| 柳正春,蔡锐,张凯丽,阮国柱,刘美莲.慢病毒介导的DKC1基因沉默对人宫颈癌HeLa细胞放射敏感性的影响[J].中华放射医学与防护杂志,2020,40(8):590-594 |
| 慢病毒介导的DKC1基因沉默对人宫颈癌HeLa细胞放射敏感性的影响 |
| Effects of lentivirus-mediated DKC1 gene silence on radiosensitivity of human cervical cancer HeLa cells |
| 投稿时间：2019-12-31 |
| DOI：10.3760/cma.j.issn.0254-5098.2020.08.003 |
| 中文关键词: DKC1基因 人宫颈癌HeLa细胞 放射敏感性 慢病毒 shRNA |
| 英文关键词:DKC1 gene Cervical cancer HeLa cells Radiosensitivity Lentivirus shRNA |
| 基金项目:广西自然科学基金面上项目（2016GXNSFAA380306） |
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| 中文摘要: |
| 目的 探讨降低角化不良蛋白基因（DKC1基因）表达对人宫颈癌HeLa细胞放射敏感性的影响。方法 以慢病毒为载体通过shRNA技术建立DKC1基因低表达的细胞模型，并利用RT-PCR、Western blot验证干扰效率。将细胞分为干扰组、空白对照组和阴性对照组，通过端粒重复序列扩增酶联免疫吸附试验（TRAP-ELISA法）检测端粒酶活性，实时PCR检测相对端粒长度，克隆形成实验计算克隆形成率，并利用单击多靶模型拟合细胞存活曲线、计算放射生物学相关参数（D0、Dq、N、SF2）及放射增敏比（SER）。结果 以慢病毒为载体的DKC1干扰序列（Lv-shDKC1）转染HeLa细胞后，与空白对照组相比，干扰组DKC1 mRNA及蛋白表达水平均明显下降，其表达抑制率分别为（71.330±4.112）%（t=25.53，P<0.05）、（35.520±3.804）%（t=4.833，P<0.05）。与空白对照组及阴性对照组相比，干扰组端粒酶活性从0.900±0.044、0.897±0.031降到0.713±0.021（F=31.44，P<0.05），相对端粒长度从4.233±0.306、4.633±0.379降到2.667±0.404（F=39.15，P<0.05），而空白对照组及阴性对照组间端粒酶活性和相对端粒长度差异均无统计学意义（P>0.05）。干扰组存活分数（SF2）（0.571±0.006）明显低于空白对照组（0.861±0.009）及阴性对照组（0.807±0.002）（F=1 812，P<0.05），SER为1.508。结论 干扰DKC1的表达对人宫颈癌HeLa细胞有放射增敏作用，可通过抑制端粒酶活性、缩短相对端粒长度而发挥放射增敏作用，有望成为新的放射增敏靶点。 |
| 英文摘要: |
| Objective To investigate the effect of decreasing DKC1 gene expression on radiosensitivity of HeLa cells. Methods A cell model with low expression of DKC1 gene was established by shRNA technology with lentivirus as vector, and the interference efficiency was verified by RT-PCR and Western blot assay. Cells were divided into two groups of interference (Lv-shDKC1) and its negative control. Telomerase activity was detected by TRAP-ELISA, and telomere length was measured by Real-time PCR. Cell survival was obtained through clone formation assay and fitted by multi-target single-hit model, and radiobiological parameters (D0, Dq, N, SF2) and radiosensitization ratio (SER) were calculated. Results After DKC1 interfering, the expression levels of mRNA and protein of DKC1 in HeLa cells were significantly decreased by (71.330±4.112)% (t=25.53,P<0.05) and (35.520±3.804)% (t=4.833, P<0.05), respectively. Compared with the blank control group and negative control group, the telomerase activity of Lv-shDKC1 group decreased significantly from 0.900±0.044 and 0.897±0.031 to 0.713±0.021 (F=31.44, P<0.05), the relative telomere length was significantly decreased from 4.233±0.306 and 4.633±0.379 to 2.667±0.404 (F=39.15, P<0.05). The telomerase activity and relative telomere length of blank control group and Lv-shDKC1 negative control group had no significant difference(P>0.05). SF2 in the interference group (0.571±0.006) was significantly lower than that of the blank control group (0.861±0.009) and the Lv-shDKC1 negative control group (0.807±0.002) (F=1812, P<0.05), and the radiosensitization ratio (SER) of shDKC1 interference was 1.508. Conclusions Downregulation of DKC1 in human cervical cancer HeLa cells enhances the radiosensitivity through inhibiting the activity of telomerase and shortening the length of telomere. DKC1 gene may become a new target of radiosensitization. |
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