BCOR突变通过PI3K/Akt/mTOR通路介导MDS细胞生物学行为改变的机制研究

BCOR Mutation Induces the Change of Cell Biological Behaviors in Myelodysplastic Syndromes via PI 3K/Akt/mTOR Pathway

目的 探讨BCOR(BCL6 co-repressor)基因突变介导骨髓增生异常综合征(myelodysplastic syndromes, MDS)细胞生物学行为的分子机制。方法 通过构建BCORP483L过表达慢病毒以及空载体病毒,转染K562细胞,采用集落形成实验检测BCOR突变对细胞克隆形成能力的影响,流式细胞术检测细胞凋亡、细胞周期,Western blot法和实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction, RT-qPCR)方法检测细胞焦亡和PI3K/Akt/mTOR通路分子水平,采用转录组测序技术(RNA-sequencing, RNA-Seq)检测分析BCORP483L突变对下游靶基因表达的影响。结果 BCORP483L突变显著地抑制细胞克隆形成能力,促进细胞凋亡,并导致细胞周期阻滞和细胞焦亡水平增加。RNA-Seq分析发现,BCORP483L突变后K562内与细胞凋亡、G2M周期检查点、慢性髓系白血病、细胞衰老、细胞内炎性反应相关的基因以及原癌基因MYC目标基因表达上调。Western blot法实验进一步证实,BCORP483L突变细胞的PI3K/Akt/mTOR通路明显受到抑制。结论 BCOR突变导致细胞PI3K/Akt/mTOR通路受到抑制,进而导致细胞凋亡增加、增殖受抑。BCOR突变能通过上调细胞衰老相关基因以及原癌基因MYC目标基因表达,提高细胞内炎性反应水平,从而促进MDS疾病进展。

Objective To explore the molecular mechanism of BCL6 co-repressor(BCOR)gene mutation in mediating the cell biological behaviors of myelodysplastic syndromes(MDS).Methods K562 cells were transfected with BCOR P483L expression lentivirus and empty vector virus,respectively.Colony formation test was used to detect the effect of BCOR P483L mutation on the clonogenic formation ability of cells.The cell apoptosis and cell cycle were analyzed by flow cytometry.Western blot and real-time quantitative polymerase chain reaction(RT-qPCR)were used to detect the levels of pyroptosis and PI 3K/Akt/mTOR pathway.And RNA-Sequencing(RNA-Seq)was used to detect the effect of BCOR P483L mutation on the expressions of downstream target genes.Results BCOR P483L mutation significantly inhibited the colony formation ability of cells,promoted cell apoptosis,blocked the cell cycle,and increased pyroptosis.RNA-Seq analysis showed that the genes related to cell apoptosis,G 2M cycle checkpoint,chronic myeloid leukemia,cell senescence,intracellular inflammatory response,and the target genes of oncogene MYC were up-regulated in the K562 cells after BCOR P483L mutation.Western blot experiment further confirmed that BCOR P483L mutation significantly inhibited the PI 3K/Akt/mTOR pathway in the K562 cells.Conclusion BCOR P483L mutation promotes cell apoptosis and inhibits cell proliferation via the inhibition of PI 3K/Akt/mTOR pathway.And BCOR P483L mutation can up-regulate the cell-senescence-related genes,oncogene MYC target genes,and increases the level of the intracellular inflammatory response,thus promoting the development of MDS.