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Methylation status, mRNA and protein expression of the SMAD4 gene in patients with non-melanocytic skin cancers

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Abstract

Background

SMAD4 is a potent tumor suppressor. SMAD4 loss increases genomic instability and plays a critical role in the DNA damage response that leads to skin cancer development. We aimed to investigate SMAD4 methylation effects on mRNA and protein expression of SMAD4 in cancer and healthy tissues from patients with basal cell carcinoma (BCC), cutaneous squamous cell carcinoma (cSCC), and basosquamous skin cancer (BSC).

Methods and results

The study included 17 BCC, 24 cSCC and nine BSC patients. DNA and RNA were isolated from cancerous and healthy tissues following punch biopsy. Methylation-specific polymerase chain reaction (PCR) and real-time quantitative PCR methods were used to examine SMAD4 promoter methylation and SMAD4 mRNA levels, respectively. The percentage and intensity of staining of the SMAD4 protein were determined by immunohistochemistry. The percentage of SMAD4 methylation was increased in the patients with BCC (p = 0.007), cSCC (p = 0.004), and BSC (p = 0.018) compared to the healthy tissue. SMAD4 mRNA expression was decreased in the patients with BCC (p˂0.001), cSCC (p˂0.001), and BSC (p = 0.008). The staining characteristic of SMAD4 protein was negative in the cancer tissues of the patients with cSCC (p = 0.00). Lower SMAD4 mRNA levels were observed in the poorly differentiated cSCC patients (p = 0.001). The staining characteristics of the SMAD4 protein were related to age and chronic sun exposure.

Conclusions

Hypermethylation of SMAD4 and reduced SMAD4 mRNA expression were found to play a role in the pathogenesis of BCC, cSCC, and BSC. A decrease in SMAD4 protein expression level was observed only in cSCC patients. This suggests that epigenetic alterations to the SMAD4 gene are associated with cSCC.

Trial Registration

The name of the trial register: SMAD4 Methylation and Expression Levels in Non-melanocytic Skin Cancers; SMAD4 Protein Positivity.

The registration number: NCT04759261 (https://clinicaltrials.gov/ct2/results?term=NCT04759261).

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Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files.

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Funding

This study was supported by the Trakya University Research Project Fund (2020/55).

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Authors and Affiliations

  1. Department of Dermatology and Venereology, Faculty of Medicine, Trakya University, Edirne, Turkey

    Yıldız Gürsel Ürün

  2. Department of Biophysics, Faculty of Medicine, Trakya University, Edirne, Turkey

    Metin Budak

  3. Department of Pathology, Faculty of Medicine, Trakya University, Edirne, Turkey

    Elif Usturalı Keskin

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  1. Yıldız Gürsel Ürün
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Contributions

YGÜ and MB designed the research study. MB and EUK performed the research. YGÜ and MB carried out the data analysis and writing—review and editing. All the authors discussed the results and contributed to the final manuscript.

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Correspondence to Yıldız Gürsel Ürün.

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The authors agree that there are no conflicts of interest to declare.

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Approval for the study was granted by the Scientific Research Ethics Committee of the Trakya University Faculty of Medicine (approval number: 18/14, date: 06/11/2019).

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All the subjects who agreed to participate in the study gave their written informed consent.

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Gürsel Ürün, Y., Budak, M. & Usturalı Keskin, E. Methylation status, mRNA and protein expression of the SMAD4 gene in patients with non-melanocytic skin cancers. Mol Biol Rep 50, 7295–7304 (2023). https://doi.org/10.1007/s11033-023-08656-2

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