5-氮杂-2'-脱氧胞苷对脑胶质瘤 U251 细胞 SLC22A18 的影响。

Effect of 5-Aza-2'-deoxycytidine on SLC22A18 in glioma U251 cells.

机构信息

Department of Neurosurgery, No. 3 People's Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, Shanghai 201900, PR China.

出版信息

Mol Med Rep. 2012 Jan;5(1):138-41. doi: 10.3892/mmr.2011.620. Epub 2011 Oct 11.

DOI:10.3892/mmr.2011.620

PMID:21993522

Abstract

SLC22A18 [solute carrier family 22 (organic cation transporter) member 18] is located within the 11p15.5 cluster, and may be a new tumor suppressor gene; evidence of SLC22A18 hypermethylation is documented in several types of human cancers. In order to determine whether SLC22A18 hypermethylation is involved in glioma, we determined the SLC22A18 gene protein expression, mRNA expression and methylation status in glioma U251 cells before and after treatment with 5-Aza-2'‑deoxycytidine (5-Aza-CdR), and observed the change in growth. Glioma U251 cells treated with 5-Aza-CdR were analyzed by flow cytometry to identify any change in their cell cycle profiles. Tumors induced via the injection of untreated U251 cells were measured. Immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR-based methylation assay were carried out to determine SLC22A18 gene protein expression, mRNA expression and methylation status in glioma U251 cells before and after treatment with 5-Aza‑CdR. The treated cells showed an increase in their proportion in G1, from 79.2 to 83.5%, and a decrease in S phase, from 12.4 to 5.8%. The apoptotic rate increased from 6.4 to 15.8%. Tumors induced via the injection of untreated U251 cells were approximately 1.46 cm³ in size, whereas the tumors induced by U251 cells treated with 5-Aza-CdR averaged 0.88 cm³ in size. The expression levels of SLC22A18 protein and mRNA in U251 cells were increased following treatment with 5x10⁻⁷ M 5-Aza‑CdR. Prior to 5-Aza-CdR treatment, the SLC22A18 gene demonstrated hypermethylation and therefore could not be cleaved by HpaII and MspI. It is known that only the DNA digested with HpaII or MspI can be amplified. Following treatment with 5-Aza‑CdR, the SLC22A18 gene became demethylated, and could then be cleaved by both of the enzymes, and this failed to be amplified. 5-Aza-cdR may induce glioma U251 cell division and apoptosis and enhance demethylation and protein and mRNA expression of SLC22A18. The hypermethylation of SLC22A18 may be related to the transcriptional silencing of this gene. The growth inhibitory effects of 5-Aza-CdR treatment in vivo remain recognizable.

摘要

SLC22A18（溶质载体家族 22（有机阳离子转运蛋白）成员 18）位于 11p15.5 簇内，可能是一个新的肿瘤抑制基因；在几种人类癌症中都有 SLC22A18 过度甲基化的证据。为了确定 SLC22A18 过度甲基化是否参与神经胶质瘤，我们在使用 5-氮杂-2'-脱氧胞苷（5-Aza-CdR）处理前后，测定了神经胶质瘤 U251 细胞中的 SLC22A18 基因蛋白表达、mRNA 表达和甲基化状态，并观察了生长的变化。用流式细胞术分析用 5-Aza-CdR 处理的神经胶质瘤 U251 细胞，以鉴定其细胞周期谱的任何变化。测量了未经处理的 U251 细胞注射诱导的肿瘤。用免疫组织化学、逆转录-聚合酶链反应（RT-PCR）和基于 PCR 的甲基化分析，测定了用 5-Aza-CdR 处理前后神经胶质瘤 U251 细胞中的 SLC22A18 基因蛋白表达、mRNA 表达和甲基化状态。处理后的细胞 G1 期比例从 79.2%增加到 83.5%，S 期从 12.4%减少到 5.8%。凋亡率从 6.4%增加到 15.8%。未经 5-Aza-CdR 处理的 U251 细胞注射诱导的肿瘤大小约为 1.46cm³，而用 5-Aza-CdR 处理的 U251 细胞注射诱导的肿瘤大小平均为 0.88cm³。用 5x10⁻⁷ M 5-Aza-CdR 处理后，U251 细胞中 SLC22A18 蛋白和 mRNA 的表达水平增加。在 5-Aza-CdR 处理之前，SLC22A18 基因表现出过度甲基化，因此不能被 HpaII 和 MspI 切割。已知只有被 HpaII 或 MspI 切割的 DNA 才能被扩增。用 5-Aza-CdR 处理后，SLC22A18 基因去甲基化，然后可以被两种酶切割，并且不能被扩增。5-Aza-CdR 可能诱导神经胶质瘤 U251 细胞分裂和凋亡，并增强 SLC22A18 的去甲基化和蛋白及 mRNA 表达。SLC22A18 的过度甲基化可能与该基因的转录沉默有关。5-Aza-CdR 治疗在体内的生长抑制作用仍然可以识别。