亚砷酸钠调控A549细胞FNDC3B表达的分子机制及其促癌功能

Molecular mechanisms and cancer-promoting roles of sodium arsenite in regulating FNDC3B expression in A549 cells

  • 摘要:
    背景 砷暴露引起细胞凋亡。含纤维连接蛋白Ⅲ型结构域3B蛋白基因(FNDC3B)可促进癌细胞增殖,但其在砷的致凋亡中尚未研究。
    目的 探讨亚砷酸钠(NaAsO2)及其代谢产物对A549细胞中FNDC3B基因表达的影响以及了解FNDC3B基因在A549细胞中的功能。
    方法 (1)A549细胞暴露于不同终浓度的NaAsO2中,48 h后通过细胞计数试剂盒-8(CCK-8)测450 nm波长处的光密度值,绘制存活率曲线,根据存活率选择最终的暴露剂量。使A549细胞暴露于高浓度(30 µmol·L−1)、中浓度(20 µmol·L−1)、低浓度(10 µmol·L−1)NaAsO2、高浓度(30 µmol·L−1)一甲基胂酸(MMA)、高浓度(30 µmol·L−1)二甲基胂酸(DMA)48 h后提取总蛋白和RNA,采用定量实时聚合酶链式反应(qRT-PCR)和蛋白质印迹法(WB)检测FNDC3B基因的mRNA表达和蛋白表达情况,采用免疫共沉淀(CO-IP)和WB实验检测FNDC3B蛋白泛素化表达情况。(2)采用siRNA干扰方法敲低A549细胞中FNDC3B基因的表达。si-FNDC3B片段转染A549细胞48 h后,采用qRT-PCR和WB法检测FNDC3B基因的mRNA和蛋白的表达情况,利用CCK-8实验检测细胞活力,Hoechest33342/碘化丙啶(PI)双重染色法和JC-1线粒体膜电位实验检测细胞早期及晚期凋亡情况,WB检测cleaved caspase3蛋白和P53信号通路相关蛋白表达。
    结果 (1)CCK-8结果发现随着NaAsO2浓度的增加,A549细胞活力下降,半数抑制浓度为38.12 µmol·L−1。qRT-PCR结果显示,与对照组相比,不同浓度NaAsO2(10、20 和 30 µmol·L−1) 均显著升高FNDC3B基因的mRNA表达(P<0.01),MMA和DMA对FNDC3B基因的mRNA表达影响差异无统计学意义(P>0.05);WB结果显示,与对照组相比,NaAsO2处理组FNDC3B基因的蛋白表达下降,FNDC3B蛋白泛素化表达水平升高,FNDC3B蛋白泛素化K48位点泛素化表达水平升高;MMA和DMA对FNDC3B基因的蛋白表达无影响。(2)特异性敲低A549细胞中FNDC3B的表达后,CCK-8实验显示与对照组相比,沉默FNDC3B组(si-FNDC3B组)细胞活力显著降低;JC-1结果显示si-FNDC3B组线粒体膜电位低于对照组;Hoechst33342/PI染色实验结果显示si-FNDC3B组细胞呈现出明显的凋亡。WB结果显示,与对照组比较,si-FNDC3B组cleaved caspase3、P53、MDM2、Bad、Bax蛋白相对表达上升。
    结论 NaAsO2促进FNDC3B蛋白泛素化的表达,进而降低FNDC3B蛋白的表达,但其主要代谢产物DMA和MMA对FNDC3B蛋白表达无影响;沉默FNDC3B后抑制A549细胞的活力和促进细胞的凋亡,该现象与激活P53信号通路有关。

     

    Abstract:
    Background Arsenic exposure has been demonstrated to induce apoptosis. The fibronectin type III structural domain 3B protein (FNDC3B) has been shown to promote cancer cell proliferation; however, its role in arsenic-induced apoptosis remains to be elucidated.
    Objective To investigate the effects of sodium arsenite (NaAsO2) and its metabolites on the expression of FNDC3B gene in A549 cells and to understand the function of FNDC3B gene in A549 cells.
    Methods (1) A549 cells were exposed to varying final concentrations of NaAsO2 and their optical density at 450 nm values were measured by Cell Counting Kit-8 (CCK-8) after 48 h. Survival curves were plotted, and a final exposure dose was selected according to the survival rate. Total protein and RNA were extracted by exposing A549 cells to high (30 µmol·L−1), medium (20 µmol·L−1), and low (10 µmol·L−1) NaAsO2 concentrations, high (30 µmol·L−1) monomethylarsinic acid (MMA), and high (30 µmol·L−1) dimethylarsinic acid for a period of 48 h. mRNA expression and the protein expression of the FNDC3B gene was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB), while the protein ubiquitination expression of the FNDC3B gene was detected by co-immunoprecipitation (CO-IP) and WB assay. (2) Knockdown of FNDC3B gene expression was achieved in A549 cells by siRNA interference. The si-FNDC3B fragment was transfected in A549 cells for 48 h. The mRNA and protein expression of FNDC3B gene was then detected by qRT-PCR and WB assay. Cell viability was determined through CCK-8 assay. Hoechst 33342/propidium iodide (PI) double staining and JC-1 mitochondrial membrane potential assay were employed to detect both early and late apoptosis, while cleaved caspase3 protein and P53 signalling pathway related protein expressions were evaluated by WB.
    Results (1) The CCK-8 results demonstrated a decline in the viability of A549 cells with an increase in NaAsO2 concentration, with an inhibitory concentration at 50% of 38.12 µmol·L−1. The qRT-PCR results demonstrated that compared to the control group, varying concentrations of NaAsO₂ (10, 20, and 30 µmol·L⁻¹) significantly upregulated the mRNA expression of FNDC3B gene (P<0.01). In contrast, MMA and DMA showed no significant effect on FNDC3B mRNA expression (P>0.05). The WB analysis revealed that the protein expression of FNDC3B was reduced in the NaAsO₂-treated group compared to the control, accompanied by elevated ubiquitination levels of FNDC3B protein, particularly at the K48 ubiquitination site. MMA and DMA exhibited no impact on FNDC3B protein expression. (2) Following the specific knockdown of FNDC3B expression in A549 cells, the CCK-8 assay demonstrated a significant reduction in cell viability in the silenced FNDC3B group (si-FNDC3B) compared to the control group. The JC-1 assay demonstrated that the mitochondrial membrane potential was diminished in the si-FNDC3B group relative to the control group. The Hoechst 33342/PI staining assay revealed that the si-FNDC3B group exhibited a notable degree of apoptosis. The si-FNDC3B group also displayed substantial apoptosis. The WB analysis indicated that the relative expressions of cleaved caspase3, P53, MDM2, Bad, and Bax proteins were elevated in the si-FNDC3B group in comparison to the control group.
    Conclusion The presence of NaAsO2 is observed to promote the ubiquitination expression of the FNDC3B protein, which in turn reduces the expression of FNDC3B protein. However, the main metabolites DMA and MMA have no effect on the expression of FNDC3B. Furthermore, the silencing of FNDC3B is observed to inhibit the viability of A549 cells and promote apoptosis, a phenomenon related to the activation of P53 signaling pathway.

     

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