Shademan Milad, Zare Khadijeh, Zahedi Morteza, Mosannen Mozaffari Hooman, Bagheri Hosseini Hadi, Ghaffarzadegan Kamran, Goshayeshi Ladan, Dehghani Hesam
Graduate Program in Physiology, Department of Basic Sciences, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran.
Stem Cell Biology and Regenerative Medicine Research Group, Research Institute of Biotechnology, Ferdowsi University of Mashhad, Azadi Square, Mashhad, 91779-48974 Iran.
Cancer Cell Int. 2020 Sep 1;20:426. doi: 10.1186/s12935-020-01511-5. eCollection 2020.
The methylation of the CpG islands of the LINE-1 promoter is a tight control mechanism on the function of mobile elements. However, simultaneous quantification of promoter methylation and transcription of LINE-1 has not been performed in progressive stages of colorectal cancer. In addition, the insertion of mobile elements in the genome of advanced adenoma stage, a precancerous stage before colorectal carcinoma has not been emphasized. In this study, we quantify promoter methylation and transcripts of LINE-1 in three stages of colorectal non-advanced adenoma, advanced adenoma, and adenocarcinoma. In addition, we analyze the insertion of LINE-1, Alu, and SVA elements in the genome of patient tumors with colorectal advanced adenomas.
LINE-1 hypomethylation status was evaluated by absolute quantitative analysis of methylated alleles (AQAMA) assay. To quantify the level of transcripts for LINE-1, quantitative RT-PCR was performed. To find mobile element insertions, the advanced adenoma tissue samples were subjected to whole genome sequencing and MELT analysis.
We found that the LINE-1 promoter methylation in advanced adenoma and adenocarcinoma was significantly lower than that in non-advanced adenomas. Accordingly, the copy number of LINE-1 transcripts in advanced adenoma was significantly higher than that in non-advanced adenomas, and in adenocarcinomas was significantly higher than that in the advanced adenomas. Whole-genome sequencing analysis of colorectal advanced adenomas revealed that at this stage polymorphic insertions of LINE-1, Alu, and SVA comprise approximately 16%, 51%, and 74% of total insertions, respectively.
Our correlative analysis showing a decreased methylation of LINE-1 promoter accompanied by the higher level of LINE-1 transcription, and polymorphic genomic insertions in advanced adenoma, suggests that the early and advanced polyp stages may host very important pathogenic processes concluding to cancer.
LINE-1启动子的CpG岛甲基化是对移动元件功能的一种严格调控机制。然而，尚未在结直肠癌的进展阶段同时对LINE-1启动子甲基化和转录进行定量分析。此外，在进展期腺瘤（结直肠癌前的癌前阶段）基因组中移动元件的插入情况尚未得到重视。在本研究中，我们对结直肠非进展期腺瘤、进展期腺瘤和腺癌三个阶段的LINE-1启动子甲基化和转录本进行了定量分析。此外，我们分析了患有结直肠进展期腺瘤患者肿瘤基因组中LINE-1、Alu和SVA元件的插入情况。
通过甲基化等位基因绝对定量分析（AQAMA）法评估LINE-1低甲基化状态。为了定量LINE-1转录本水平，进行了定量逆转录聚合酶链反应（qRT-PCR）。为了发现移动元件插入情况，对进展期腺瘤组织样本进行了全基因组测序和MELT分析。
我们发现进展期腺瘤和腺癌中LINE-1启动子甲基化显著低于非进展期腺瘤。相应地，进展期腺瘤中LINE-1转录本的拷贝数显著高于非进展期腺瘤，而腺癌中的LINE-1转录本拷贝数显著高于进展期腺瘤。结直肠进展期腺瘤的全基因组测序分析表明，在此阶段，LINE-1、Alu和SVA的多态性插入分别占总插入的约16%、51%和74%。
我们的相关性分析表明，LINE-1启动子甲基化降低伴随着LINE-1转录水平升高，以及进展期腺瘤中的多态性基因组插入，这表明息肉的早期和进展期阶段可能存在导致癌症的非常重要的致病过程。
