Department of Respiratory Medicine, Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu, 212002, People's Republic of China.
Department of Internal Medicine, Clinical Medicine College of Jiangsu University, Zhenjiang, Jiangsu, 212013, People's Republic of China.
Virol J. 2017 Oct 3;14(1):190. doi: 10.1186/s12985-017-0852-z.
The aim of this study were to investigate the possible pro-apoptotic mechanisms of the recombinant Newcastle disease virus (NDV) strain rL-RVG, which expresses the rabies virus glycoprotein, in A549 lung adenocarcinoma cells via the regulation of alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) and to analyze the relationships between α7 nAChR expression in lung cancer and the clinical pathological features.
α7 nAChR expression in A549, LΑ795, and small-cell lung carcinoma (SCLC) cells, among others, was detected using reverse transcription polymerase chain reaction (RT-PCR). The optimal α7 nAChR antagonist and agonist concentrations for affecting A549 lung adenocarcinoma cells were detected using MTT assays. The α7 nAChR expression in A549 cells after various treatments was assessed by Western blot, immunofluorescence and RT-PCR analyses. Apoptosis in the various groups was also monitored by Western blot and TUNEL assays, followed by the detection of cell migration via transwell and scratch tests. Furthermore, α7 nAChR expression was examined by immunohistochemistry in lung cancer tissue samples from 130 patients and 40 pericancerous tissue samples, and the apoptotis in lung adenocarcinoma tissue was detected by Tunel assay, Then, the expression levels and clinicopathological characteristics were analyzed.
Of the A549, LΑ795, SCLC and U251 cell lines, the A549 cells exhibited the highest α7 nAChR expression. The cells infected with rL-RVG exhibited high RVG gene and protein expression. The rL-RVG group exhibited weaker α7 nAChR expression compared with the methyllycaconitine citrate hydrate (MLA, an α7 nAChR antagonist) and NDV groups. At the same time, the MLA and rL-RVG treatments significantly inhibited proliferation and migration and promoted apoptosis in the lung cancer cells (P < 0.05). The expression of α7 nAChR was upregulated in lung cancer tissue compared with pericancerous tissue (P = 0.000) and was significantly related to smoking, clinical tumor-node-metastases stage, and histological differentiation (P < 0.05). The AI in lung adenocarcinoma tissue in high-medium differentiation group was lower than that in low differentiation group (p < 0.01).
An antagonist of α7 nAChR may be used as a molecular target for lung adenocarcinoma therapy. Recombinant NDV rL-RVG enhances the apoptosis and inhibits the migration of A549 lung adenocarcinoma cells by regulating α7 nAChR signaling pathways.
本研究旨在通过调节α7 烟碱型乙酰胆碱受体（α7 nAChR）来探讨表达狂犬病病毒糖蛋白的重组新城疫病毒（NDV）株 rL-RVG 在 A549 肺腺癌细胞中诱导细胞凋亡的可能机制，并分析肺癌中α7 nAChR 表达与临床病理特征的关系。
采用逆转录聚合酶链反应（RT-PCR）检测 A549、LΑ795 和小细胞肺癌（SCLC）等细胞中α7 nAChR 的表达。用 MTT 法检测影响 A549 肺腺癌细胞的最佳α7 nAChR 拮抗剂和激动剂浓度。通过 Western blot、免疫荧光和 RT-PCR 分析检测 rL-RVG 处理后 A549 细胞中α7 nAChR 的表达。通过 Western blot 和 TUNEL 分析监测各组细胞凋亡，然后通过 Transwell 和划痕试验检测细胞迁移。此外，采用免疫组织化学法检测 130 例肺癌组织标本和 40 例癌旁组织标本中α7 nAChR 的表达，并采用 Tunel 法检测肺腺癌组织中的凋亡，然后分析其表达水平和临床病理特征。
在 A549、LΑ795、SCLC 和 U251 细胞系中，A549 细胞中α7 nAChR 的表达最高。感染 rL-RVG 的细胞表现出高 RVG 基因和蛋白表达。rL-RVG 组的α7 nAChR 表达弱于甲基氯化烟碱柠檬酸酯水合物（MLA，一种α7 nAChR 拮抗剂）和 NDV 组。同时，MLA 和 rL-RVG 处理明显抑制了肺癌细胞的增殖和迁移，促进了细胞凋亡（P<0.05）。与癌旁组织相比，肺癌组织中α7 nAChR 的表达上调（P=0.000），且与吸烟、临床肿瘤-淋巴结-转移分期和组织学分化显著相关（P<0.05）。中高分化组肺腺癌组织的 AI 低于低分化组（p<0.01）。
α7 nAChR 的拮抗剂可作为肺腺癌治疗的分子靶点。重组 NDV rL-RVG 通过调节α7 nAChR 信号通路增强 A549 肺腺癌细胞的凋亡，抑制其迁移。
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