摘要
长链非编码RNAs(long noncoding RNAs,lncRNAs)可在表观遗传水平、转录水平和转录后水平调节基因的表达,对细胞功能起着重要的调节作用。RNA结合蛋白可与很多的RNA结合,并在转录后水平发挥重要的调节作用。然而,RNA结合蛋白是否可以在细胞内广泛结合lncRNAs对其发挥调节作用,仍需进一步证实。本研究通过RNA结合蛋白免疫沉淀技术联合高通量测序(RNA binding protein immunoprecipitation-high throughput sequencing,RIP-Seq)的方法在人肝癌细胞株HepG2中,鉴定与人抗原R(human antigen R,HuR)蛋白相结合的lncRNA分子,并进行了初步的验证。首先,通过HuR-RIP实验分离与HuR蛋白结合的RNA分子,然后高通量测序及生物信息学分析。根据分析结果,鉴定出HepG2细胞中361条与HuR蛋白结合的lncRNAs分子,包括基因间lncRNA(large intergenic noncoding RNA,LincRNA)、内含子lncRNA、与编码基因正义链有重叠的lncRNA和与编码基因反义链有重叠lncRNA(antisense lncRNA)等。并进一步通过RIP-qPCR技术,对其中20条LincRNA分子进行了定量检测,验证测序结果。在HepG2细胞中敲低HuR基因表达,发现这些LincRNA分子中,11条LincRNA分子表达水平显著降低(P<0.05),2条LincRNA显著升高(P<0.05),剩余7条LincRNA表达量未发生变化(P>0.05)。本研究结果说明,HuR在细胞内可以广泛结合lncRNA分子,并且可能对结合的lncRNA分子的表达量产生影响,这也为进一步研究这些lncRNA的功能和HuR调控网络的研究提供了基础。
Long noncoding RNAs（ lncRNAs） can regulate gene expression at epigenetic,transcriptional and post-transcriptional levels and play an important role in the regulation of cell function. RNA-binding proteins can bind to RNAs and play an important regulatory role at post-transcriptional level. However,whether RNA-binding proteins can widely bind to lncRNAs and regulate their expression in cells remains to be confirmed. In this study,we used the RNA binding protein immunoprecipitation-high throughputsequencing（ RIP-Seq） technique to identify lncRNAs which bind to human antigen R（ HuR） protein in HepG2 cells. The experiments were first performed to extract RNAs that bound to the HuR protein,followed by high-throughput sequencing and bioinformatics analysis. According to the results,totally 361 lncRNAs binding to HuR protein in HepG2 cells were identified,including large intergenic lncRNA（ LincRNA）,Intron lncRNA,lncRNAs overlapping with the sense or antisense strands of coding genes,and so on. Furthermore, 20 LincRNAs were quantitatively detected by RIP-qPCR to verify the sequencing results. After knockdown of HuR gene expression in HepG2 cells,we found that eleven of the twenty lincRNAs were significantly down-regulated（ P〈 0. 05） and two of lincRNAs were upregulated（ P 〈0. 05）,the remainder seven LincRNAs had no significant changes（ P 〈0. 05）. The results suggested that HuR could bind extensively to lncRNAs in cells and might affect the expression of lncRNAs. These findings provided information for the further study of the function of these lncRNAs and the HuR regulatory network.
作者
黄亚洲
刘叶文
王玲玲
金晶
陈茜
李伟
HUANG Ya-Zhou;LIU Ye-Wen;WANG Ling-Ling;JIN Jing;CHEN Qian;LI Wei(Wenzhou Medical University School of Laboratory Medicine and Life Sciences;Key Laboratory of Medical Genetics of Zhejiang Province, Wenzhou 325035, Zhejiang, China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2018年第4期417-426,共10页
Chinese Journal of Biochemistry and Molecular Biology
基金
浙江省自然科学基金资助(No.LY13H070006)~~
