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DNA损伤是肿瘤发生的基础,细胞内应答DNA损伤的分子信号通路是阻挡癌症发生的屏障。近年来发现Smad1在DNA损伤应答的分子信号通路中具有重要的作用。本实验旨在探究结直肠癌患者组织中Smad1的序列突变及其生物学意义。本实验提取结直肠癌患者肿瘤组织和正常组织的总RNA并逆转录成c DNA,分段扩增Smad1基因并进行克隆和双向测序,测序结果进行比对,发现Smad1的MH1区域与MH2区域的错义突变较少,6个主要的错义突变均集中在Linker区域,其氨基酸残基改变分别是:N147H、P167S、T202A、M218I、M247V和A248V,其中Thr202是已知的GSK3作用位点;正常组织及肿瘤组织中的突变情况不同,T202A和M218I分别是仅存在于正常组织和肿瘤组织中的突变,提示Smad1 Linker区域的突变可能影响其生物学功能。并利用RNAfold和SOPMA在线服务器分别预测发现错义突变对Smad1的RNA二级结构和蛋白质二级结构造成不同程度的影响。
DNA damage is the basis of tumorigenesis. The molecular signaling pathway that responds to DNA damage in the cell is a barrier that blocks the development of cancer. In recent years, Smad1 has been found to play an important role in the molecular signaling pathway of DNA damage response. The purpose of this experiment was to explore the sequence mutation of Smad1 in the tissues of patients with colorectal cancer and its biological significance. In this study, total RNA was extracted from tumor tissues and normal tissues of patients with colorectal cancer and reverse transcribed into c DNA. The Smad1 gene was amplified and cloned by two-way sequencing. The sequencing results were compared and found that the MH1 region of Smad1 and MH2 region The six major missense mutations were concentrated in the Linker region. The changes of amino acid residues were: N147H, P167S, T202A, M218I, M247V and A248V, among which Thr202 was the known site of GSK3. Mutations in normal tissues and tumor tissues are different. T202A and M218I are mutations that exist only in normal tissues and tumor tissues, respectively, suggesting that mutations in the Smad1 Linker region may affect their biological functions. And RNAfold and SOPMA online server were used to predict the missense mutation to Smad1 RNA secondary structure and protein secondary structure to varying degrees.