gga-miR-26a 靶向 NEK6 并抑制马立克氏病淋巴瘤细胞增殖。

gga-miR-26a targets NEK6 and suppresses Marek's disease lymphoma cell proliferation.

机构信息

Department of Animal Genetics and Breeding, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.

出版信息

Poult Sci. 2014 May;93(5):1097-105. doi: 10.3382/ps.2013-03656.

DOI:10.3382/ps.2013-03656

PMID:24795301

Abstract

MicroRNA (miRNA) are a class of highly conserved, small noncoding RNA that emerge as key posttranscriptional regulators in various neoplastic transformations. Our previous study profiling the miRNA transcriptome in Marek's disease virus (MDV)-induced lymphoma revealed many novel and differentially expressed miRNA, including gga-miR-26a, which was downregulated in MDV-infected spleens of chickens. In this study, differential expression of gga-miR-26a between MDV-infected and noninfected spleens at 4, 7, 14, 21, and 28 d postinfection was analyzed by real-time PCR. The results showed gga-miR-26a were downregulated in MDV-infected spleens at cytolytic infection, latency, and tumor transformation phases. Subsequent cell proliferation assay revealed cell viability was lower in gga-miR-26a mimic transfection group than that in negative controls. Target genes of gga-miR-26a were identified by luciferase reporter gene assay. The results showed significant interaction between gga-miR-26a and Never In Mitosis Gene A (NIMA)-related kinase 6 (NEK6) gene. Subsequent gain of function experiment and Western blot assay showed that mRNA and protein levels of NEK6 were downregulated after gga-miR-26 mimic was transfected into MDV-transformed lymphoid cell line (MSB-1), indicating that NEK6 was modulated by gga-miR-26a. The expression of NEK6 showed a higher trend in MDV-infected samples including tumorous spleen and MD lymphoma from liver than that in noninfected controls. The results suggested that gga-miR-26a inhibited MSB-1 cell proliferation. Gga-miR-26a and its direct target, NEK6, might play important roles in MDV infection.

摘要

微小 RNA（miRNA）是一类高度保守的小型非编码 RNA，作为各种肿瘤转化中关键的转录后调控因子出现。我们之前的研究对马立克氏病病毒（MDV）诱导的淋巴瘤中的 miRNA 转录组进行了分析，发现了许多新的和差异表达的 miRNA，包括在 MDV 感染的鸡脾脏中下调的gga-miR-26a。在这项研究中，通过实时 PCR 分析了 MDV 感染和未感染的脾脏在感染后 4、7、14、21 和 28 天之间gga-miR-26a 的差异表达。结果表明，在细胞溶解感染、潜伏期和肿瘤转化阶段，gga-miR-26a 在 MDV 感染的脾脏中下调。随后的细胞增殖试验显示，gga-miR-26a 模拟转染组的细胞活力低于阴性对照组。通过荧光素酶报告基因试验鉴定了 gga-miR-26a 的靶基因。结果表明，gga-miR-26a 与有丝分裂不进入基因 A（NIMA）相关激酶 6（NEK6）基因之间存在显著的相互作用。随后的功能获得实验和 Western blot 分析表明，在将 gga-miR-26 模拟物转染到 MDV 转化的淋巴样细胞系（MSB-1）后，NEK6 的 mRNA 和蛋白水平下调，表明 NEK6 受 gga-miR-26a 调节。在感染了 MDV 的样本（包括肿瘤脾脏和来自肝脏的 MD 淋巴瘤）中，NEK6 的表达呈上升趋势，高于未感染的对照组。结果表明，gga-miR-26a 抑制了 MSB-1 细胞的增殖。gga-miR-26a 及其直接靶标 NEK6 可能在 MDV 感染中发挥重要作用。