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中国癌症防治杂志

中国癌症防治杂志 ›› 2014, Vol. 6 ›› Issue (4): 342-347.doi: 10.3969/j.issn.1674-5671.2014.04.05

• 基础研究 • 上一篇    下一篇

人端粒酶逆转录酶基因小片段干扰RNA 慢病毒表达载体的构建及其在子宫颈癌caski 细胞的表达

  

  1. 广西医科大学附属肿瘤医院妇瘤科；广西医科大学研究生学院
  • 出版日期:2014-12-25 发布日期:2015-01-12
  • 通讯作者: 莫凌昭 E-mail:molingzhao@hotmail.com
  • 基金资助:

    广西自然科学基金资助项目（2013GXNSFAAO19254）

Construction of a plasmid expressing small hairpin RNA against human telomerase reverse transcriptase and functional analysis in the CaSki cervical cancer line

  • Online:2014-12-25 Published:2015-01-12

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被引次数

5

摘要： 目的 构建及鉴定携带沉默人端粒酶逆转录酶基因（human telomerase reverse transcriptase，hTERT ）慢病毒（lentivirus，LV）表达载体，并观察其在子宫颈癌caski 细胞的表达情况。方法 以Designer3.0（Genepharma）软件设计靶向hTERT 基因特异性的小片段RNA（shRNA）干扰序列，将hTERT-shRNA基因片段插入重组慢病毒pGLV3/H1/GFP+Puro，构建慢病毒表达质粒LV3-shRNA-hTERT，酶切、DNA 测序验证hTERT 片段准确性。LV3-shRNA-hTERT与包装质粒共转染293T 细胞，浓缩上清液并测定病毒滴度获得重组慢病毒后感染caski 细胞。将细胞分为空白对照组（未感染病毒的caski 细胞）、阴性对照组（感染空载病毒LV3-shNC的caski 细胞）和hTERT 干扰组（感染携带LV3-shhTERT慢病毒的caski 细胞）。绿色荧光蛋白（GFP ）的表达判断转染结果并估计转染效率，流式细胞术仪检测病毒感染率，实时荧光定量PCR 法（qRT-PCR）检测转染后基因hTERT mRNA 的表达情况，CCK-8法检测caski 细胞增殖情况。结果 将目的序列成功连接到载体上，并经测序分析证实载体构建成功；成功包装成高滴度的慢病毒，且能有效感染子宫颈癌caski 细胞。荧光定量PCR 检测结果证实构建的hTERT-小片段干扰RNA 慢病毒表达载体可显著抑制hTERT 基因的表达。hTERT 干扰组细胞增殖受抑制，生长速度较阴性对照组生长缓慢。结论 成功构建了携带hTERT-shRNA慢病毒表达载体LV3-shRNA-hTERT，并稳定转染子宫颈癌caski 细胞。该载体能够有效抑制hTERT 的表达，使子宫颈癌caski 细胞增殖缓慢，为进一步探讨hTERT 基因在子宫颈癌的发病机制和体外基因干预治疗奠定基础。

关键词: 子宫颈肿瘤, 人端粒酶逆转录酶基因, 慢病毒载体, 小片段RNA, caski细胞

Abstract: Objective To construct a plasmid expressing a small hairpin RNA（shRNA）to knockdown expression of human telomerase reverse transcriptase （hTERT），and to observe its effects on hTERT expression in caski cervical cancer cells. Methods Designer   3.0 software （Genepharma）was used to design an shRNA targeting hTERT. The shRNA was cloned into the pGLV3/Hi/GFP+Puro   vector and confirmed by sequencing. Cells were divided into blank control group，negative control group and hTERT interference   group. The resulting plasmid was transfected into caski cervical cancer cells and expression of the GFP reporter was analyzed. In   addition，expression of chromosomal hTERT mRNA was quantified using RT-PCR. Cell proliferation in the presence and absence of shRNA was measured using the cck-8 assay.Results A plasmid expressing an shRNA targeting hTERT was constructed and used to generate recombinant lentivirus. Lentiviral infection of caski cells led to lower expression of hTERT mRNA and slower cell   proliferation than in controls. Conclusion A plasmid expressing shRNA targeting hTERT can effectively knockdown endogenous   hTERT expression. This reagent may prove useful for understanding the role of telomerase in the pathogenesis of cervical cancer.

Key words: Cervical neoplasm, Human telomerase reverse transcriptase gene （hTERT）, Lentivirus-based vectors, Small hairpin RNA, Caski cell