PinX1差异表达乳腺癌中LncRNA的筛选及其对肿瘤发生的作用机制研究

Our previous study found that, as a potential tumor suppressor gene, although PinX1 decreased in the majority of breast cancer specimens, the mRNA expression level of PinX1 varied greatly in different samples. Furthermore, LncRNA expression profile changes could be induced after we regulated the expression level of PinX1 in MCF-7 breast cancer cell line, which suggesting some interaction may exist between LncRNAs and PinX1, and the LncRNAs may affect the molecular function of PinX1 through after transcription level and protein level regulation. This project intends to screen out the differentially expressed LncRNAs between low and high expressed PinX1 breast cancer specimens through the whole transcriptome library construction and Solexa sequencing technology. The differentially expressed LncRNAs screened out are going to be annotated, predicted for target genes and transcription factor binding sites. The differentially expressed LncRNAs and mRNAs will then be applied for a co-expression analysis, a co-expression network is going to be constructed and the key node LncRNAs of the network will be confirmed by the 5'and 3'RACE amplification. Quantitative PCR will be applied to verify the expression level of the LncRNAs. ChIRP and RNA pull-down are going to be used to verify the interaction of the LncRNAs with PinX1 and other target moleculars. Furthermore, the LncRNAs screened out will be overexpressed or knocked down in breast cancer cell MCF-7 to study their regulation on the tumor cell proliferation. Thus to confirm the molecular function of PinX1, as well as providing new theoretical basis for the regulation of tumorigenesis of breast cancer by the non-coding RNAs.

我们前期研究发现，PinX1作为潜在抑癌基因，在大多数乳腺癌中明显降低，但其mRNA表达水平在不同标本间差异较大。调控乳腺癌MCF-7细胞的PinX1表达水平，可引起LncRNA表达谱改变，提示可能存在LncRNA与PinX1的互作，并通过转录后水平及蛋白水平等调控作用，影响其分子功能的实现。本项目拟采用全转录组Solexa测序技术，筛选出PinX1低表达与高表达乳腺癌标本的差异LncRNA，对其重注释，预测靶基因、转录因子结合位点，与差异mRNA共表达分析筛选出关键节点LncRNA，5'及3'RACE扩增确认，定量PCR验证其表达水平， ChIRP及RNA pull-down验证其与PinX1及下游靶分子的互作，进而构建乳腺癌MCF-7细胞的LncRNA过表达及敲减模型，体内外实验研究其对肿瘤细胞增殖的影响，为确认PinX1分子功能，探讨乳腺癌发生的非编码RNA调控机制提供新的理论依据。

PinX1在大多数乳腺癌中表达降低，具有类似抑癌基因的功能。调控乳腺癌MCF-7细胞中PinX1表达水平，可引起LncRNA表达谱的改变，PinX1可能通过与LncRNA的互作，在转录后水平及蛋白水平上发挥调控作用。本项目采用全转录组Solexa测序技术建立了PinX1表达差异的乳腺癌标本转录组学数据库，筛选出PinX1低表达vs. PinX1高表达乳腺癌标本中差异表达的LncRNA及mRNA、以及cSNP突变、结构变异转录本、低丰度及未知转录本等，初步揭示了少数乳腺癌临床标本中PinX1表达水平并不降低的原因，确认了PinX1作为潜在性抑癌基因的分子功能。随后，通过生物信息学方法，对增强子样LncRNA、Hox基因簇内的LncRNA、基因间的Rinn lincRNAs进行了分类分析。筛选在乳腺癌发生中具有重要调控作用的分子，以及与其可能存在共表达调控关系的LncRNA，构建了PinX1表达水平差异的乳腺癌标本LncRNA-mRNA共表达调控网络，对关键节点 LncRNA分子进行RACE克隆和确认工作、以及LncRNA互作蛋白的RNA pull-down及质谱鉴定工作、PinX1相关分子TMEM40及其非编码突变体TMEM40-AS3的体内、外功能进行研究，为从非编码RNA水平上进一步完善PinX1在乳腺癌发生中的作用及分子机制提供了重要的实验依据。