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中华肺部疾病杂志(电子版)

中华肺部疾病杂志(电子版) ›› 2024, Vol. 17 ›› Issue (04) : 512 -518. doi: 10.3877/cma.j.issn.1674-6902.2024.04.002

论著

过表达小GTP酶Rab32抑制非小细胞肺癌细胞侵袭性生长
赵蒙蒙1, 黄洁1, 余荣环1, 王葆青2,†()   
  1. 1. 200031　上海，上海市徐汇区中心医院，复旦大学附属中山医院徐汇医院呼吸与危重症医学科
    2. 200031　上海，上海市徐汇区中心医院，复旦大学附属中山医院徐汇医院呼吸与危重症医学科；200032　上海，复旦大学附属中山医院呼吸与危重症医学科
  • 收稿日期:2024-06-13 出版日期:2024-08-25
  • 通信作者: 王葆青
  • 基金资助:
    上海市徐汇区卫生系统高峰学科建设项目(SHXHZDXK202312)

Overexpression of the small GTPase Rab32 inhibits the invasive growth of non-small cell lung cancer cells

Mengmeng Zhao1, Jie Huang1, Ronghuan Yu1, Baoqing Wang2,†()   

  1. 1. Department of Respiratory Medicine, Shanghai Xuhui Central Hospital, Zhongshan-Xuhui Hospital, Fudan University, Shanghai 200031, China
    2. Department of Respiratory Medicine, Shanghai Xuhui Central Hospital, Zhongshan-Xuhui Hospital, Fudan University, Shanghai 200031, China; Department Respiratory Medicine, Zhongshan Hospital, Fudan University, Shanghai 200032, China
  • Received:2024-06-13 Published:2024-08-25
  • Corresponding author: Baoqing Wang
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赵蒙蒙, 黄洁, 余荣环, 王葆青. 过表达小GTP酶Rab32抑制非小细胞肺癌细胞侵袭性生长[J]. 中华肺部疾病杂志(电子版), 2024, 17(04): 512-518.

Mengmeng Zhao, Jie Huang, Ronghuan Yu, Baoqing Wang. Overexpression of the small GTPase Rab32 inhibits the invasive growth of non-small cell lung cancer cells[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2024, 17(04): 512-518.

目的

分析Rab32对非小细胞肺癌(non-small cell lung cancer, NSCLC)细胞增殖、迁移及侵袭的影响及作用机制；

方法

通过生物信息学判断NSCLC中Rab32的表达；用qRT-PCR和Western blot检测NSCLC细胞中Rab32 mRNA和蛋白表达水平；应用Rab32过表达慢病毒感染A549和H1299细胞，构建Rab32稳转A549和H1299细胞株为观察组，空载体慢病毒感染A549和H1299细胞后构建的为对照组；利用CCK-8和集落形成实验判断Rab32对NSCLC细胞影响增殖能力；通过划痕和Transwell实验检测Rab32对A549和H1299细胞迁移和侵袭能力的影响；利用Western blot检测不同组细胞中EMT标志物(E-cadherin、N-cadherin和Vimentin)蛋白的表达水平变化。

结果

通过生物信息学分析显示，肺腺癌(Lung adenocarcinoma, LUAD)和肺鳞癌(lung squamous cell carcinoma, LUSC)中Rab32的mRNA表达较对照组显著降低(P<0.05)；qRT-PCR及Western blot结果表明A549、H1299和HCC827中Rab32 mRNA和蛋白表达水平显著低于正常人支气管上皮细胞Beas-2B，其中A549和H1299细胞中Rab32 mRNA和蛋白较Beas-2B下降显著。A549、H1299细胞中，观察组的Rab32 mRNA和蛋白表达水平显著高于空载体对照组(P<0.05)。CCK-8和集落形成实验结果表明观察组的细胞增殖和克隆能力较对照组显著下降(P<0.05)；划痕和Transwell实验结果提示观察组细胞的迁移和侵袭能力明显下降(P<0.05)；Western blot结果显示观察组细胞的上皮表型标志物E-cadherin蛋白表达水平显著高于对照组，而间皮表型标志物N-cadherin和Vimentin表达较对照组明显升高(P<0.05)。

结论

Rab32抑制NSCLC细胞的增殖、迁移和侵袭，可作为潜在临床治疗NSCLC患者新途径。

关键词: 非小细胞肺癌, Rab32, 细胞增殖, 细胞迁移, 侵袭能力
Objective

To investigate the role and novel mechanism of Rab32 on the proliferation, migration, and invasion of non-small cell lung cancer (NSCLC) cells.

Methods

The expression of Rab32 in NSCLC was assessed using bioinformatics. The mRNA and protein expression levels of Rab32 in NSCLC cells were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis. Stable Rab32-overexpressing A549 and H1299 cell lines were constructed using a lentivirus system, with the overexpression group serving as the observation group. The effects of Rab32 on the proliferation of NSCLC cells were evaluated by CCK-8 and colony formation assays. The impact of Rab32 on the migration and invasion capabilities of A549 and H1299 cells was examined using scratch and Transwell assays. The expression levels of epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and Vimentin) in different groups of cells were measured by Western blot.

Results

Bioinformatics analysis indicated that the mRNA expression of Rab32 was significantly reduced in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) compared to the control group(P<0.05). qRT-PCR and Western blot results revealed that the mRNA and protein expression levels of Rab32 in A549, H1299, and HCC827 were significantly lower than those in normal human bronchial epithelial cells (Beas-2B), with more pronounced decreases in A549 and H1299 cells. In A549 and H1299 cells, the mRNA and protein expression levels of Rab32 in the observation group were significantly higher than those in the empty vector control group (P<0.05). The CCK-8 and colony formation assay results showed that the cell proliferation and cloning capacity of the observation group were significantly reduced compared to the control group (P<0.05). The scratch and Transwell assay results suggested a marked decrease in the migration and invasion capabilities of the observation group cells (P<0.05). Western blot analysis showed that the protein expression level of the epithelial phenotype marker E-cadherin in the observation group cells was significantly higher than that in the control group, while the expression of mesenchymal phenotype markers N-cadherin and Vimentin was markedly increased compared to the control group (P<0.05).

Conclusion

Rab32 inhibits the proliferation, migration, and invasion of NSCLC cells and may serve as a potential new avenue for the clinical treatment of NSCLC patients.

Key words: Non-small cell lung cancer, Rab32, Cell proliferation, Cell migration, Invasive capacity
图1 Rab32在NSCLC中表达情况。注：A：基于UCSC Xena数据库分析Rab32在LUAD和LUSC类型NSCLC及对照组中表达水平；B：qPCR检测Rab32 mRNA在正常起到上皮细胞Beas-2B和NSCLC细胞HCC827、H1299、A549中的表达水平；C: Western blot检测Beas-2B、HCC827、H1299和A549细胞中Rab32蛋白的表达水平。***：P<0.001
图2 获得稳定过表达Rab32的A549和H1299细胞。注：A：Western blot分别检测A549观察组和A549对照组、H1299观察组和H1299对照组中Rab32蛋白的表达水平；B：qPCR分别检测A549观察组和、A549对照组(左侧图)、H1299观察组和H1299对照组中Rab32 mRNA的表达水平。***：P<0.001
图3 Rab32显著抑制A549、H1299细胞增殖能力。注：A：CCK-8分别检测A549观察组和A549对照组(左侧图)、H1299观察组和H1299对照组(右侧图)的细胞增殖情况；B：克隆形成实验判断A549观察组和A549对照组、H1299观察组和H1299对照组克隆能力。左侧图为代表性图片，右侧图为克隆形成数目的统计分析。***：P<0.001
图4 Rab32抑制A549、H1299细胞迁移及侵袭。注：A：划痕实验A549观察组和A549对照组、H1299观察组和H1299对照组的细胞迁移情况。左图代表性图片，右图细胞迁移率统计分析；B：Transwell实验A549观察组和A549对照组、H1299观察组和H1299对照组细胞侵袭情况。左侧图为代表性图片，右侧图为细胞侵袭能力半定量的统计分析。***：P<0.001
图5 Rab32抑制对A549、H1299细胞迁移及侵袭。注：A：Western blot检测A549观察组和A549对照组、H1299观察组和H1299对照组中E-cadherin(E-cad)、N-cadherin(N-cad)、Vimentin(Vim)的蛋白表达水平；B：对A图的半定量统计分析。***, P<0.001
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