Nm23-H1核内定位对人肺腺癌A549细胞增殖的影响

发布时间：2018-07-27 18:23

【摘要】：背景与目的现有研究发现Nm23-H1还存在胞核表达,而既往的研究都是以过表达或抑制胞浆Nm23-H1为研究手段,由于Nm23-H1本身缺乏核引导序列,其研究结果并不能真实反映或重复临床中Nm23-H1以胞核定位为主的实际生物学效应。因此,本研究通过构建带有核引导序列的Nm23-H1载体并转染A549细胞以探讨Nm23-H1从胞浆向胞核转位对肺癌细胞增殖的影响。方法采用基因重组技术构建带核定位信号序列的p Lentis-CMV-NME1-IRES2-PURO慢病毒载体,酶切和测序鉴定正确后,稳定转染A549细胞后用Western blot和激光共聚焦检测Nm23-H1蛋白的定位和表达,用CCK-8法检测细胞的增殖,流式细胞术检测细胞周期变化。结果成功构建了核内定向表达Nm23-H1的慢病毒载体。转染组在72 h、96 h和120 h时增殖率与空载体组相比均显著升高(P0.000,1)。空载体组A549细胞在G0期/G1期所占比例为35.69%,高于转染组的28.28%(t=1.461,P=0.217);而转染组细胞在G2期/M期所占比例为58.7%,空载体组为31.30%(t=4.560,P=0.010)。结论 Nm23-H1在人肺腺癌A549细胞的核内过表达使细胞主要分布在G2期/M期并促进了细胞的体外增殖。
[Abstract]:Background & AIM existing studies have found that Nm23-H1 still exists nuclear expression, but previous studies have been carried out by overexpression or inhibition of cytoplasmic Nm23-H1, due to the lack of nucleocapsid sequence in Nm23-H1 itself. The results can not truly reflect or repeat the actual biological effects of nuclear localization of Nm23-H1 in clinical practice. Therefore, we constructed a Nm23-H1 vector with nucleocapsid sequence and transfected A549 cells to investigate the effect of Nm23-H1 translocation from cytoplasm to nucleus on the proliferation of lung cancer cells. Methods the vector of p Lentis-CMV-NME1-IRES2-PURO lentivirus with nuclear localization signal sequence was constructed by gene recombination technique. After confirmed by restriction endonuclease digestion and sequencing, the localization and expression of Nm23-H1 protein were detected by Western blot and confocal laser scanning after stable transfection into A549 cells. CCK-8 assay was used to detect cell proliferation and flow cytometry was used to detect cell cycle changes. Results Lentivirus vector expressing Nm23-H1 in nucleus was successfully constructed. The proliferation rate of the transfected group was significantly higher than that of the blank vector group at 72 h and 120 h (P0.0001). The percentage of A549 cells in G _ 0 / G _ 1 phase was 35.699.It was higher than that in transfection group (28.28%), while the proportion of transfected A549 cells in G _ 2 / M phase was 58.7% and 31.30% in empty vector group (t _ (4.560) P _ (0.010). Conclusion overexpression of Nm23-H1 in the nucleus of human lung adenocarcinoma A549 cells makes the cells mainly distribute in G2 phase and promote the proliferation of A549 cells in vitro.
【作者单位】：第三军医大学大坪医院野战外科研究所肿瘤中心;
【基金】：国家自然科学基金面上项目(No.81272599)资助~~
【分类号】：R734.2

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