PFNA 유발 복제 스트레스와 p53 의존 G1기 세포주기 정지 기전 분석 = Analysis of PFNA-induced replication stress and p53-dependent G1 cell cycle arrest mechanisms

다국어 초록 (Multilingual Abstract)

Per- and Polyfluoroalkyl Substances (PFAS) are characterized by strong carbon-fluorine bonds, which grant them high chemical stability and heat resistance. Since the 1940s, PFAS have been widely used in various industrial products such as food packaging, water-repellent coatings, and firefighting foams. However, PFAS are highly persistent in the environment and within organisms, causing accumulation in soil, water systems, wildlife, and human blood and tissues. Continuous exposure to these substances has been linked to multiple health risks including immunosuppression, endocrine disruption, liver toxicity, developmental toxicity, reproductive impairment, and increased cancer risk. Despite this, the molecular toxic mechanisms of Perfluorononanoic acid (PFNA) on lung epithelial cells remain unclear, highlighting the need for further lung toxicity assessments.
In this study, RNA sequencing was performed to investigate transcriptomic changes induced by PFNA in the human lung epithelial cell line A549. Differentially expressed genes (DEGs) were identified and analyzed using KEGG (Kyoto Encyclopedia of Genes and Genomes) and IPA (Ingenuity Pathway Analysis). The analysis revealed alterations in pathways related to the cell cycle, DNA replication, and cellular stress responses. These findings demonstrate that PFNA induces cell cycle arrest following DNA replication inhibition due to cellular stress, accompanied by changes in the expression of related genes. To determine the specific cell cycle phase affected, Propidium Iodide (PI) staining and flow cytometry were conducted. As a result, The proportion of cells in the G1 phase was significantly increased (13.11 ± 0.88%) by PFNA treatment, confirming G1 phase cell cycle arrest.
Further, The expression changes of DNA damage response genes—such as p53, CDKN1A, GADD45A, and MDM2—and of genes in the p53 signaling pathway were detected by qPCR analysis. Additionally, Alterations were observed in key regulators of the G1 phase cell cycle control, including p21, cyclin, and CDK. These findings indicate that PFNA affects the p53-p21-pRb-E2F cell cycle regulatory pathway.
In conclusion, The effect of PFNA, a toxic environmental PFAS compound, on lung cell cycle arrest was specifically elucidated, and the underlying mechanism of this arrest was systematically characterized.

목차 (Table of Contents)

• 1. INTRODUCTION 3
• 2. MATERIALS AND METHODS 7
• 2.1. Materials 7
• 2.2. Cell culture 7
• 2.3. PFNA treatment 7
• 2.4. RNA extraction and transcriptome analysis 8
• 2.4.1. RNA extraction and precipitation 8
• 2.4.2. Library preparation and sequencing 9
• 2.4.3. Data processing and analysis 9
• 2.5. Cell cycle analysis 10
• 2.6. Quantitative real-time PCR 10
• 2.7. Pathway and functional analysis 11
• 2.8. Statistical analysis 11
• 3. RESULT 12
• 3.1. Effects of PFNA Treatment on A549 Cell Viability and morphological Characteristics 12
• 3.2. PFNA-Induced Transcriptomic and Pathway Changes 12
• 3.3. Cell Cycle Arrest Location in A549 Cells Following PFNA Treatment: Flow Cytometric Analysis with PI Staining 14
• 3.4. Differential Expression of p53 Target Genes Following PFNA Treatment (qPCR Analysis) 15
• 3.5. Suppression of Cell Cycle-Related E2F Target Genes by PFNA 16
• 3.6. Validation of RNA-seq Data by qPCR and Correlation Analysis 17
• 4. DISCUSSION 19
• 5. CONCLUSION 23
• REFERENCES 25
• TABLES 30
• FIGURES 34
• LEGEND OF FIGURES 46
• ABSTRACT (KOREAN) 50