Department of Pathology, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou, Fujian 363000, P.R. China.
Department of Hematology, Zhangzhou Affiliated Hospital of Fujian Medical University, Zhangzhou, Fujian 363000, P.R. China.
Int J Mol Med. 2017 Aug;40(2):319-328. doi: 10.3892/ijmm.2017.3032. Epub 2017 Jun 19.
Lysine-specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics; however, the pathological roles of its dysfunction in mantle cell lymphoma (MCL) and T-cell acute lymphoblastic leukemia remain to be elucidated. In this study, we evaluated LSD1, and histone H3 lysine 4 (H3K4)me1 and H3K4me2 expression in patients with MCL and silenced LSD1 in JeKo‑1 and MOLT‑4 cells, in order to define its role in JeKo‑1 and MOLT‑4 cell proliferation and apoptosis. We retrospectively analyzed the protein expression of LSD1, and mono- and dimethylated H3K4 (H3K4me1 and H3K4me2), and cyclin D1 and Ki67 in 30 cases of MCL by immunohistochemistry. The correlation of LSD1, H3K4me1 and H3K4me2 with Ki67 was determined by statistical analysis. LSD1 was silenced by small interfering RNA (siRNA). Cell apoptosis and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol‑2-yl)‑2,5-diphenyltetrazolium bromide (MTT) assay. The protein expression levels of LSD1, histone methylated H3K4, histone acetylated H3, cyclin D1, apoptotic proteins, p15 and DNA methyltransferase 1 (DNMT1) were examined by western blot analysis. We demonstrated that LSD1 was upregulated, and that H3K4me1 and H3K4me2 were downregulated in the cases with MCL, compared to those with proliferative lymphadenitis (p<0.05). LSD1 positively correlated with Ki67 in MCL [Cohen's kappa (κ)=0.667, p<0.01]. There was no significant correlation between H3K4me1 and H3K4me2, and Ki67 (κ=-0.182, p>0.05, κ=-0.200, p>0.05). The silencing of LSD1 decreased the levels of the apoptosis-related proteins, Bcl-2, pro-caspase-3 and C-myc, and decreased those of DNMT1 and increased p15, and resulted in the loss of cell viability and the induction apoptosis. The silencing of LSD1 increased the expression of H3K4me1 and H3K4me2, and histone acetylated H3 in the JeKo‑1 and MOLT‑4 cells. LSD1 siRNA also decreased cyclin D1 expression in the JeKo‑1 cells. On the whole, our findings demonstrate that the overexpression of LSD1 may be associated with the pathogenesis in MCL. We demonstrated that the silencing of LSD1 is capable of removing the mono- and dimethyl groups from H3K4, and upregulating the histone acetylation of H3 in JeKo‑1 and MOLT‑4 cells. The silencing of LSD1 inhibited cell growth and induced cell apoptosis. Of note, in JeKo‑1 cells, the silencing of LSD1 decreased cyclin D1 expression, which is one of the genes that contribute to the pathogenesis of MCL. LSD1 may thus be a possible therapeutic target in MCL and acute lymphoblastic leukemia MOLT‑4 cells.
赖氨酸特异性脱甲基酶 1（LSD1）已在表观遗传学中被鉴定和生化表征；然而，其在套细胞淋巴瘤（MCL）和 T 细胞急性淋巴细胞白血病中的功能障碍的病理作用仍有待阐明。在这项研究中，我们评估了 LSD1 以及组蛋白 H3 赖氨酸 4（H3K4）单甲基化（H3K4me1）和 H3K4 二甲基化（H3K4me2）在 MCL 患者中的表达，并在 JeKo-1 和 MOLT-4 细胞中沉默 LSD1，以确定其在 JeKo-1 和 MOLT-4 细胞增殖和凋亡中的作用。我们通过免疫组织化学法回顾性分析了 30 例 MCL 患者的 LSD1、单甲基化和二甲基化 H3K4（H3K4me1 和 H3K4me2）、细胞周期蛋白 D1 和 Ki67 的蛋白表达。通过统计分析确定 LSD1 与 Ki67 的相关性。通过小干扰 RNA（siRNA）沉默 LSD1。通过流式细胞术和 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）测定检测细胞凋亡和细胞增殖。通过 Western blot 分析检测 LSD1、组蛋白甲基化 H3K4、组蛋白乙酰化 H3、细胞周期蛋白 D1、凋亡蛋白、p15 和 DNA 甲基转移酶 1（DNMT1）的蛋白表达水平。我们证明与增生性淋巴结炎相比，MCL 病例中 LSD1 上调，H3K4me1 和 H3K4me2 下调（p<0.05）。LSD1 在 MCL 中与 Ki67 呈正相关[Cohen's kappa（κ）=0.667，p<0.01]。H3K4me1 和 H3K4me2 与 Ki67 之间无显著相关性（κ=-0.182，p>0.05，κ=-0.200，p>0.05）。LSD1 的沉默降低了凋亡相关蛋白 Bcl-2、原半胱天冬酶-3 和 C-myc 的水平，并降低了 DNMT1 和增加了 p15 的水平，导致细胞活力丧失和诱导凋亡。LSD1 的沉默增加了 JeKo-1 和 MOLT-4 细胞中 H3K4me1 和 H3K4me2 的表达以及组蛋白乙酰化 H3。LSD1 siRNA 还降低了 JeKo-1 细胞中细胞周期蛋白 D1 的表达。总的来说，我们的研究结果表明 LSD1 的过表达可能与 MCL 的发病机制有关。我们证明 LSD1 的沉默能够从 H3K4 上去除单甲基和二甲基基团，并上调 JeKo-1 和 MOLT-4 细胞中 H3 的组蛋白乙酰化。LSD1 的沉默抑制细胞生长并诱导细胞凋亡。值得注意的是，在 JeKo-1 细胞中，LSD1 的沉默降低了细胞周期蛋白 D1 的表达，这是导致 MCL 发病机制的基因之一。因此，LSD1 可能是 MCL 和急性淋巴细胞白血病 MOLT-4 细胞的潜在治疗靶点。
