Department of Hematology, Shenzhen Longhua People's Hospital, Shenzhen, P.R. China.
Department of Hematology, Daqing Oilfield General Hospital, Daqing, P.R. China.
Oncol Res. 2019 Aug 8;27(8):935-944. doi: 10.3727/096504019X15555388198071. Epub 2019 Apr 17.
Acute lymphoblastic leukemia (ALL) is the most prevalent of pediatric cancers. Neuroepithelial cell-transforming 1 (NET1) has been associated with malignancy in a number of cancers, but the role of NET1 in ALL development is unclear. In the present study, we investigated the effect of NET1 gene in ALL cell proliferation and chemoresistance. We analyzed GEO microarray data comparing bone marrow expression profiles of pediatric B-cell ALL samples and those of age-matched controls. MTT and colony formation assays were performed to analyze cell proliferation. ELISA assays, Western blot analyses, and TUNEL staining were used to detect chemoresistance. We confirmed that NET1 was targeted by miR-206 using Western blot and luciferase reporter assays. We identified NET1 gene as one of the most significantly elevated genes in pediatric B-ALL. MTT and colony formation assays demonstrated that NET1 overexpression increases B-ALL cell proliferation in Nalm-6 cells. ELISA assays, Western blot analyses, and TUNEL staining showed that NET1 contributes to ALL cell doxorubicin resistance, whereas NET1 inhibition reduces resistance. Using the TargetScan database, we found that several microRNAs (miRNAs) were predicted to target NET1, including microRNA-206 (miR-206), which has been shown to regulate cancer development. To determine whether miR-206 targets NET1 in vitro, we transfected Nalm-6 cells with miR-206 or its inhibitor miR-206-in. Western blot assays showed that miR-206 inhibits NET1 expression and miR-206-in increases NET1 expression. Luciferase assays using wild-type or mutant 3'-untranslated region (3'-UTR) of NET1 confirmed these findings. We ultimately found that miR-206 inhibits B-ALL cell proliferation and chemoresistance induced by NET1. Taken together, our results provide the first evidence that NET1 enhances proliferation and chemoresistance in B-ALL cells and that miR-206 regulates these effects by targeting NET1. This study therefore not only contributes to a greater understanding of the molecular mechanisms underlying B-ALL progression but also opens the possibility for developing curative interventions.
急性淋巴细胞白血病（ALL）是儿童中最常见的癌症。神经上皮细胞转化 1（NET1）已与多种癌症的恶性肿瘤相关，但 NET1 在 ALL 发展中的作用尚不清楚。在本研究中，我们研究了 NET1 基因对 ALL 细胞增殖和化疗耐药性的影响。我们分析了 GEO 微阵列数据，比较了儿科 B 细胞 ALL 样本和年龄匹配对照的骨髓表达谱。通过 MTT 和集落形成实验分析细胞增殖。ELISA 检测、Western blot 分析和 TUNEL 染色用于检测化疗耐药性。我们使用 Western blot 和荧光素酶报告基因检测证实 NET1 是 miR-206 的靶标。我们确定 NET1 是儿科 B-ALL 中上调最显著的基因之一。MTT 和集落形成实验表明，NET1 过表达可增加 Nalm-6 细胞中 B-ALL 细胞的增殖。ELISA 检测、Western blot 分析和 TUNEL 染色表明，NET1 有助于 ALL 细胞对阿霉素的耐药性，而 NET1 抑制则降低耐药性。使用 TargetScan 数据库，我们发现几种 microRNAs（miRNAs）被预测为 NET1 的靶标，包括已被证明可调节癌症发展的 microRNA-206（miR-206）。为了确定 miR-206 是否在体外靶向 NET1，我们用 miR-206 或其抑制剂 miR-206-in 转染 Nalm-6 细胞。Western blot 分析表明，miR-206 抑制 NET1 表达，miR-206-in 增加 NET1 表达。使用野生型或突变型 NET1 3'-非翻译区（3'-UTR）的荧光素酶实验证实了这些发现。我们最终发现，miR-206 抑制 NET1 诱导的 B-ALL 细胞增殖和化疗耐药性。综上所述，我们的研究结果首次表明，NET1 增强了 B-ALL 细胞的增殖和化疗耐药性，miR-206 通过靶向 NET1 调节这些作用。因此，本研究不仅有助于更深入地了解 B-ALL 进展的分子机制，而且为开发治疗干预措施提供了可能。
