<i>circCCDC138</i>在炭黑纳米粒子诱导人肺上皮细胞早期恶性转化中的作用

李润锋; 马丽纯; 覃舒琳; 刘雯

doi:10.11836/JEOM24393

circCCDC138在炭黑纳米粒子诱导人肺上皮细胞早期恶性转化中的作用

Role of circCCDC138 in early malignant transformation of human lung epithelial cells induced by carbon black nanoparticles

摘要

摘要:
背景随着炭黑纳米粒子（CBNPs）的大规模生产和应用，职业和人群暴露呈明显上升趋势。相关研究表明，CBNPs暴露会诱导氧化应激、炎症和DNA损伤。
目的构建CBNPs暴露诱导的人肺上皮细胞（BEAS-2B）恶性转化细胞（C-BEAS-2B）模型，探究circCCDC138在CBNPs诱导BEAS-2B细胞恶性转化过程中的作用和机制。
方法设置0、10、20、40、80 μg·mL⁻¹ CBNPs染毒剂量，采用CCK8试验检测细胞活力；将BEAS-2B细胞暴露于20 mg·mL⁻¹ CBNPs 3个月，构建CBNPs诱导的BEAS-2B细胞恶性转化模型；采用细胞划痕和Transwell实验检测细胞的迁移和侵袭能力；采用定量PCR检测BEAS-2B细胞和C-BEAS-2B细胞中circCCDC138的表达量，并通过消化抗性试验验证其稳定性；构建干扰、过表达circCCDC138的细胞模型，采用定量PCR检测细胞中circCCDC138的表达量，通过流式细胞仪测定细胞周期和细胞凋亡情况；采用Western blot分析相关细胞中p53蛋白的表达量。
结果电镜下CBNPs为球形颗粒，呈现链状结构。20 μg·mL⁻¹ CBNPs染毒条件下BEAS-2B活力的降低相对较小（10%）。与对照组细胞相比，20 μg·mL⁻¹ CBNPs暴露组在24 h、48 h表现出更明显的细胞迁移和侵袭能力，表明CBNPs暴露诱导了BEAS-2B细胞早期恶性转化（P<0.01）。测定暴露于CBNPs后0、7、15、30 d的C-BEAS-2B中circCCDC138的表达量，提示circCCDC138上调且呈时间依赖性增加。与C-BEAS-2B细胞相比，过表达circCCDC138的C-BEAS-2B细胞表现出S期进展受阻（36.9%）与凋亡抗性增加（P<0.01），同时细胞中p53蛋白表达下调（P<0.01），而干扰circCCDC138的C-BEAS-2B细胞则出现相反的结果（P<0.01）。
结论持续暴露于CBNPs（20 μg·mL⁻¹）的BEAS-2B细胞具有显著增强的迁移和侵袭能力，表现出早期恶性转化特性。circCCDC138在C-BEAS-2B细胞中呈高表达，具有RNase R消化抗性，并随CBNPs暴露呈时间依赖性增加且可能通过抑制p53蛋白表达来促进细胞恶性转化。

Abstract:
Background With the large-scale production and application of carbon black nanoparticles (CBNPs), occupational and general exposure is obviously increasing. Related studies have shown that exposure to CBNPs can induce oxidative stress, inflammation, and DNA damage.
Objective To establish a CBNPs-induced malignant transformation (C-BEAS-2B) model of human lung epithelial cells (BEAS-2B) and explore the role and mechanism of circCCDC138 in the malignant transformation process.
Methods At 0, 10, 20, 40 and 80 μg·mL⁻¹ CBNPs concentrations, cell viability was detected by CCK8 assay. BEAS-2B cells were exposed to 20 mg·mL⁻¹ CBNPs for three months, and a malignant transformation model of BEAS-2B induced by CBNPs was constructed. The migration and invasion abilities of the cells were detected by cell scratch and Transwell assays. The expressions of circ-CCDC138 in BEAS-2B and C-BEAS-2B were detected by qRT-PCR, and its stability was verified by a digestive resistance test. A cell model with interference or overexpression of circCCDC138 was constructed, and the expression of circCCDC138 in the cells was detected by quantitative reverse transcription-PCR. The cell cycle and apoptosis were determined by flow cytometry. Western blot was used to analyze the expression of p53 protein.
Results The CBNPs used in the experiment were spherical particles with a chain-like structure. In the 20 μg·mL⁻¹ CBNPs group, the reduction in the viability of BEAS-2B cells was relatively small (10%). Compared with the control cells, the 20 μg·mL⁻¹ CBNPs group showed more obvious cell migration and invasion at 24 h and 48 h, indicating that the exposure to CBNPs induced early malignant transformation of BEAS-2B cells (P<0.01). The circCCDC138 expression in C-BEAS-2B was upregulated in a time-dependent manner after exposure to CBNPs. Compared with the C-BEAS-2B cells, the C-BEAS-2B cells over-expressing circCCDC138 exhibited arrested S phase progression (36.9%) and apoptosis resistance (P<0.01), along with down regulation of p53 protein expression in the cells (P<0.01), while the C-BEAS-2B cells interfering with circCCDC138 showed the opposite results (P<0.01).
Conclusion BEAS-2B cells exposed to CBNPs (20 μg·mL⁻¹) have significantly enhanced migration and invasion abilities, showing early malignant transformation characteristics. In addition, circCCDC138 is highly expressed in C-BEAS-2B cells with RNase R digestive resistance and increases in a time-dependent manner with CBNPs exposure. More importantly, circCCDC138 may promote the induction of malignant transformation of cells by inhibiting p53 protein expression.

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