Feng Xiangling, Ren Caiping, Zhou Wen, Liu Weidong, Zeng Liang, Li Guifei, Wang Lei, Li Min, Zhu Bin, Yao Kaitai, Jiang Xingjun
Key Laboratory for Carcinogenesis of Chinese Ministry of Health, Key Laboratory for Carcinogenesis and Cancer Invasion of Chinese Ministry of Education, Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, Hunan, P.R., China.
Mol Carcinog. 2014 Nov;53(11):858-70. doi: 10.1002/mc.22044. Epub 2013 Jun 12.
Previous studies have shown that promoter hypermethylation plays a key role in DLC-1 inactivation in nasopharyngeal carcinoma (NPC). However, DLC-1 mutation in NPC has not been reported, and there remain some discrepancies in methods and results between different groups. Here, we examined the mRNA and protein expression of DLC-1 in chronic nasopharyngitis (CN) and NPC tissues by reverse transcription-polymerase chain reaction/qPCR and immunohistochemistry, respectively. DLC-1 mRNA was undetectable in all the seven widely used NPC cell lines and absent or significantly down-regulated in 70% of NPC tissues. DLC-1 protein level was reduced in 74.3% of NPCs when compared with CN tissues, and significantly lower in NPC samples at advanced clinical stages than that at early stages. Then, we purified the same batch of specimens by microdissection and analyzed the possible mechanisms of DLC-1 downregulation with mutation and allelic loss analysis, methylation-specific PCR and bisulfite genomic sequencing. Only one mutation was detected at codon 693 of exon 8 in 3.3% of NPCs and five single nucleotide polymorphisms (SNPs) were identified. Loss of DLC-1 was detected in 23.3% of NPC tissues. The 100% of NPC cell lines, 80% of primary NPC and 22.2% of CN tissues showed methylation in DLC-1 promoter, while DLC-1 expression was recovered in seven NPC cell lines after 5-aza-dC treatment. Patched methylation assay confirmed that promoter methylation could repress DLC-1 expression. This report demonstrates that DLC-1 is negatively associated with NPC carcinogenesis, and promoter hypermethylation along with loss of heterozygosity, but not mutation, contributes to inactivation of DLC-1 in NPC.
以往研究表明，启动子高甲基化在鼻咽癌（NPC）中DLC-1失活过程中起关键作用。然而，尚未见NPC中DLC-1突变的报道，且不同研究组在方法和结果上仍存在一些差异。在此，我们分别采用逆转录-聚合酶链反应/qPCR和免疫组化方法检测了慢性鼻咽炎（CN）和NPC组织中DLC-1的mRNA和蛋白表达。在所有七种广泛使用的NPC细胞系中均未检测到DLC-1 mRNA，且在70%的NPC组织中缺失或显著下调。与CN组织相比，74.3%的NPC中DLC-1蛋白水平降低，且晚期临床阶段的NPC样本中DLC-1蛋白水平显著低于早期阶段。然后，我们通过显微切割纯化了同一批标本，并通过突变和等位基因缺失分析、甲基化特异性PCR和亚硫酸氢盐基因组测序分析了DLC-1下调的可能机制。在3.3%的NPC中仅在外显子8的密码子693处检测到一个突变，并鉴定出五个单核苷酸多态性（SNP）。在23.3%的NPC组织中检测到DLC-1缺失。100%的NPC细胞系、80%的原发性NPC和22.2%的CN组织在DLC-1启动子中显示甲基化，而5-氮杂-2'-脱氧胞苷（5-aza-dC）处理后七个NPC细胞系中DLC-1表达得以恢复。patched甲基化分析证实启动子甲基化可抑制DLC-1表达。本报告表明，DLC-1与NPC致癌呈负相关，启动子高甲基化以及杂合性缺失而非突变导致NPC中DLC-1失活。
