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Abstract <p>Tyrosinase-related protein 1 (TRP-1) has been known to play an important role in melanogenesis. While we had studied TRP-1 gene knockout using the CRISPR/Cas9 system, it was found that <i>TRP-1</i> gene knockout could induce apoptosis of human fibrosarcoma cells (HT1080). The purpose of this study was to investigate the effect of <i>TRP-1</i> knockout on the induction of apoptosis in HT1080 cells. The <i>TRP-1</i> KO cells were successfully established using the CRISPR/Cas9 system, and the frameshift mutation was verified by the Sanger DNA sequencing analysis. The modified <i>TRP-1</i> structure by frameshift mutation caused a decrease in gene and protein expression in <i>TRP-1</i> KO cells. The <i>TRP-1</i> KO cells were phenotypically changed and induced apoptosis. The flow cytometry analysis showed the early apoptosis phase. Furthermore, the <i>TRP-1</i> KO cells decreased the scavenging activity of hydrogen peroxide, compared to normal cells. DNA fragmentation and TUNEL assays confirmed the apoptotic cell death in these cells. The expression levels of proteins such as Caspase 3, Bax, and Bcl-2 were positively modulated in the apoptosis induction of <i>TRP-1</i> KO cells in a mitochondrial-dependent pathway. The expression levels of Catalase, SOD1, and SOD2 were remarkably decreased in the these cells, compared to normal cells. The expression levels of MAPKs kinase such as p38, ERK1/2, JNK, and NF-κB related to cell proliferation were also reduced in the <i>TRP-1</i> KO cells. Therefore, our findings suggest that TRP-1 could play an important role in apoptosis in addition to melanin production.</p>

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Tyrosinase-related Protein 1 Gene Knockout by CRISPR/Cas9 System Induces Apoptosis in Human Fibrosarcoma Cells

  • 期刊论文

  • 发表日期：

    2025年1月20日

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  • Natchanok Talapphet,
  • Moon-Moo Kim

摘要

Abstract

Tyrosinase-related protein 1 (TRP-1) has been known to play an important role in melanogenesis. While we had studied TRP-1 gene knockout using the CRISPR/Cas9 system, it was found that TRP-1 gene knockout could induce apoptosis of human fibrosarcoma cells (HT1080). The purpose of this study was to investigate the effect of TRP-1 knockout on the induction of apoptosis in HT1080 cells. The TRP-1 KO cells were successfully established using the CRISPR/Cas9 system, and the frameshift mutation was verified by the Sanger DNA sequencing analysis. The modified TRP-1 structure by frameshift mutation caused a decrease in gene and protein expression in TRP-1 KO cells. The TRP-1 KO cells were phenotypically changed and induced apoptosis. The flow cytometry analysis showed the early apoptosis phase. Furthermore, the TRP-1 KO cells decreased the scavenging activity of hydrogen peroxide, compared to normal cells. DNA fragmentation and TUNEL assays confirmed the apoptotic cell death in these cells. The expression levels of proteins such as Caspase 3, Bax, and Bcl-2 were positively modulated in the apoptosis induction of TRP-1 KO cells in a mitochondrial-dependent pathway. The expression levels of Catalase, SOD1, and SOD2 were remarkably decreased in the these cells, compared to normal cells. The expression levels of MAPKs kinase such as p38, ERK1/2, JNK, and NF-κB related to cell proliferation were also reduced in the TRP-1 KO cells. Therefore, our findings suggest that TRP-1 could play an important role in apoptosis in addition to melanin production.