■犬传染性性病肿瘤(CTVT),一种独特的狗传染性癌症,会影响外生殖器,并可能扩散到身体的其他部位。虽然致癌和肿瘤抑制基因的体细胞突变与CTVT的发展有关,DNA甲基化的影响,影响基因表达,尚不清楚。这项研究探讨了CTVT中MYC癌基因和CDKN2B抑癌基因启动子区域的DNA甲基化是否与其表达相关。在基因和蛋白质水平。
■为了研究CTVT中MYC和CDKN2B的启动子DNA甲基化,我们分析了生殖器CTVT(GTVT)和生殖器外CTVT(ETVT)的冷冻组织样本.提取基因组DNA,亚硫酸氢盐处理,并使用亚硫酸氢盐聚合酶链反应(PCR)和测序进行分析。还提取了MYC和CDKN2B的信使RNA和蛋白质,并通过实时PCR和Western印迹进行了评估。匹配福尔马林固定,石蜡包埋块用于免疫组织化学染色,以显示GTVT和ETVT组织中的蛋白分布.
■尽管GTVT和ETVT样本均显示MYC启动子甲基化,甲基化程度差异显著。GTVT表现出更高的甲基化程度,与ETVT相比,在GTVT中观察到的MYC基因表达的下调和c-MYC蛋白水平的降低可能解释。我们的数据揭示了两种样品类型的CDKN2B启动子中普遍存在的超甲基化模式。然而,DNA甲基化,预期会有抑制作用,与基因/蛋白质表达无关。尽管CDKN2B表达显著降低,GTVT仍显示出高水平的蛋白。相反,ETVT维持CDKN2B正常表达,但蛋白产量减少,提示在这些肿瘤中甲基化和表达之间复杂的相互作用。
■MYC证明了其启动子甲基化状态之间的明确关联,基因表达,和蛋白质水平;然而,CDKN2B缺乏这种相关性,这意味着甲基化非依赖性调节机制的参与,并强调需要进一步研究。
UNASSIGNED: Canine transmissible venereal tumor (CTVT), a unique transmissible cancer in dogs, affects the external genitalia and potentially spreads to other parts of the body. While somatic mutations in oncogenic and tumor-suppressing genes are linked to CTVT development, the impact of DNA methylation, which affects gene expression, remains unclear. This study explored whether DNA methylation in the promoter regions of the
MYC oncogene and CDKN2B tumor suppressor genes in CTVTs is associated with their expression, both at the gene and protein levels.
UNASSIGNED: To investigate promoter DNA methylation of
MYC and CDKN2B in CTVTs, we analyzed frozen tissue samples from genital CTVT (GTVTs) and extragenital CTVT (ETVTs). Genomic DNA was extracted, bisulfite-treated, and analyzed using bisulfite polymerase chain reaction (PCR) and sequencing. The messenger RNA and protein of
MYC and CDKN2B were also extracted and assessed by real-time PCR and Western blotting. Matching formalin-fixed, paraffin-embedded blocks were used for immunohistochemical staining to visualize protein distribution in GTVT and ETVT tissues.
UNASSIGNED: Although both GTVT and ETVT samples showed
MYC promoter methylation, the extent of methylation differed significantly. GTVTs displayed a much higher degree of methylation, potentially explaining the more pronounced downregulation of MYC gene expression and reduction in c-
MYC protein levels observed in GTVTs compared with ETVTs. Our data revealed a prevalent hypermethylation pattern in the CDKN2B promoter across both sample types. However, DNA methylation, which was expected to have a suppressive effect, did not correlate with gene/protein expression. GTVTs displayed high protein levels despite significantly reduced CDKN2B expression. Conversely, ETVTs maintained regular CDKN2B expression but exhibited reduced protein production, suggesting a complex interplay between methylation and expression in these tumors.
UNASSIGNED: MYC demonstrated a clear association between its promoter methylation status, gene expression, and protein levels; however, CDKN2B lacked this correlation, implying the involvement of methylation-independent regulatory mechanisms and highlighting the need for further investigation.