Molecular Genetics Laboratory, Department of Biotechnology & Bioinformatics, North-Eastern Hill University, Shillong, Meghalaya 793022, India.
Department of Biotechnology, Royal School of Biosciences, The Assam Royal Global University, Guwahati, Assam 781035, India.
Mutagenesis. 2022 Oct 26;37(3-4):182-190. doi: 10.1093/mutage/geac015.
Research over the years revealed that precocious anaphase, securin overexpression, and genome instability in both target and nontarget cells are significantly associated with the increased risk of areca nut (AN) and lime-induced oral, esophageal, and gastric cancers. Further, hyperphosphorylation of Rb and histone H3 epigenetic modifications both globally and in the promoter region of the securin gene were demonstrated after AN + lime exposure. This study aims whether the extract of raw AN + lime relaxes chromatin structure which further facilitates the histone H3 epigenetic modifications during the initial phase of carcinogenesis. Three groups of mice (10 in each group) were used. The treated group consumed 1 mg/day/mice of AN extract with lime ad libitum in the drinking water for 60 days. The dose was increased by 1 mg every 60 days. Isolated nuclei were digested with DNaseI and 2 kb and below DNA was eluted from the agarose gel, purified and PCR amplified by using securin and GAPDH primers. Securin and E2F1 expression, pRb phosphorylation, and histone epigenetic modifications were analyzed by immunohistochemistry. The number of DNA fragments within 2 kb in size after DNaseI treatment was higher significantly in AN + lime exposed tissue samples than in the untreated one. The PCR result showed that the number of fragments bearing securin gene promoter and GAPDH gene was significantly higher in AN + lime exposed DNaseI-treated samples. Immunohistochemistry data revealed increased Rb hyperphosphorylation, upregulation of E2F1, and securin in the AN + lime-treated samples. Increased trimethylation of histone H3 lysine 4 and acetylation of H3 lysine 9 and 18 were observed globally in the treated samples. Therefore, the results of this study have led to the hypothesis that AN + lime exposure relaxes the chromatin, changes the epigenetic landscape, and deregulates the Rb-E2F1 circuit which might be involved in the upregulation of securin and some other proto-oncogenes that might play an important role in the initial phases of AN + lime mediated carcinogenesis.
多年的研究表明，早熟后期、securin 过表达以及靶细胞和非靶细胞的基因组不稳定性与槟榔（AN）和石灰诱导的口腔、食管和胃癌风险增加显著相关。此外，在 AN+石灰暴露后，Rb 的过度磷酸化和 securin 基因启动子区域的组蛋白 H3 表观遗传修饰都得到了证明。本研究旨在探讨生槟榔+石灰提取物是否能放松染色质结构，从而在致癌作用的初始阶段进一步促进组蛋白 H3 的表观遗传修饰。使用了三组小鼠（每组 10 只）。实验组每天每只小鼠消耗 1mg 的 AN 提取物，同时在饮用水中添加石灰，持续 60 天。每 60 天增加 1mg 剂量。用 DNaseI 消化分离的核，并从琼脂糖凝胶中洗脱 2kb 及以下的 DNA，通过 securin 和 GAPDH 引物进行纯化和 PCR 扩增。通过免疫组织化学分析 securin 和 E2F1 表达、pRb 磷酸化和组蛋白表观遗传修饰。DNaseI 处理后，AN+石灰暴露组织样本中大小在 2kb 内的 DNA 片段数量明显高于未处理样本。PCR 结果显示，在 AN+石灰暴露的 DNaseI 处理样本中，携带 securin 基因启动子和 GAPDH 基因的片段数量显著增加。免疫组织化学数据显示，在 AN+石灰处理的样本中，Rb 过度磷酸化、E2F1 上调和 securin 上调。在处理样本中观察到组蛋白 H3 赖氨酸 4 的三甲基化和 H3 赖氨酸 9 和 18 的乙酰化在全球范围内增加。因此，本研究的结果导致了这样的假设，即 AN+石灰暴露放松染色质，改变表观遗传景观，并使 Rb-E2F1 电路失活，这可能与 securin 和其他一些原癌基因的上调有关，这些基因可能在 AN+石灰介导的致癌作用的初始阶段发挥重要作用。
