MicroRNA应答一碳单位代谢主要组分缺乏并调控错配修复通路核心基因表达的机制研究

One-carbon metabolism(OCM) containing methyl chemicals/ coenzymes is critical for DNA synthesis, integrity and methylation reactions. Our previous research indicated that the major components of OCM such as folate, choline and vitamin B12 deficiency induced genomic structural damage and chromosomal nonsegregation, global genomic methylation aberration and specific gene expression change. The disfunction of OCM is a fundamental cause of imprinting disorders, tumorigenesis, neurological disorders, cardiovascular disease and autoimmune diseases. MicroRNAs(miRNAs), a class of small non-coding RNAs, are fundamental for the post-transcriptional regulation of gene expression. miRNAs are able to sense the internal/external environmental changing and genotoxic/ epigenetic carcinogen exposures , modulate the relative biological processes, affect the metabolism and health status. The research will focus on the response of miRNAs to the functional variation of OCM and the regulation of DNA mismatch repair (MMR). Human colon epithelial cell line CRL-1790 is going to be intervened by the modified RPMI 1640 medium with deficiency/sufficient concentrations of major OCM components( folic acid, FA; 5-methyltetrahydrofolate,5-MeTHF; Choline, CL; Vitamin 12, VB12). Bioinformatics analysis, sRNA-seq, miRNA luciferase reporter gene assay, qRT-PCR and western blotting will be employed to analyze the alteration of the core genes of MMR(hMSH2、hMSH6、hMLH1 and hPMS2) expression, of miRNA expression profile under the deficiency/sufficient of major OCM components. The experiment will also explore the mechanisms of post-transcriptional regulation of miRNAs which target core genes of MMR and response the OCM variation simultaneously. The research will reveal the mechanisms of miRNAs response OCM variation and regulating the DNA damage repair under the genotoxic stress. It could be benefit to establish some new linkers for genomic stability and the diagnosis/ treatment of gene-nutrition interaction related disease from the angle of miRNA.

一碳单位代谢（OCM）中的甲基化合物/辅酶与DNA合成和甲基化息息相关。前期工作证实叶酸、胆碱、维生素B12等任一OCM组分缺乏导致基因组损伤与甲基化模式改变。OCM紊乱是多数肿瘤、基因组印记性和退行性疾患的病因之一。miRNA可感应细胞内外环境变化、调节相应生物学过程并影响代谢与健康。研究围绕miRNA应答OCM变异并调控DNA错配修复（MMR）的主题，以不同OCM组分缺乏/充足条件分别干预人MMR阳性细胞，借助生物信息学、miRNA表达谱分析、荧光素酶报告基因检测、qRT-PCR、western blotting等技术，探讨OCM主要组分浓度变化引起的MMR通路MutS、MutL家族核心基因表达和miRNA表达谱改变；筛选并确认既应答OCM变异又靶向MMR核心基因的miRNA；揭示它们对MMR核心基因的转录后调控机制，解析miRNA响应OCM变异并影响机体应对遗传损伤胁迫的修复模式。

一碳单位代谢(OCM)是以各种不同形式叶酸为基点的一碳基团的微营养素代谢网络，涉及DNA合成与损伤修复、DNA甲基化等遗传学和表观遗传学过程。OCM的紊乱是多数肿瘤、基因组印记和退行性疾患的病因之一。miRNA可感应包括微营养环境在内的细胞内外环境变化、调节相应生物学过程并影响代谢与健康。研究围绕①OCM核心微营养组分浓度变化对miRNA表达谱的影响、②靶向错配修复(MMR)核心基因和OCM代谢关键基因的miRNAs确证、③OCM 核心组分浓度变化对MMR通路核心基因表达的影响、④OCM 核心组分浓度变化对靶向MMR核心基因的miRNAs表达的影响、⑤目标 miRNAs对MMR核心基因表达的调控机制等问题开展了全面研究、⑥研究还拓展性地探讨了既响应OCM重要组分缺乏、又靶向调控内质网应激标志基因HSPA5/生长阻滞和 DNA 损伤诱导基因Gadd45α的一组miRNAs。.研究揭示了OCM 核心微营养素叶酸浓度改变引起的miRNA和被靶向基因的转录谱改变；验证了靶向hMLH1、hMSH2、 HSPA5、Gadd45α等一系列与DNA损伤修复、自噬与凋亡、生长阻滞和 DNA 损伤诱导等相关基因的miRNAs；解析了相关miRNAs对hMLH1、hMSH2、 HSPA5、Gadd45α等基因的调控机制，从而在多个层面探讨了OCM微营养素尤其叶酸的缺乏对DNA损伤形成、修复、细胞周期的调控、细胞死亡等途径的影响，为以微营养素供给状态对基因组损伤与修复的关联提出了进一步的科学依据；研究还发现不同病理生理状态下的细胞对OCM 组分尤其叶酸缺乏具有不同的响应与生物学结局：叶酸缺乏对正常细胞的基因组稳定性、损伤修复、细胞死亡等机制具有胁迫效应；而相同的叶酸缺乏浓度对于肿瘤细胞则出现不同的效应，研究为个性化的营养干预与miRNA介导的肿瘤治疗提供了新的思考。