摘要
目的探讨转化生长因子β(TGF-β)调控人胆管癌细胞系RBE细胞增殖的关键微RNA(miRNA)及其潜在的机制。方法该研究起止时间为2020年1月至2022年1月。磷酸盐缓冲液(PBS)处理为对照组,TGF-β处理为TGF-β组,TGF-β抗体处理为抗体组。检测三组RBE细胞的增殖水平。miRNA高通量测序检测三组RBE细胞的miRNA调控变化,并进行miRNA模拟物过表达筛选鉴定受TGF-β调控的影响RBE细胞增殖水平的关键miRNA。miRNA数据库(miRDB)在线分析miRNA的潜在底物,并通过小干扰RNA(siRNA)敲低筛选鉴定影响RBE细胞增殖水平的关键底物。结果相比于对照组,TGF-β组RBE细胞的增殖水平上升(1.62±0.07比2.35±0.09,P<0.05),抗体组RBE细胞的增殖水平下降(1.62±0.07比1.11±0.08,P<0.05)。过表达微RNA-196a-1-3p(miR-196a-1-3p)时,RBE细胞的增殖水平下降(P<0.05)。敲低Ras响应元件结合蛋白(RREB1)时,RBE细胞的增殖水平下降(P<0.05)。过表达miR-196a-1-3p后,RBE细胞中RREB1的信使RNA(mRNA)和蛋白水平下降(P<0.05)。敲低miR-196a-1-3p后,RBE细胞中RREB1与SMAD家族蛋白3(SMAD3)的相互作用增加。敲低SMAD3后,RBE细胞的增殖水平下降(P<0.05)。与仅敲低SMAD3相比,敲低SMAD3的同时过表达RREB1的RBE细胞的增殖水平无显著变化,并且同时敲低SMAD3和miR-196a-1-3p的RBE细胞的增殖水平无显著变化。结论TGF-β能够通过miR-196a-1-3p/RREB1/SMAD3轴促进RBE细胞增殖;miR-196a-1-3p和RREB1可作为潜在的治疗胆管癌的靶标,为针对该靶标的新药研发奠定了基础。
Objective To investigate the key microRNAs(miRNAs)of transforming growth factorβ(TGF-β)regulating cholangiocarcinoma cells(RBE)proliferation and their potential mechanisms.Methods The starting and ending time of this study was from January 2020 to January 2022.Phosphate buffer(PBS)treatment was used as the control group,TGF-βtreatment as the TGF-βgroup,and TGF-βantibody treatment as the antibody group.The proliferation levels of RBE cells in the three groups were detected.miRNA highthroughput sequencing(miRNA-seq)was performed to detect miRNA regulatory changes in the three groups of RBE cells,and miRNA mimics overexpression screening was performed to identify key miRNAs regulated by TGF-βthat affect the proliferation levels of RBE cells.miRNA database(miRDB)was analyzed online for potential substrates of miRNAs,and small interfering RNA(siRNA)knockdown screen to identify key substrates affecting the proliferation level of RBE cells.Results Compared to the control group,the proliferation level of RBE cells was increased in the TGF-βgroup(1.62±0.07 vs.2.35±0.09,P<0.05)and decreased in the antibody group(1.62±0.07 vs.1.11±0.08,P<0.05).The proliferation level of RBE cells was decreased upon overexpression of miR-196a-1-3p(P<0.05).The proliferation level of RBE cells was decreased when Ras response element binding protein(RREB1)was knocked down(P<0.05).The messenger RNA(mRNA)and protein levels of RREB1 were decreased in RBE cells after overexpression of miR-196a-1-3p(P<0.05).The interaction of RREB1 with SMAD3 was increased in RBE cells after knockdown of miR-196a-1-3p.The proliferation level of RBE cells decreased after knockdown of SMAD3 (P<0.05). There was no significant change in the proliferation level of RBE cells with knockdown of SMAD3 and overexpression of RREB1 compared to knockdown of SMAD3 alone, and there was no significant change in the proliferation level of RBE cells with both SMAD3 and miR-196a-1-3p knockdown.Conclusion TGF- β can promote RBE cell proliferation through miR-196a-1-3p/RREB1/SMAD3 axis. miR-196a-1-3p and RREB1 can be used as potential targets for the treatment of cholangiocarcinoma, laying the foundation for the development of new drugs against this target.
作者
丁敬健
张升涛
郭永锋
王尚毓
罗孔亮
董伟
DING Jingjian;ZHANG Shengtao;GUO Yongfeng;WANG Shangyu;LUO Kongliang;DONG Wei(Department of General Surgery,Xi´an Ninth Hospital,Xi´an,Shaanxi 710054,China;Hepatobiliary Department,First Affiliated Hospital of Air Force Military Medical University,Xi´an,Shaanxi 710032,China;Department of General Surgery,XD Group Hospital,Xi´an,Shaanxi 710077,China)
出处
《安徽医药》
CAS
2024年第7期1399-1403,I0004,共6页
Anhui Medical and Pharmaceutical Journal
基金
西安市卫生厅科研基金项目(20A038)。
