Zaravinos Apostolos, Kanellou Peggy, Baritaki Stavroula, Bonavida Benjamin, Spandidos Demetrios A
Laboratory of Virology, Medical School, University of Crete, Heraklion, Crete, Greece.
Cell Cycle. 2009 May 1;8(9):1402-8. doi: 10.4161/cc.8.9.8308. Epub 2009 May 26.
Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). However, little is known about the implication of BRAF and RKIP expression, or about the incidence of BRAF mutations in the formation of these cutaneous diseases. The RAS oncogene has been proposed to significantly contribute to skin cancer development. Moreover, numerous BRAF mutations have been detected in melanoma biopsy specimens and cell lines.
This study aimed to measure the mRNA levels of the genes BRAF and RKIP in AK, as well as their possible implication in the progress of AK to SCC. All biopsy specimens were also screened for BRAF mutations within exons 11 and 15.
Significant downregulation was noted for both genes in SCC, compared to normal tissue (for BRAF, p = 0.002; for RKIP, p < 0.001). RKIP expression levels were significantly higher than the corresponding levels of BRAF (p < 0.001), whereas the two genes showed a negative correlation not only in AK and SCC, but in the adjacent phenotypically normal skin tissue, as well. No mutation was detected, either in AK or SCC, within exons 11 and 15 of the BRAF gene.
Expression levels of the genes BRAF and RKIP were examined in 16 AKs and 12 SCCs by RT-qPCR. A novel allele-specific qPCR method, in combination with direct DNA sequencing, was performed in order to inspect the frequency of the V600E mutation in exon 15, as well as to examine the mutation status of the gene within exon 11.
Both BRAF and RKIP expression levels exhibit a decrease from normal skin tissue and AK, going to SCC. The decrease of RKIP mRNA levels in SCC, suggests one novel mechanism in the deregulation of the BRAF signaling pathway. Our results further indicate that BRAF does not appear to be frequently mutated either in pre-cancerous skin lesions (AK) or in non-melanoma skin tumors (SCC).
光化性角化病（AK）是一种公认的癌前皮肤病变，有发展为鳞状细胞癌（SCC）的可能。然而，关于BRAF和RKIP表达的意义，或BRAF突变在这些皮肤疾病形成中的发生率，人们了解甚少。RAS癌基因已被认为对皮肤癌的发展有显著作用。此外，在黑色素瘤活检标本和细胞系中已检测到许多BRAF突变。
本研究旨在测量AK中BRAF和RKIP基因的mRNA水平，以及它们在AK发展为SCC过程中的可能作用。所有活检标本还进行了第11和15外显子内BRAF突变的筛查。
与正常组织相比，SCC中这两个基因均有显著下调（BRAF，p = 0.002；RKIP，p < 0.001）。RKIP表达水平显著高于相应的BRAF水平（p < 0.001），而这两个基因不仅在AK和SCC中呈负相关，在相邻的表型正常皮肤组织中也是如此。在BRAF基因的第11和15外显子中，无论是AK还是SCC均未检测到突变。
通过RT-qPCR检测了16例AK和12例SCC中BRAF和RKIP基因的表达水平。采用一种新的等位基因特异性qPCR方法并结合直接DNA测序，以检测第15外显子中V600E突变的频率，并检查第11外显子内基因的突变状态。
BRAF和RKIP的表达水平从正常皮肤组织、AK到SCC均呈下降趋势。SCC中RKIP mRNA水平的降低提示了BRAF信号通路失调的一种新机制。我们的结果进一步表明，BRAF在癌前皮肤病变（AK）或非黑色素瘤皮肤肿瘤（SCC）中似乎并不经常发生突变。
