Autophagy Research Center and Department of Biochemistry, Shiraz University of Medical Sciences, Shiraz, Iran.
Colorectal Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
BMC Cancer. 2020 Apr 25;20(1):350. doi: 10.1186/s12885-020-6706-x.
ARID1A has been described as a tumor suppressor gene, participating in chromatin re-modeling, epithelial-mesenchymal-transition and many other cellular and molecular processes. It has been cited as a contribute in tumorigenesis. The role of ARID1A in CRC is not yet defined.
To investigate the role of ARID1A methylation and CNV in its expression in CRC cell lines and to examine the relationship between ARID1A status with survival and clinicopathologic characteristics in patients with CRC.
We used RT-PCR to determine both CNV and expression of ARID1A from six CRC cell lines. We used MSP to evaluate methylation of ARID1A. IHC was used to assess ARID1A protein expression. We also evaluated MSI and EMAST status in 18 paired CRC and adjacent normal tissues. 5AzadC was used to assess effect of DNA demethylation on ARID1A expression. Statistical analysis was performed to establish correlations between ARID1A expression and other parameters.
Among the 18 CRC tumors studied, 7 (38.8%) and 5 tumors (27.7%) showed no or low ARID1A expression, respectively. We observed no significant difference in ARID1A expression for overall patient survival, and no difference between clinicopathological parameters including MSI and EMAST. However, lymphatic invasion was more pronounced in the low/no ARID1A expression group when compared to moderate and high expression group (33% VS. 16.6% respectively. ARID1A promoter methylation was observed in 4/6 (66%) cell lines and correlated with ARID1A mRNA expression level ranging from very low in SW48, to more pronounced in HCT116 and HT-29/219. Treatment with the methyltransferase inhibitor 5-Azacytidine (5-aza) resulted in a 25.4-fold and 6.1-fold increase in ARID1A mRNA expression in SW48 and SW742 cells, respectively, while there was no change in SW480 and LS180 cells. No ARID1A CNV was observed in the CRC cell lines.
ARID1A expression is downregulated in CRC tissues which correlates with it being a tumor suppressor protein. This finding confirms ARID1A loss of expression in CRC development. Our in-vitro results suggest high methylation status associates with reduced ARID1A expression and contributes to CRC tumorigenesis. However, there was no significant association between ARID1A loss of expression and clinicopathological characteristics. Future in-vivo analysis is warranted to further establish ARID1A role in colorectal neoplastic transformation.
ARID1A 被描述为一种肿瘤抑制基因，参与染色质重塑、上皮-间充质转化和许多其他细胞和分子过程。它被认为是肿瘤发生的原因之一。ARID1A 在 CRC 中的作用尚不清楚。
研究 ARID1A 甲基化和 CNV 对 CRC 细胞系中其表达的影响，并探讨 ARID1A 状态与 CRC 患者生存和临床病理特征的关系。
我们使用 RT-PCR 从 6 种 CRC 细胞系中确定 ARID1A 的 CNV 和表达。我们使用 MSP 评估 ARID1A 的甲基化。免疫组化用于评估 ARID1A 蛋白表达。我们还评估了 18 对 CRC 和相邻正常组织中的 MSI 和 EMAST 状态。5AzadC 用于评估 DNA 去甲基化对 ARID1A 表达的影响。进行统计学分析以确定 ARID1A 表达与其他参数之间的相关性。
在研究的 18 个 CRC 肿瘤中，分别有 7 个（38.8%）和 5 个肿瘤（27.7%）表现出无或低 ARID1A 表达。我们观察到 ARID1A 表达对总患者生存无显著影响，并且在包括 MSI 和 EMAST 在内的临床病理参数之间无差异。然而，与中度和高表达组相比，低/无 ARID1A 表达组的淋巴浸润更为明显（分别为 33%和 16.6%）。在 6 种细胞系中观察到 ARID1A 启动子甲基化，与 ARID1A mRNA 表达水平相关，从 SW48 的极低水平到 HCT116 和 HT-29/219 的更显著水平。用甲基转移酶抑制剂 5-氮杂胞苷（5-aza）处理后，SW48 和 SW742 细胞中 ARID1A mRNA 表达分别增加了 25.4 倍和 6.1 倍，而 SW480 和 LS180 细胞没有变化。在 CRC 细胞系中未观察到 ARID1A CNV。
CRC 组织中 ARID1A 的表达下调，这与它作为一种肿瘤抑制蛋白有关。这一发现证实了 ARID1A 在 CRC 发展过程中的表达缺失。我们的体外研究结果表明，高甲基化状态与 ARID1A 表达降低相关，并有助于 CRC 肿瘤发生。然而，ARID1A 表达缺失与临床病理特征之间没有显著关联。需要进一步的体内分析来进一步确定 ARID1A 在结直肠肿瘤发生中的作用。
