P53 expression, Genome-wide transcriptome profiling and LGS test (a blood test to detect cancer): comparison of UVA exposed lymphocytes from malignant melanoma patients and healthy controls.

Abstract

This study aimed to evaluate the expression of the P53 gene following exposure to varying doses of UVA radiation, using lymphocytes as surrogates. Lymphocytes from malignant melanoma (MM) patients (n = 20) with positive sentinel nodes were compared to healthy controls (HC)(volunteers) (n = 20). These samples were processed by Comet assay following the Lymphocyte Genome Sensitivity (LGS) test, quantitative real-time Polymerase Chain Reaction (qPCR), western blotting and whole genome transcriptome profiling. LGS test evaluates the level of alterations in lymphocytes resulting from continuous exposure to various physical and chemical insults in the blood, promoting DNA damage, ultimately leading to oxidative stress. It is believed that in cancer, the circulatory tumour cells, exosomes and cytokines impact peripheral lymphocytes. The Comet assay performed within the LGS test indicated a significant difference between the lymphocytes from two groups of HC and MM patients. The qPCR data demonstrated an overall 43.8-fold increase in TP53 gene expression in lymphocytes from MM patients after treatment with 0.2mW/cm2 UVA intensity radiation, compared to healthy and untreated controls. Western blotting was used to confirm gene expression. The p53 protein expression was significantly increased in MM lymphocytes after UVA exposure compared to healthy individuals (p-value < 0.05). The genome transcriptome profiling data also displayed differences in gene expression between the UV-treated lymphocytes from healthy groups as compared to melanoma samples. Nine out of the 23 (~ 40%) genes displaying differences in gene expression were mitochondrial genes, which were increased in lymphocytes from MM compared to HCs. The genes that play an important role in oxidative phosphorylation, such as MT-CYB, MT-CO2, MT-ND2, MT-ND6 and MTRNR2L12, were upregulated in lymphocytes from MM patients compared to HCs. The down-regulated genes in lymphocytes from MM, such as MYH9, RN7SL2, ACTB, AHNAK and FLNA, are related to cell structure, migration and tumour metastasis. Peripheral lymphocytes from MM patients are more sensitive and susceptible to the genotoxic effects of UVA compared to healthy individuals. Our previous studies showed that UVA exposure in various intensities distinguishes differences in the level of DNA damage between lymphocytes from cancer patients compared to HCs through the LGS test. The current results provide further credibility to the LGS assay as a screening test for detecting cancer. This feature could be a promising blood biopsy biomarker for staging and preventing carcinomas at early stages.