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目的:前期工作发现补体应答基因32(RGC-32)是转化生长因子β(TGF-β)诱导的肾小管上皮细胞-间充质转化(EMT)过程中Smad信号下游的关键调控分子。本研究拟进一步明确TGF-β诱导肾小管EMT过程中RGC-32的转录调控机制。方法:体外培养NRK-52E细胞,利用荧光素酶报告基因实验检测RGC-32启动子活性以确定Smad结合元件(SBE)是否为TGF-β诱导的RGC-32转录调控位点;通过凝胶迁移率实验(EMSA)明确与之结合的转录调控分子。结果:(1)转染野生型SBE片段的NRK-52E细胞荧光素酶活性明显增高,而转染突变的SBE片段的细胞无此表现,表明SBE是调控TGF-β诱导的RGC-32转录启动的关键位点。(2)合成32P标记的包含SBE的寡核苷酸探针行EMSA可见TGF-β诱导的核蛋白与探针复合物生成,且加入包含SBE序列的竞争片段可以抑制此核蛋白-探针复合物的形成,表明有蛋白与SBE结合并调控RGC-32的转录。(3)当加入不同的Smad抗体行超迁移实验发现Smad2和Smad3抗体可以和复合物相互作用,表明Smad2和Smad3可能是TGF-β诱导的转录复合物的组成部分。(4)将Smad2和Smad3分别与p-1500RGCluc共同转染NRK-52E细胞,Smad3可以显著增加RGC-32启动子活性,表明Smad3是TGF-β诱导的转录复合物的组成部分,而不是Smad2。结论:在TGF-β诱导肾小管EMT过程中,Smad3通过与RGC-32启动子区SBE结合,从而调控肾小管EMT过程。
OBJECTIVE: Previous work found that complement response gene 32 (RGC-32) is a key regulatory molecule downstream of Smad in transforming growth factor β (TGF-β) -induced tubulointerstitial-mesenchymal transition (EMT). This study was to further clarify the transcriptional regulation of RGC-32 during TGF-β-induced tubular EMT. Methods: The NRK-52E cells were cultured in vitro and the activity of RGC-32 promoter was detected by luciferase reporter assay to determine if the Smad binding element (SBE) was the TGF-β-induced RGC-32 transcriptional regulatory site. Rate Assay (EMSA) A transcriptional regulator that binds specifically to it. Results: (1) The luciferase activity of NRK-52E cells transfected with wild-type SBE fragments was significantly higher than that of the transfected SBE fragments, indicating that SBE regulates TGF-β-induced RGC-32 transcriptional activation The key site. (2) Synthesis of 32P labeled SBE-containing oligonucleotide probes EMSA showed that TGF-β-induced nucleoprotein and probe complex formation, and adding a competitive fragment containing SBE sequence can inhibit this nuclear protein - probe complex The formation of the substance indicates that the protein binds to SBE and regulates the transcription of RGC-32. (3) Smad2 and Smad3 antibodies interacted with the complex when added with different Smad antibodies, indicating that Smad2 and Smad3 may be part of the TGF-β-induced transcription complex. (4) Smad2 and Smad3 co-transfected with p-1500RGCluc, respectively, and NRK-52E cells, Smad3 can significantly increase the activity of RGC-32 promoter, indicating that Smad3 is part of TGF-βinduced transcription complex, but not Smad2. Conclusion: TGF-β induces tubular EMT during renal tubular EMT through binding Smad3 with SBE in RGC-32 promoter region.