RNA 结合蛋白 QKI 吸附海绵载体的构建及其对肝癌细胞增殖的影响

Construction of the Sponge Adsorption Vector of RNA Binding Pro-tein QKI and Its Inhibition Effect on the Proliferation of HepG2 Cells

目的：构建针对 RNA 结合蛋白 quaking （QKI）的特异性吸附海绵载体（QRE-sponge）,验证其对肝癌细胞系 HepG2中内源性 QKI 分子的抑制作用。方法利用人源 QKI 的特异性识别效应原件（qua-king responsive element, QRE）序列,重复13个拷贝,串联克隆至 pEGFP-C2真核表达载体中,转染 HepG2肝癌细胞及293 T 细胞。通过 Real time PCR,双荧光素酶报告基因检测及 MTT 实验进一步验证该吸附海绵的效果及其对肝癌细胞系 HepG2增殖的影响。结果 pEGFP-C2-QRE-SP 载体经测序证实构建成功,可以成功的在 HepG2细胞中表达,其绿色荧光呈阳性,且转染24 h 后显著影响 HepG2细胞中 QKI 下游靶基因的表达（P ﹤0.05）。双荧光素酶报告基因系统显示, pGL3-QRE-SP 载体可以显著抑制外源性 QKI 的活性（P﹤0.05）。 MTT 结果显示 pEGFP-C2-QRE-SP 载体促进 HepG2细胞的增殖。结论 QKI 吸附海绵载体策略构建的 pEGFP-C2-QRE-SP 载体可以成功的诱骗吸附 QKI,并促进肝癌细胞系 HepG2增殖,为进一步研究 QKI的功能研究提供了良好工具。

Objective To construct a sponge adsorption vector specifically targeting RNA binding protein QKI, and to investigate the inhibitory role of the quaking responsive element （QRE） -sponge vector in the QKI of hepatocellular carcinoma HepG2 cells. Methods Thirteen copies of human QRE sequence were serially cloned to pEGFP-C2 vector and then transfected into HepG2 and 293T cells. The efficacy of QRE-sponge and its effect on the proliferation of HepG2 cells were measured by real-time PCR, MTT assay and dual luciferase reporter gene system. Results The construction of pEGFP-C2-QRE-SP vector was confirmed by sequencing. QRE was expressed in HepG2 by pEGFP-C2-QRE-SP clone and GFP was positive. Real time PCR showed that the expres-sion of various QKI downstream genes were significantly affected by this vector （P ﹤ 0. 05） . Dual luciferase reporter gene system showed that exogenous QKI was significantly inhibited by pEGFP-C2-QRE-SP vector （P ﹤ 0. 05） . MTT showed a notable promotion of cell proliferation of HepG2 by this＆amp;nbsp;vector. Conclusion QRE-sponge could successfully decoy absorption of QKI. pEGFP-C2-QRE-SP was able to inhibit the function of QKI in HepG2 cells and further promote cell proliferation, which provides a novel tool for QKI function study.