[Effect of Nm23-H1 Nuclear Localization on Proliferation of Human Lung Adenocarcinoma Cell Line A549]

Abstract

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.

1

重组质粒pLentis-CMV-NME1-IRES2-PURO酶切鉴定结果 Results enzyme digestion analysis of recombinant plasmid. pLentis-CMV-NME1-IRES2-PURO. M: 1, 000 bp DNA Marker. Lane 1-2: pLentis-CMV-NME1-IRES2-PURO

2

pLentis-CMV-NME1-IRES2-PURO表达载体 Recombinant plasmid pLentis-CMV-NME1-IRES2-PURO

3

载体pLentis-CMV-NME1-IRES2-PURO转染后A549细胞中Nm23-H1蛋白的定位及表达。A：Nm23-H1蛋白在A549细胞中以胞浆表达为主，转染组带有HA标签的Nm23-H1蛋白在胞浆和胞核均检测到，但主要以胞核表达为主。β-tublin和Lamin A/C作为内对照；B：Nm23-H1蛋白主要位于A549细胞的胞浆，转染后其在细胞核表达明显增多；1：A549；2：A549空载体组；3：A549 nNME1转染组 Nm23-H1 positioning and expression after stably transfected A549 cells by pLentis-CMV-NME1-IRES2-PURO. A: Nm23-H1 was mainly localized in the cytoplasm of A549 cells, Nm23-H1 with HA tag was detected in cytoplasm and nucleus of A549 nNME1 transfected and mainly localized in nucleus. β-tublin and Lamin A/C served as an internal control; B: Nm23-H1 was mainly localized in the cytoplasm of A549 cells and its nuclear localization significantly increased after transfected; 1: A549; 2: A549 vector only transfected; 3: A549 nNME1 transfected

4

CCK-8法检测Nm23-H1核过表达对A549细胞增殖的促进作用。***P < 0.000, 1，n=3 The nuclear Nm23-H1 promotes A549 cells proliferation in vitro was detected by cell counting kit-8 (CCK-8). ***P < 0.000, 1, n=3

5

A549、A549空载体组和A549 nNME1转染组的细胞周期检测。A：A549、A549空载体组和A549 nNME1转染组细胞的流式细胞术周期检测结果，a：A549；b: A549空载体组；c：A549 nNME1转染组；B：转染组细胞在G₂期/M期所占比例（58.7%）明显高于空载体组（31.30%）和未转染组（30.7%）。* P < 0.05 Changes in the level of cell cycle in A549, A549 vector only transfected and A549 nNME1 transfected group cells. A: The flow cytometry results showed the cell cycle of A549, A549 vector only transfected and A549 nNME1 transfected group cells; a: A549; b: A549 vector only transfected; c: A549 nNME1 transfected; B:The cell circle distribution of A549 nNME1 transfected (58.7%) was significantly higher than those of A549 vector only transfected (31.30%) and A549 cells (30.7%). *P < 0.05