目的 :探讨人类泛素偶联酶E2C(ubiquitin-conjugating enzyme E2C,UBE2C)基因沉默对去势抵抗性前列腺癌PC3细胞增殖和侵袭的影响。方法:应用RT-PCR及蛋白质印迹法检测前列腺癌LNCap、PC3、DU145和C4-2细胞中UBE2C m RNA及蛋白的表达水...目的 :探讨人类泛素偶联酶E2C(ubiquitin-conjugating enzyme E2C,UBE2C)基因沉默对去势抵抗性前列腺癌PC3细胞增殖和侵袭的影响。方法:应用RT-PCR及蛋白质印迹法检测前列腺癌LNCap、PC3、DU145和C4-2细胞中UBE2C m RNA及蛋白的表达水平。将UBE2C si RNA转染入PC3细胞后,应用RT-PCR及蛋白质印迹法检测UBE2C m RNA及蛋白的表达水平,MTT法检测细胞的增殖能力,Transwell侵袭实验检测细胞的侵袭能力,蛋白质印迹法检测细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2,ERK1/2)、磷酸化ERK1/2(phospho-ERK1/2,p-ERK1/2)、蛋白激酶B(protein kinase B,PKB,又称Akt)和磷酸化Akt(phospho-Akt,p-Akt)蛋白的表达水平。结果:PC3、DU145和C4-2细胞中UBE2C m RNA和蛋白的表达水平显著高于LNCap细胞(P值均<0.05)。UBE2C si RNA转染后,PC3细胞中UBE2C m RNA及蛋白的表达水平均被抑制(P值均<0.05);UBE2C si RNA转染组PC3细胞的增殖能力和侵袭能力显著下降(P值均<0.01),p-ERK1/2和p-Akt蛋白的表达水平被显著抑制(P值均<0.01)。结论 :UBE2C基因沉默能够显著抑制去势抵抗性前列腺癌PC3细胞的增殖和侵袭,这一作用可能与下调ERK信号通路蛋白的活化有关。展开更多
UBE2C(Ubiquitin conjugating enzyme E2 C), a key regulator of cell cycle progression, is a promising target for discovery of antitumor agents. However, it is challenging to develop inhibitors of UBE2C owing to its lack...UBE2C(Ubiquitin conjugating enzyme E2 C), a key regulator of cell cycle progression, is a promising target for discovery of antitumor agents. However, it is challenging to develop inhibitors of UBE2C owing to its lack of “druggable” pockets. Bio PROTACs(biological proteolysis targeting chimeras) are a kind of protein-based degraders by fusing an adaptor to a subunit of E3 ligase for ubiquitination and subsequent proteasome-dependent degradation of target protein. We report herein the design and biological evaluation of a UBE2C-targeting bio PROTAC based on the NEL(novel E3 ligase) domain of bacterial E3 ligase Ipa H9.8 and the UBE2C-binding WHB(winged-helix B) domain of APC_(2)(anaphase promoting complex subunit 2). The in vitro ubiquitination test and Mass Spectrometry analysis showed that the bio PROTAC could transfer ubiquitin to surface exposed lysines on UBE2C and catalyzed the formation of polyubiquitin chains. In addition, the transient co-expression experiment showed that the bio PROTAC could promote proteasomal degradation of heterologous UBE2C and rescue its downstream substrates in mammalian cells.展开更多
文摘目的将构建好的UBE2C干扰质粒转入SMMC-7721细胞中,观察其对SMMC-7721细胞生物学行为的影响。方法采用脂质体法将构建好的UBE2C干扰质粒转入SMMC-7721细胞株中,潮霉素筛选4周,并经Western blot鉴定,选出干扰效果最佳的低表达稳定株(shUBE2C组)用于后续实验。用MTT法检测细胞增殖,流式细胞仪检测细胞周期,软琼脂克隆形成实验检测克隆形成能力。结果 Western blot检测显示,转shUBE2C组UBE2C的蛋白表达水平低于转阴性对照shNC组(P<0.05)。shUBE2C组的SMMC-7721细胞生长曲线明显右移,显示靶向抑制UBE2C可降低SMMC-7721细胞的增殖能力。与shNC组相比,shUBE2C组细胞的G1期细胞比例明显增高(P﹤0.05),而S期的比例明显降低(P﹤0.05),提示其延缓细胞周期进程。shUBE2C组的克隆形成率明显低于shNC组(P﹤0.05),提示其降低克隆形成能力。结论 UBE2C低表达可改变SMMC-7721细胞的生物学行为。
文摘目的 :探讨人类泛素偶联酶E2C(ubiquitin-conjugating enzyme E2C,UBE2C)基因沉默对去势抵抗性前列腺癌PC3细胞增殖和侵袭的影响。方法:应用RT-PCR及蛋白质印迹法检测前列腺癌LNCap、PC3、DU145和C4-2细胞中UBE2C m RNA及蛋白的表达水平。将UBE2C si RNA转染入PC3细胞后,应用RT-PCR及蛋白质印迹法检测UBE2C m RNA及蛋白的表达水平,MTT法检测细胞的增殖能力,Transwell侵袭实验检测细胞的侵袭能力,蛋白质印迹法检测细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2,ERK1/2)、磷酸化ERK1/2(phospho-ERK1/2,p-ERK1/2)、蛋白激酶B(protein kinase B,PKB,又称Akt)和磷酸化Akt(phospho-Akt,p-Akt)蛋白的表达水平。结果:PC3、DU145和C4-2细胞中UBE2C m RNA和蛋白的表达水平显著高于LNCap细胞(P值均<0.05)。UBE2C si RNA转染后,PC3细胞中UBE2C m RNA及蛋白的表达水平均被抑制(P值均<0.05);UBE2C si RNA转染组PC3细胞的增殖能力和侵袭能力显著下降(P值均<0.01),p-ERK1/2和p-Akt蛋白的表达水平被显著抑制(P值均<0.01)。结论 :UBE2C基因沉默能够显著抑制去势抵抗性前列腺癌PC3细胞的增殖和侵袭,这一作用可能与下调ERK信号通路蛋白的活化有关。
基金supported by the National Natural Science Foundation of China (No. 21907006 to Yuxin Ye)the Natural Science Foundation of Guangdong Province (No. 2020A1515011544to Yuxin Ye)the Shenzhen Science and Technology Innovation Committee (No. JCYJ20180302150357309 to Yuxin Ye, and No.JCYJ20200109140401752 to Hao Huang)。
文摘UBE2C(Ubiquitin conjugating enzyme E2 C), a key regulator of cell cycle progression, is a promising target for discovery of antitumor agents. However, it is challenging to develop inhibitors of UBE2C owing to its lack of “druggable” pockets. Bio PROTACs(biological proteolysis targeting chimeras) are a kind of protein-based degraders by fusing an adaptor to a subunit of E3 ligase for ubiquitination and subsequent proteasome-dependent degradation of target protein. We report herein the design and biological evaluation of a UBE2C-targeting bio PROTAC based on the NEL(novel E3 ligase) domain of bacterial E3 ligase Ipa H9.8 and the UBE2C-binding WHB(winged-helix B) domain of APC_(2)(anaphase promoting complex subunit 2). The in vitro ubiquitination test and Mass Spectrometry analysis showed that the bio PROTAC could transfer ubiquitin to surface exposed lysines on UBE2C and catalyzed the formation of polyubiquitin chains. In addition, the transient co-expression experiment showed that the bio PROTAC could promote proteasomal degradation of heterologous UBE2C and rescue its downstream substrates in mammalian cells.
