ABSTRACT
@#[摘 要] 目的：探讨lncRNA FAM95B1对胶质瘤细胞增殖和迁移能力的影响并探究其相关作用机制。方法： 选取2018年1月至2020年8月于合肥市第三人民医院行手术治疗的38例胶质瘤患者的胶质瘤组织及癌旁组织标本，利用qPCR检测胶质瘤组织与4种细胞系中FAM95B1的表达水平，以表达最低的胶质瘤LN382细胞为研究对象，转染空载质粒（对照组）或pcDNA3.1-FAM95B1质粒（实验组）。MTT法和划痕实验检测FAM95B1对LN382细胞增殖和迁移能力的影响。生物信息学分析技术和双荧光素酶基因报告实验预测并验证FAM95B1与miR-26a-5p及PTEN之间的相互作用机制，应用qPCR和WB法检测FAM95B1对miR-26a-5p和PTEN表达的影响。结果： FAM95B1在胶质瘤组织中的表达明显低于癌旁组织（P<0.01）。FAM95B1在多种胶质瘤细胞系中的表达均明显低于正常脑胶质细胞（均P<0.01）。过表达FAM95B1可以下调LN382细胞的增殖（P<0.05）和迁移能力（P<0.01）。FAM95B1能够靶向结合miR-26a-5p（P<0.01），miR-26a-5p能够靶向结合PTEN mRNA（P<0.01）。过表达FAM95B1可下调LN382细胞中miR-26a-5p的表达（P<0.01），促进PTEN mRNA的表达（P<0.01）。结论：在胶质瘤组织和细胞系中异常低表达的FAM95B1通过发挥竞争性内源RNA的功能抑制miR-26a-5p的表达而增强PTEN蛋白表达，抑制胶质瘤细胞系LN382的增殖和迁移。
ABSTRACT
Background: The dried sclerotium of medicinal fungus Polyporus umbellatus (Pers.) Fries has many pharmacological functions such as diuretic and anticancer activity, in which high-content polysaccharides may play an important role. However, RNA isolation is difficult in filamentous fungi and lacking in P. umbellatus. Results: Five methods for RNA extraction from five strains collected from four provinces were assessed for their ability to recover a high-quality RNA applicable for sequence-related amplification polymorphism (SRAP) PCR and GDP-D-mannose pyrophosphorylase (GMP) gene expression profiles. Both A260/A280 and A260/A230 ratios of the best Trizol Plus + RNAiso-mate for Plant Tissue method are around 2 with a yield of 1122.00 +/- 0.21 ng ul-1. The Trizol method also showed good quality with the yield 469.60 ng ul-1. The SRAP PCR amplified clear and polymorphic bands in all five cDNA samples transcribed from RNA by using primer Me4-Em4. GMP gene fragment (1251 bp) was successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. Conclusion: All these results showed that the total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications.