contr. Compared with the ol group, the apoptotic index of the cells in the ATG5 silencing group increased significantly (F=394.876, P0.01). The results of the clone formation experiment showed that the cell survival fraction of the ATG5 group decreased (P0.05) in the ATG5 group than that in the Control and NC groups. The two linear model fitted the dose survival curve to show that the silence of the ATG5 gene could increase the radiation of CNE-2 cells in nasopharyngeal carcinoma. The results of tumor formation in nude mice showed that after 10Gy ray irradiation, the growth of the transplanted tumor in the group ATG5 was significantly slower than that of the Control and NC groups, and the weight of the ATG5 group was significantly smaller than that of the other two groups (F=4.035, P0.05).
[Conclusion]
The silencing of autophagy related gene ATG5 can increase the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma and preliminarily determine that autophagy is a protective mechanism in the radiation process of nasopharyngeal carcinoma CNE-2 cells.
Part 2 Effect of PARP-1 gene silencing on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
This part uses the slow virus mediated RNA interference technique to inhibit the expression of gene PARP-1, and observe the effect of its silence on the autophagy and the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma, and further explore the relationship between PARP-1 and the radiosensitivity of the autophagy and nasopharyngeal carcinoma, and provide a theoretical basis for the search for the radiosensitivity pathway of nasopharyngeal carcinoma.
[method]
3 interfering targets designed and synthesized by gene PARP-1 were designed and packed in lentivirus. RT-PCR and Western-blot experiments were used to screen the best target of silence effect. The.Western-blot experiment of cells with stable and silent PARP-1 gene from infected target cells was selected to detect the CNE-2 cell shoot of PARP-1 and ATG5 gene silencing by.Western-blot. The changes of LC3- II, ATG5 and PARP-1 protein before and after line irradiation were used to study the relationship between PARP-1 and autophagy. In addition, the changes of radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma after]PARP-1 gene silencing were detected by CCK-8, flow cytometry and cloning formation. [results]
The results of Western-blot experiment showed that the relative expression of LC3- II in the PARP-1 gene silencing group was significantly lower than that in the simple irradiation group (F=34.856, P0.01), and the phenomenon was also similar in the ATG5 silence group. At the same time, the relative expression of the PARP-1 protein decreased (F=14.853, P0.01) after the silence of PARP-1 gene (F=14.853, P0.01). The expression of protein was also reduced (F=10.863, P0.01), while the expression of ATG5 protein was reduced after the silence of autophagy related gene ATG5, but the expression of PARP-1 was not affected by the.CCK-8 experimental results. The survival rate of the cells in the experimental groups decreased (P0.05) after the reduction of the expression of PARP-1, and the survival curve of the cells was reduced to PARP-1. The expression of CNE-2 cells increased the radiosensitivity of the cells. The results of flow cytometry showed that after the 6Gy ray irradiation, the apoptosis rate of the cells in the PARP-1 silence group was significantly increased (F=501.048, P0.01) compared with the NC group and the Control group (F=501.048, P0.01). The results of the clone formation experiment showed that the survival fraction of the cells of each dose point PARP-1 sink group decreased (P0.05), two times. Linear model fitting dose survival curve showed that down-regulation of PARP-1 expression could increase radiosensitivity of nasopharyngeal carcinoma CNE-2 cells.
[Conclusion]
Radiation can induce PARP-1 activation.PARP-1 to participate in the autophagy of nasopharyngeal carcinoma CNE-2 cells induced by ray, and the decrease of.PARP-1 gene expression may be in the corresponding signal pathway to increase the sensitivity of CNE-2 cells to radiation, that is to say, PARP-1 plays a protective role in the death of nasopharyngeal carcinoma CNE-2 cells caused by radiation.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.63
本文编号：2174275
[Abstract]:Part I Effect of silencing autophagy-related gene ATG5 on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
In this part, the expression of autophagy related gene ATG5 was suppressed by lentivirus mediated RNA interference technique, and the effects of ATG5 silence on the proliferation, apoptosis and radiosensitivity of nasopharyngeal carcinoma CNE-2 cells were observed in order to explore the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma CNE-2 cells, and to provide new targets for the radiation sensitization and gene therapy of nasopharyngeal carcinoma.
[method]
3 interference targets were designed and synthesized for autophagy related gene ATG5, and the best silent fragment was screened for lentivirus package, CNE-2 cells of nasopharyngeal carcinoma were infected and the cell lines stable and silent by flow sorting. The interference effect was verified by RT-PCR and Western-blot experiments. The CCK-8 method, flow cytometry and clone formation test were used to detect autophagy. The changes in the radiosensitivity of the nasopharyngeal carcinoma CNE-2 cells after the ATG5 gene was silenced. At the same time, the nude mice were tested in vivo to observe the effect of radiation on the growth of xenografts in nude mice after the ATG5 gene silencing, and to further verify the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma.
[results]
The results of CCK-8 experiment showed that compared with the untransfected Control group and the negative control NC group, the cell survival rate of each point ATG5 silencing group decreased significantly (P0.05). The cell survival curve could be shown to increase the radiosensitivity of the CNE-2 cells after the expression of the down-regulation of ATG5. The results of flow cytometry showed that after the 6Gy ray irradiation, it was associated with the NC group and Contr. Compared with the ol group, the apoptotic index of the cells in the ATG5 silencing group increased significantly (F=394.876, P0.01). The results of the clone formation experiment showed that the cell survival fraction of the ATG5 group decreased (P0.05) in the ATG5 group than that in the Control and NC groups. The two linear model fitted the dose survival curve to show that the silence of the ATG5 gene could increase the radiation of CNE-2 cells in nasopharyngeal carcinoma. The results of tumor formation in nude mice showed that after 10Gy ray irradiation, the growth of the transplanted tumor in the group ATG5 was significantly slower than that of the Control and NC groups, and the weight of the ATG5 group was significantly smaller than that of the other two groups (F=4.035, P0.05).
[Conclusion]
The silencing of autophagy related gene ATG5 can increase the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma and preliminarily determine that autophagy is a protective mechanism in the radiation process of nasopharyngeal carcinoma CNE-2 cells.
Part 2 Effect of PARP-1 gene silencing on radiosensitivity of nasopharyngeal carcinoma CNE-2 cells
[Objective]
This part uses the slow virus mediated RNA interference technique to inhibit the expression of gene PARP-1, and observe the effect of its silence on the autophagy and the radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma, and further explore the relationship between PARP-1 and the radiosensitivity of the autophagy and nasopharyngeal carcinoma, and provide a theoretical basis for the search for the radiosensitivity pathway of nasopharyngeal carcinoma.
[method]
3 interfering targets designed and synthesized by gene PARP-1 were designed and packed in lentivirus. RT-PCR and Western-blot experiments were used to screen the best target of silence effect. The.Western-blot experiment of cells with stable and silent PARP-1 gene from infected target cells was selected to detect the CNE-2 cell shoot of PARP-1 and ATG5 gene silencing by.Western-blot. The changes of LC3- II, ATG5 and PARP-1 protein before and after line irradiation were used to study the relationship between PARP-1 and autophagy. In addition, the changes of radiosensitivity of CNE-2 cells in nasopharyngeal carcinoma after]PARP-1 gene silencing were detected by CCK-8, flow cytometry and cloning formation. [results]
The results of Western-blot experiment showed that the relative expression of LC3- II in the PARP-1 gene silencing group was significantly lower than that in the simple irradiation group (F=34.856, P0.01), and the phenomenon was also similar in the ATG5 silence group. At the same time, the relative expression of the PARP-1 protein decreased (F=14.853, P0.01) after the silence of PARP-1 gene (F=14.853, P0.01). The expression of protein was also reduced (F=10.863, P0.01), while the expression of ATG5 protein was reduced after the silence of autophagy related gene ATG5, but the expression of PARP-1 was not affected by the.CCK-8 experimental results. The survival rate of the cells in the experimental groups decreased (P0.05) after the reduction of the expression of PARP-1, and the survival curve of the cells was reduced to PARP-1. The expression of CNE-2 cells increased the radiosensitivity of the cells. The results of flow cytometry showed that after the 6Gy ray irradiation, the apoptosis rate of the cells in the PARP-1 silence group was significantly increased (F=501.048, P0.01) compared with the NC group and the Control group (F=501.048, P0.01). The results of the clone formation experiment showed that the survival fraction of the cells of each dose point PARP-1 sink group decreased (P0.05), two times. Linear model fitting dose survival curve showed that down-regulation of PARP-1 expression could increase radiosensitivity of nasopharyngeal carcinoma CNE-2 cells.
[Conclusion]
Radiation can induce PARP-1 activation.PARP-1 to participate in the autophagy of nasopharyngeal carcinoma CNE-2 cells induced by ray, and the decrease of.PARP-1 gene expression may be in the corresponding signal pathway to increase the sensitivity of CNE-2 cells to radiation, that is to say, PARP-1 plays a protective role in the death of nasopharyngeal carcinoma CNE-2 cells caused by radiation.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.63
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