Faculty of Medicine and Health Sciences, Department of Paraclinical Sciences, Universiti Malaysia Sarawak, Kota Samarahan, Sarawak, Malaysia.
BMC Med Genomics. 2019 Jan 15;12(1):9. doi: 10.1186/s12920-018-0465-4.
It has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC.
Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed.
In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient.
Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.
慢性鼻-鼻窦炎（CRS）会增加罹患鼻咽癌（NPC）的风险。胃食管反流（GOR）可导致 CRS，而 GOR 可能会波及鼻咽部。反流物的主要成分胆汁酸（BA）已被证实具有致癌性和遗传毒性。BA 诱导的细胞凋亡与多种癌症相关。我们先前的研究表明，BA 可诱导鼻咽上皮细胞发生凋亡和基因断裂。染色体断裂发生在凋亡和染色体重排的早期阶段。有研究提示，染色体断裂往往聚集在富含基质结合区/支架附着区（MAR/SAR）的区域。本研究假设 BA 可能在 MAR/SAR 导致 NPC 中的染色体断裂，引起染色体异常。本研究针对位于 9p22 的 AF9 基因，因为 9p22 是 NPC 中的缺失热点。
使用 MAR/SAR 预测工具预测 AF9 基因中的潜在 MAR/SAR 位点。用 BA 处理中性和酸性 pH 值下的正常鼻咽上皮细胞（NP69）和 NPC 细胞（TWO4）。反转聚合酶链反应（IPCR）用于鉴定 SAR 区（包含 MAR/SAR）和非 SAR 区（不包含 MAR/SAR）中的染色体断裂。为了确定 AF9 SAR 和非 SAR 区内的染色体断裂点，进行 DNA 测序。
在 AF9 SAR 区，BA 处理的 NP69 和 TWO4 细胞的基因断裂频率明显高于未处理对照组。对于 AF9 非 SAR 区，未处理和 BA 处理细胞的断裂频率无显著差异。在 SAR 区检测到的少数断裂点定位于之前报道的与混合谱系白血病（MLL）基因易位的急性淋巴细胞白血病（ALL）患者中的 AF9 区域内。
我们的研究结果提示，MAR/SAR 可能参与了 BA 诱导的染色体断裂的位置定义。我们的报告首次揭示了这些 BA 诱导的染色体断裂与 AF9 染色质结构的关系。
