Cloning of the XPD gene and its function in malignant melanoma cells
- Yue Wang
- Youyou Zhou
- Yanfu Wang
- Cong Peng
- Mingyang Gao
Affiliations: Department of Dermatology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, P.R. China, Department of Dermatology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China
- Published online on: June 9, 2020 https://doi.org/10.3892/ol.2020.11708
Copyright: © Wang
et al. This is an open access article distributed under the
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The xeroderma pigmentosum group D (XPD) gene is a member of the transcription factor IIH complex and serves an important role in gene repair. Previous studies have suggested that genetic variants of the XPD gene may be associated with an increased risk of cutaneous melanoma. However, the exact mechanism remains unclear. In the present study, the XPD gene was cloned, and its localization and function in malignant melanoma cells were investigated. The human full length XPD gene was cloned via reverse transcription‑PCR using the total RNA extracted from human cervical squamous cell carcinoma epithelial HeLa cells. Subsequently, the gene was inserted into a plasmid fused to green fluorescent protein (GFP; pEGFP‑N1/XPD), and pEGFP‑N1/XPD and pcDNA3.1(+)/XPD were transfected into human malignant melanoma A375 cells using Lipofectamine® 2000. The expression levels of XPD were detected by western blotting. The Golgi marker GM130 and the endoplasmic reticulum membrane protein marker KDEL were used for immunofluorescence staining, and the subcellular localization of XPD was observed under a fluorescence microscope. Cell proliferation was measured using an MTT assay. The recombinant pEGFP‑N1/XPD plasmid expressing the human wild‑type XPD gene was successfully constructed by restriction enzyme digestion and assessed by gene sequencing. XPD was localized in the endoplasmic reticulum of malignant melanoma A375 cells, as confirmed by immunofluorescence staining. Furthermore, MTT assays indicated that XPD inhibited the proliferation of malignant melanoma A375 cells. The present study provides a basis for further investigation of the biological effects and functions of XPD in malignant melanoma cells.