Department cum National Centre for Human Genome Studies and Research (NCHGSR), Panjab University, Chandigarh, 160 014, India.
Mol Cell Biochem. 2019 Jul;457(1-2):31-40. doi: 10.1007/s11010-019-03509-8. Epub 2019 Feb 21.
Angiotensin II type 1 receptor can activate number of signalling pathways upon stimulation and consequently its involvement in cancer progression have also been revealed. But which epigenetic mechanisms are involved in its regulation, need to be further explored. In-silico analysis revealed a promoter CpG island (CGI) which was cloned and assayed for functional activity using reporter gene system. The effect of methylation on this CGI was analysed through varying degree of methyltransferase treatment of cloned fragment. Results thus obtained were validated by direct sequencing. To further establish the status of this effect, in-vivo analysis was done through screening of methylation pattern in the targeted region among hypertensives (HTN) and normotensives (NTN) using PCR-Bisulphite sequencing. Additionally, clinical details of all participants, biochemical parameters and lifestyle related information was also collected and statistically evaluated. Reporter gene assay assigned functional activity to the cloned promoter CpG island. Increased dose and durations of methyltransferase treatment decreased the expression of reporter gene indicating the role of promoter DNA methylation. Among all the human samples screened, only one of the HTN individual was found to have single hemi-methylated CpG site at a position which happens to be a part of Sp1 transcription factor binding site. To conclude, CpG island in the promoter region of AT1R (CpG.P.AT1R) gets regulated through epigenetic mechanism of DNA methylation.
血管紧张素 II 型 1 型受体（AT1R）在受到刺激时可以激活多种信号通路，因此其在癌症进展中的作用也已被揭示。但哪些表观遗传机制参与了其调节，仍需要进一步探索。计算机分析揭示了一个启动子 CpG 岛（CGI），我们克隆了该 CpG 岛并通过报告基因系统检测其功能活性。通过改变甲基转移酶处理克隆片段的程度来分析甲基化对该 CGI 的影响。通过直接测序验证了由此获得的结果。为了进一步确定这种影响的状态，我们通过 PCR-亚硫酸氢盐测序在高血压（HTN）和正常血压（NTN）个体中筛选靶向区域的甲基化模式，进行了体内分析。此外，还收集了所有参与者的临床详细信息、生化参数和与生活方式相关的信息，并进行了统计评估。报告基因检测赋予克隆启动子 CpG 岛功能活性。增加甲基转移酶处理的剂量和时间会降低报告基因的表达，表明启动子 DNA 甲基化的作用。在所筛选的所有人类样本中，只有一名 HTN 个体在一个位置上发现了单一半甲基化的 CpG 位点，而该位置恰好是 Sp1 转录因子结合位点的一部分。总之，AT1R 启动子区域的 CpG 岛（CpG.P.AT1R）通过 DNA 甲基化的表观遗传机制进行调节。
