环境污染物As及SHP2D61G/＋激活对小鼠成纤维母细胞miRNA表达谱的影响

Effect of environmental pollutant-As and SHP2^(D61G/+) activation on miRNA expression profiles in MEFs

目的建立环境致癌物与基因突变联合因素影响下的miRNA表达谱,为肿瘤发生机制的进一步研究提供新线索。方法同等条件下,首先制备SHP2+/+野生型(wild type,Wt)及SHP2D61G/+激活突变(gain-of-function mutation,GOF mutation)小鼠永生化的成纤维母细胞(mouse embryonic fibroblasts,MEFs)细胞,之后以0.5μmol/L的三氧化二砷急性处理MEFs细胞48 h后提取细胞总RNA,用小鼠miRNA芯片杂交/分析,建立miRNA(microRNA,miRNA,miR)表达谱,筛选差异表达显著者,用RT-PCR进行验证。结果重金属化合物三氧化二砷处理SHP2+/+野生型和SHP2D61G/+突变的MEFs细胞48h后,数十个miRNAs出现差异性表达,miRNA在三氧化二砷诱导前后的细胞或SHP2激活突变与否的细胞出现表达增加或减少,且发现受三氧化二砷和SHP2激活突变影响变化一致的miRNAs,如mmu-miR-100(表达下降)、mmu-miR-1907(表达升高)等,RT-PCR检测发现mmu-miRNA-100、mmu-miRNA-1907的表达趋势与芯片结果是一致的。结论基因突变与环境污染可以影响MEFs细胞的miRNA表达谱出现明显改变,这些改变可能是细胞恶性转化及肿瘤发生的基础。

Objective Establish the miRNA expression profiling under environmental carcinogens and gene mutations, to provide some new clues for further study on the mechanism of tumorgencsis. Methods Under the same condition, mice were first prepared in SHP2 ^＋/＋ normal （wild type, Wt）and SHP2^D61G/＋ activating mutation （gain-of- function mutation, GOF mutation）of immortalized fibroblast cells（ mouse embryonic fibroblasts, MEFs）, acute treat MEFs cells with 0. 5μLmoL/L As2 03 48 hours, then extract the cell total RNA. Use the analysis of miRNA hybridization to establish the miRNA （microRNA, miRNA, miR）expression profiling, screening the miRNAs which differential expressionsignificantly, then validated by RT-PCR. Results After treated the SHP2 ^＋/＋ MEFs and SHP2^D61G/＋ MEFs with As203, dozens of miRNAs showed differential expression. The expression of miRNAs increase or decrease in different SHP-2^ ＋/＋ MEFs or SHP-2^D61G/＋ MEFs when treat with As203 or not, at the same time, we discovered some miRNAs show the same change along with the As203 treating and SHP2^D61G/＋ activating mutation, such as mmu-miRNA-100 （down regulation） and mmu-miRNA-1907（ np regulation）. Conclusion Gene mutation and environmental pollution can effect the miRNAs expression profiling of MEFs significantly, these change maybe the basis of cells malignant transformation and tumourgenesis.