Department of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Hematology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Anal Cell Pathol (Amst). 2023 Feb 22;2023:2951519. doi: 10.1155/2023/2951519. eCollection 2023.
Acute myeloid leukemia (AML) is a heterogeneous malignancy with a low long-term survival rate. The aim of this study was to investigate the effects of decitabine (DAC) treatment cell proliferation and apoptosis in AML and role of the expression of LINC00599 and, consequently, miR-135a-5p.
Human promyelocytic leukemia cells (HL-60) and human acute lymphatic leukemia (CCRF-CEM) cells were treated with various concentrations of DAC. Cell proliferation in each group was detected using the cell counting kit 8. For each group, apoptosis and reactive oxygen species (ROS) levels were detected using flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) was performed to examine the expression of lncRNA LINC00599. The expression of apoptosis-related proteins was analyzed using western blotting. The regulatory relationship between miR-135a-5p and LINC00599 was verified by constructing miR-135a-5p mimics, miR-135a-5p inhibit, wild type LINC00599 3'-untranslated region (UTR), and mutant LINC00599 3'-UTR. Ki-67 expression in the tumor tissues of nude mice was detected using immunofluorescent assays.
Both DAC and LINC00599 Inhibit groups were able to significantly reduce the proliferation of HL60 and CCRF-CEM cells, increase apoptosis, upregulate the expression of Bad, cleaved caspase-3, and miR-135a-5p, downregulate the expression of Bcl-2, and elevate ROS levels in cells, with these effects being more pronounced after combined treatment with DAC and LINC00599 Inhibit. In comparison to mimic NC, the miR-135a-5p mimic group significantly decreased the relative fluorescence activity ratio of LINC00599 3'-UTR wild-type CCRF-CEM cells. The LINC00599 Inhibit and miR-135a-5p mimic groups exhibited substantially reduced proliferation of HL60 and CCRF-CEM cells, increased apoptosis, upregulated Bad, cleaved caspase-3, and miR-135a-5p expression, along with downregulated Bcl-2 and LINC00599 expression and increased ROS levels in cells; these effects were more pronounced after LINC00599 Inhibit was combined with miR-135a-5p mimics. In vivo experiments revealed that both DAC and LINC00599 Inhibit were able to considerably reduce the long diameter, short meridian, volume, and mass of tumors, increase miR-135a-5p expression, and decrease LINC00599 and ki-67 expression in tumor tissues of nude mice. This effect was more pronounced when the DAC and LINC00599 Inhibit were used in combination.
DAC regulates the expression of miR-135a-5p by regulating the expression of LINC00599, which in turn affects cell proliferation, apoptosis, and tumor proliferation. Our findings provide a theoretical basis for improving the clinical outcome of AML.
急性髓系白血病（AML）是一种异质性恶性肿瘤，其长期生存率较低。本研究旨在探讨地西他滨（DAC）治疗对 AML 细胞增殖和凋亡的影响，以及 LINC00599 的表达及其对 miR-135a-5p 的影响。
用不同浓度的 DAC 处理人早幼粒细胞白血病细胞（HL-60）和人急性淋巴细胞白血病（CCRF-CEM）细胞。用细胞计数试剂盒 8 检测各组细胞的增殖情况。用流式细胞术检测各组细胞的凋亡和活性氧（ROS）水平。采用逆转录聚合酶链反应（RT-PCR）检测 LINC00599 的表达。用 Western blot 分析凋亡相关蛋白的表达。通过构建 miR-135a-5p 模拟物、miR-135a-5p 抑制剂、野生型 LINC00599 3'UTR 和突变型 LINC00599 3'UTR 验证 miR-135a-5p 与 LINC00599 之间的调控关系。用免疫荧光法检测裸鼠肿瘤组织中的 Ki-67 表达。
DAC 和 LINC00599 抑制组均能显著降低 HL60 和 CCRF-CEM 细胞的增殖，增加细胞凋亡，上调 Bad、cleaved caspase-3 和 miR-135a-5p 的表达，下调 Bcl-2 和 LINC00599 的表达，升高细胞内 ROS 水平，联合应用 DAC 和 LINC00599 抑制后，这些作用更为明显。与 mimic NC 相比，miR-135a-5p 模拟物组显著降低了 CCRF-CEM 细胞野生型 LINC00599 3'UTR 的相对荧光活性比值。LINC00599 抑制和 miR-135a-5p 模拟物组均能显著降低 HL60 和 CCRF-CEM 细胞的增殖，增加细胞凋亡，上调 Bad、cleaved caspase-3 和 miR-135a-5p 的表达，下调 Bcl-2 和 LINC00599 的表达，升高细胞内 ROS 水平；联合应用 LINC00599 抑制和 miR-135a-5p 模拟物后，这些作用更为明显。体内实验结果表明，DAC 和 LINC00599 抑制均能显著降低裸鼠肿瘤的长径、短径、体积和质量，增加 miR-135a-5p 的表达，降低肿瘤组织中 LINC00599 和 Ki-67 的表达。当 DAC 和 LINC00599 抑制联合应用时，这种效果更为明显。
DAC 通过调节 LINC00599 的表达来调节 miR-135a-5p 的表达，进而影响细胞增殖、凋亡和肿瘤增殖。我们的研究结果为改善 AML 的临床疗效提供了理论依据。
