Background: Recent studies have indicated that Nm23-H1 is found in the nucleus, but previous studies have been based on the overexpression or suppression of Nm23-H1 in the cytoplasm. Due to the lacking nuclear localization signal of Nm23-H1, these results cannot reflect or repeat cells in which Nm23-H1 mainly positioned in nuclei and whether they cause clinical biological effects. Therefore, to explore the effects of transposing Nm23-H1 from the cytoplasm to the nucleus during lung cancer cell proliferation, a vector with a nuclear localization signal of Nm23-H1 was constructed and A549 cells were transfected.
Methods: Gene recombination technology was used to construct pLentis-CMV-NME1-IRES2-PURO lentiviral vectors using a nuclear localization signal sequence, and the recombinant plasmid was verified using restriction enzyme analysis and sequencing. Nm23-H1 positioning and expression were performed after the stably transfected A549 cells were assessed by Western blot and confocal laser scanning microscope. The A549 cell proliferation was assessed using a cell counting kit-8. Flow cytometry was performed to assess the cell cycle distribution of A549 cells.
Results: The directional Nm23-H1 lentiviral vector was successfully constructed within the nucleus. Compared with that of the empty vector group, the proliferation rates of the transfection groups at 72 h, 96 h, and 120 h were remarkably increased (P<0.000,1). Moreover, the empty vector group of A549 cells in the G0/G1 phase proportion was 35.69%, which was higher than the 28.28% of the transfection group (t=1.461, P=0.217); furthermore, the transfection group of A549 cells in the G2/M phase proportion was 58.7% and that of the empty vector group was 31.30% (t=4.560, P=0.010).
Conclusions: Human lung adenocarcinoma cell line A549 cells of Nm23-H1 nuclear localized mainly in the G2/M phase and the nuclear Nm23-H1 promoted A549 cell proliferation in vitro.
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pLentis-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（P<0.000,1）。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。.