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苯并[a]芘诱导的恶性转化细胞中DNA聚合酶β高表达的机制研究

Mechanism of DNA Polymerase Beta Hyper-expression in Malignant Transformation Induced by Benzo[a] Pyrene

  • 摘要: 【摘要】 目的 探讨苯并[a]芘(BaP)诱导的恶性转化细胞中DNA聚合酶β(polβ)表达增加的可能机制。方法 采用RT-PCR-单链构象多态性 (RT-PCR-SSCP) 分析及基因测序检测BaP诱导的恶性转化细胞(polβ-T细胞)中polβ基因外显子序列;基因测序检测polβ基因启动子序列。采用RT-PCR和Western blot检测polβ-T细胞和未经BaP处理的对照细胞(polβ细胞)中蛋白精氨酸甲基转移酶6 (PRMT6) mRNA及蛋白质表达水平。结果 RT-PCR-SSCP和基因测序未发现polβ-T细胞中polβ基因外显子序列改变,但其启动子区域经基因测序证实5′端上游-10位和-61位分别存在插入突变(插入G)和点突变(C→A)。此外,polβ-T细胞中PRMT6 mRNA和蛋白表达量均较对照polβ细胞增高(P<0.05)。结论 BaP诱导的恶性转化细胞中polβ基因的高表达不伴随其外显子序列的突变,但与启动子序列遗传学突变密切相关,PRMT6还可能通过表观遗传学途径导致polβ的高表达。

     

    Abstract: 【Abstract】 Objective To explore the mechanism of the hyper-expression of DNA polymerase beta (polβ) in benzo[a]pyrene (BaP) induced malignant transformed cell (polβ-T). Methods The mutation of polβ gene exon and promoter were examined using reverse transcriptase-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) and gene sequencing. The expression of protein-arginine N-methyhransferase 6 (PRMT6) mRNA and protein in polβ-T cell and control cell (polβ cell) were investigated by RT-PCR and Western blot. Results RT-PCR-SSCP and gene sequencing revealed that the hyper-expression of polβ in polβ-T cell was not associated with the mutation of polβ gene exon while insert mutation (G) and point mutation (C→A) were found located in the core region of polβ gene promoter. Furthermore, the expression of PRMT6 mRNA and protein also increased in polβ-T cell compared with control cell (P<0.05). Conclusion The enhancement of expression of polβ in polβ-T cell might be attributed to the mutations locating in polβ gene promoter on transcription level of polβ gene, and PRMT6 might also enhance the expression of polβ in polβ-T cell through relative epigenetic pathways.

     

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