Table_2_CRISPR/Cas9-Mediated Whole Genomic Wide Knockout Screening Identifies Specific Genes Associated With PM2.5-Induced Mineral Absorption in Liver Toxicity.XLSX

PM2.5, also known as fine particles, refers to particulate matter with a dynamic diameter of ≦2.5 μm in air pollutants, that carries metals (Zn, Co, Cd) which can pass through the alveolar epithelium and enter the circulatory system and tissues. PM2.5 can cause serious health problems, such as non-alcoholic fatty liver and hepatocellular carcinoma, although the underlying mechanisms of its toxic effect are poorly understood. Here, we exposed L02 cells to PM2.5 and performed a pooled genome−wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) to assess loss of function and identify new potential PM2.5targets. Enrichr and KEGG pathway analyses were performed to identify candidate genes associated with PM2.5 toxicity. Results revealed that four key genes, namely ATPase Na+/K+ transporting subunit alpha 2 (ATP1A2), metallothionein 1M (MT1M), solute carrier family 6 members 19 (SLC6A19) and transient receptor potential cation channel subfamily V member 6 (TRPV6) were associated with PM2.5 toxicity, mainly in regulating the mineral absorption pathway. Downregulating these genes increased cell viability and attenuated apoptosis in cells exposed to PM2.5. Conversely, overexpressing TRPV6 exacerbated cell apoptosis caused by PM2.5, while a reactive oxygen species (ROS) inhibitor N-acetyl-l-cysteine (NAC) alleviated PM2.5-induced apoptosis. In conclusion, ATP1A2, MT1M, SLC6A19 and TRPV6 may be contributing to absorption of metals in PM2.5 thereby inducing apoptosis mediated by ROS. Therefore, they hold potential as therapeutic targets for PM2.5-related diseases.

PM2.5（细颗粒物，fine particles）是指空气污染物中空气动力学直径≤2.5 μm的颗粒物，其携带的锌（Zn）、钴（Co）、镉（Cd）等金属可透过肺泡上皮屏障，进入循环系统与人体组织。PM2.5可引发多种严重健康问题，例如非酒精性脂肪肝与肝细胞癌，但其介导毒性作用的潜在分子机制目前仍未被完全阐明。本研究将L02细胞暴露于PM2.5环境中，并开展混合全基因组聚集性规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）筛选实验，以进行功能缺失性评估并识别潜在的PM2.5作用靶点。本研究通过Enrichr工具与京都基因与基因组百科全书（KEGG）通路富集分析，筛选与PM2.5毒性相关的候选基因。结果显示，ATP酶Na+/K+转运亚基α2（ATPase Na+/K+ transporting subunit alpha 2，ATP1A2）、金属硫蛋白1M（metallothionein 1M，MT1M）、溶质载体家族6成员19（solute carrier family 6 members 19，SLC6A19）以及瞬时受体电位阳离子通道亚家族V成员6（transient receptor potential cation channel subfamily V member 6，TRPV6）这4个关键基因与PM2.5毒性密切相关，其主要参与调控矿物质吸收通路。下调上述基因的表达可提升细胞存活率，并减轻PM2.5暴露诱导的细胞凋亡；反之，过表达TRPV6则会加重PM2.5引发的细胞凋亡。此外，活性氧（reactive oxygen species，ROS）抑制剂N-乙酰-L-半胱氨酸（N-acetyl-l-cysteine，NAC）可有效缓解PM2.5诱导的细胞凋亡。综上，ATP1A2、MT1M、SLC6A19与TRPV6可能通过参与PM2.5所载金属的吸收过程，进而介导活性氧依赖的细胞凋亡通路。因此，这些基因有望成为PM2.5相关疾病的潜在治疗靶点。