结节性硬化症细胞株TSC2(-/-) MEFs和正常细胞株TSC2(＋/＋) MEFs微小RNA表达谱的差异分析

MicroRNA differential expression profile in tuberous sclerosis complex cell line TSC2^(-/-) MEFs and normal cell line TSC2^(+/+) MEFs

目的:结节性硬化症是由于TSC1和TSC2基因突变导致累及多个器官、系统的常染色体显性遗传疾病,TSC2突变更为常见而且临床表型更为严重。为探讨结节性硬化症细胞株TSC2^(-/-)MEFs与正常细胞株TSC2+/+MEFs中微小RNA(microRNA,miRNA)表达谱的差异,以进一步研究相关miRNA在结节性硬化症发病机制中的作用奠定基础。方法:体外培养结节性硬化症细胞株TSC2^(-/-)MEFs和正常细胞株TSC2+/+MEFs,各选取3个样本分别作为实验组和对照组。用TRizol法提取细胞的总RNA,以紫外吸收测定法和变性琼脂糖凝胶电泳对RNA进行质量检测。采用miRNA芯片筛选两者之间差异表达的miRNA,样品间表达变化标准以上调至原2倍或下调至原1/2作为阈值范围,并以实时定量PCR验证miRNA芯片结果的可靠性。CCK-8法测定细胞增殖活性。结果:在结节性硬化症细胞株TSC2^(-/-)MEFs中,上调≥原2倍差异表达的miRNA分子有14个,下调≤原1/2差异表达的miRNA分子有26个。miRNA-199a-5p在TSC2^(-/-)MEFs显著低表达,过表达miRNA-199a-5p可显著抑制TSC2^(-/-)MEFs细胞增殖。结论:结节性硬化症细胞株TSC2^(-/-)MEFs和正常细胞株TSC2+/+MEFs存在差异表达miRNA分子。miRNA-199a-5p参与结节性硬化症的发生、发展,可能是防治结节性硬化症的重要分子靶点。

Objective： Tuberous sclerosis complex（TSC） is a multisystem genetic disorder caused by mutations in the TSC1 and TSC2 genes,but the molecular events contributing to TSC are not well understood. However,little is known about the role of microRNAs in TSC. To explore the microRNA differential expression profile between tuberous sclerosis complex cell line TSC2^（-/-）MEFs and normal type cell line TSC2^（＋/＋）MEFs,and to provide new clues to study the mechanism of microRNA function in tuberous sclerosis complex. Methods： TSC2^（-/-）MEFs and TSC2^（＋/＋）MEFs cell lines were cultured in vitro,each with three samples chosen as the experimental group and the control group respectively. Total RNA was isolated using TRizol and purified with RNeasy mini kit according to manufacturer＇s instructions. RNA quality and quantity were measured by using nanodrop spectrophotometer and RNA integrity was determined by gel electrophoresis. Total RNAs were extracted by TRizol,followed by RNA quantification and quality control. MicroRNA profiles were analyzed by microarray and the threshold value used to screen up-regulated more than 2-fold change or down-regulated less than 0. 5-fold change compared with controls. Real-time PCR was used to validate the reliability of microarray. Cell counting kit-8（CCK-8） assay was performed to evaluate the proliferation. Results： Fourteen microRNAs,including miR-18a-5p,miR-376c-3p, miR-136-5p, miR-467c-5p, miR-467b-5p, miR-5104, miR-3098-3p, miR-30a-3p,miR-302b-3p,miR-18a-3p,miR-19b-1-5p,miR-19a-5p,miR-20a-5p,miR-155-5p,were up-regulated,while twenty-six microRNAs,including miR-200b-3p,miR-450a-1-3p,miR-542-5p,miR-199b-5p,miR-10a-5p,miR-466c-5p,miR-450a-5p,miR-450b-5p,miR-542-3p,miR-351-5p,miR-322-3p,miR-199a-3p,miR-335-5p,miR-10b-5p,miR-351-3p,miR-155-3p,miR-497a-5p,miR-503-5p,miR-148a-3p, miR-1843a-5p, miR-199a-5p, miR-490-5p, miR-450a-2-3p, miR-322-5p, miR-214-3p,miR-450b-3p,were down-regulated in tuberous sclerosis complex cell line TSC2^（-/-）MEFs compared with normal type cell line TSC2^（＋/＋）MEFs（P 0. 05）. Real-time PCR confirmed the expressions of miR-136-5p,miR-30a-3p,miR-302b-3p,miR-10b-5p,miR-148a-3p,miR-199a-5p consistent with the microarray data（P 0. 05）. Furthermore,the overexpression of miR-199a-5p significantly inhibited cell proliferation（P 0. 05）. Conclusion： There are differences in the expression of miRNA between the tuberous sclerosis complex cell line TSC2^（-/-）MEFs and normal cell line TSC2^（＋/＋）MEFs. MiRNA-199a-5p plays an important role in tuberous sclerosis complex,which may be developed as an important molecular target for the treatment of tuberous sclerosis complex.