【作者】 汪志强；

【导师】 王明华；

【作者基本信息】 苏州大学 ， 生物化学与分子生物学， 2023， 硕士

【摘要】 背景:肝内胆管癌(iCC)发病机制复杂,恶性程度高,严重危害国民的健康与安全。实验室前期研究发现,在肝内胆管癌中E3泛素连接酶Tripartite Motif Containing 58(TRIM58)是受高甲基化调控的抑癌基因,但TRIM58发挥抑癌作用的分子机制尚不清楚。在此基础上,本文筛选并鉴定了在肝内胆管癌中受TRIM58泛素化修饰降解的下游靶蛋白 Kinesin Family Member C1(KIFC1),探究 TRIM58 对 KIFC1 的具体调控机制,明确TRIM58-KIFC1信号轴在肝内胆管癌中的生物学功能,以期对肝内胆管癌的发病机制有更深入的认识。方法:(1)通过Co-IP、Western Blot、免疫荧光实验对候选互作蛋白进行鉴定,并通过RT-qPCR检测底物蛋白的mRNA水平。(2)联合使用蛋白合成抑制剂CHX、蛋白酶体抑制剂MG132、溶酶体抑制剂CQ处理肝内胆管癌细胞系,利用Western Blot检测KIFC1蛋白水平变化来鉴定KIFC1的降解途径类型。(3)通过CHX追踪试验,鉴定TRIM58在肝内胆管癌细胞中对KIFC1的稳定性影响,以及通过蛋白泛素化检测实验验证TRIM58对KIFC1泛素化水平影响。(4)构建TRIM58与KIFC1的结构域缺失型表达载体,并转染HEK293T细胞,利用Co-IP对两者的结合结构域进行鉴定。(5)通过TRIM58剂量依赖性实验与蛋白泛素化检测实验,探究功能结构域的缺失在TRIM58介导的KIFC1泛素化修饰降解过程中的影响。(6)利用定点突变技术构建7种赖氨酸突变的泛素蛋白表达载体(K6R,K11R,K27R,K29R,K33R,K48R,K63R),鉴定TRIM58泛素化修饰KIFC1过程中介导的多聚泛素链类型。(7)构建敲低KIFC1的肝内胆管癌稳定株,通过体外细胞生物学实验阐明KIFC1对肝内胆管癌细胞系生物学功能影响。(8)构建过表达KIFC1和KIFC1-ΔKISc的肝内胆管癌稳定株,探究KIFC1功能域缺失对肝内胆管癌细胞系生物学功能影响。(9)构建同时过表达TRIM58与KIFC1的肝内胆管癌稳定株,鉴定TRIM58是否影响KIFC1在肝内胆管癌细胞系中的生物学效应。结果:(1)Co-IP、Western Blot等实验结果表明,TRIM58可结合KIFC1并下调其蛋白表达水平而不影响其mRNA水平。(2)使用CHX、MG132、CQ药物处理肝内胆管癌细胞后,Western Blot结果表明可以通过添加MG132而不是CQ来恢复CHX处理后KIFC1蛋白表达量的下降。(3)CHX追踪、蛋白泛素化检测等实验结果表明,TRIM58可在肝内胆管癌细胞中降低KIFC1的稳定性并提高其泛素化修饰水平。(4)成功构建结构域缺失型表达载体TRIM58-ΔN(RING)、TRIM58-ΔC(PRY/SPRY)、KIFC1-ΔSMC、KIFC1-ΔKISc,Co-IP 结果表明,TRIM58 可通过PRY/SPRY结构域识别结合KIFC1的SMC结构域。(5)Western Blot实验显示,TRIM58对KIFC1蛋白降解具有剂量依赖效应,与全长型TRIM58相比,RING结构域与PRY/SPRY结构域缺失型TRIM58对KIFC1的蛋白降解能力显著下降。由KIFC1蛋白泛素化水平检测结果可知,TRIM58的RING结构域缺失使得KIFC1泛素化水平降低最为明显。(6)当Ub蛋白的第48与63位的赖氨酸突变后,TRIM58对底物KIFC1的泛素化修饰水平显著降低。(7)敲低KIFC1抑制了肝内胆管癌细胞的增殖、克隆、划痕愈合与迁移能力。(8)功能域KISc的缺失削弱了 KIFC1对肝内胆管癌细胞划痕愈合与迁移能力的促进效应。(9)TRIM58可通过降低KIFC1蛋白水平来削弱KIFC1对肝内胆管癌的促癌效应。结论:(1)在肝内胆管癌细胞中,TRIM58可与KIFC1相互结合,并通过泛素化修饰介导KIFC1的蛋白酶体途径降解。(2)在TRIM58泛素化修饰KIFC1过程中,其RING结构域具有E3泛素连接酶活性,PRY/SPRY结构域可识别并结合KIFC1的SMC结构域,且介导的多聚泛素链类型为K48及K63型。(3)KIFC1在肝内胆管癌中作为促癌因子发挥作用,其发挥促癌功能需借助KISc结构域,并且TRIM58可显著降低KIFC1的蛋白水平并削弱KIFC1对肝内胆管癌的促癌效应。更多还原

【Abstract】 Background:Intrahepatic cholangiocarcinoma(iCC)is a highly malignant tumor with a complex pathogenesis that poses a serious threat to public health and safety.Previous laboratory studies have shown that the E3 ubiquitin ligase Tripartite Motif Containing 58(TRIM58),which is regulated by high methylation,functions as a tumor suppressor in iCC,but the molecular mechanisms by which TRIM58 exerts its tumor-suppressive effects remain unclear.Based on this,our study screened and identified the downstream target protein Kinesin Family Member C1(KIFC1)that is degraded by TRIM58 ubiquitination in iCC,explored the specific regulatory mechanism of TRIM58 on KIFC1,and clarified the biological function of the TRIM58-KIFC1 signaling axis in iCC,in order to have a deeper understanding of the pathogenesis of iCC.Methods:(1)Candidate interacting proteins were identified and confirmed by Co-IP,Western blot and immunofluorescence assays,and the mRNA levels of substrate proteins were detected by RT-qPCR.(2)The protein synthesis inhibitor CHX,proteasome inhibitor MG132 and lysosome inhibitor CQ were combined to treat iCC cell lines.Western Blot was used to detect the changes of KIFC1 protein level to identify the degradation pathway of KIFC1.(3)The effect of TRIM58 on the stability of KIFC1 in iCC cells was identified by CHX chase assay,and the effect of TRIM58 on the ubiquitination level of KIFC1 was verified by protein ubiquitination detection assay.(4)Structure domain-deleted expression vectors of TRIM58 and KIFC1 were constructed,and HEK293T cells were transfected to identify the binding structure domains of both proteins by Co-IP.(5)TRIM58 dose-dependent assay and protein ubiquitination assay were used to investigate the influence of the deletion of functional domain on TRIM58-mediated ubiquitination modification and degradation of KIFC1.(6)Seven ubiquitin protein expression vectors with lysine mutations(K6R,K11R,K27R,K29R,K33R,K48R,K63R)were constructed using site-directed mutagenesis technology to identify the type of polyubiquitin chain mediated by TRIM58 during KIFC1 ubiquitination modification.(7)Stable iCC cell lines with knockdown of KIFC1 were constructed,and the biological functions of iCC cell lines were elucidated by in vitro cell biology experiments.(8)Stable iCC cell lines overexpressing KIFC1 and KIFC1-ΔKISc were constructed to explore the effect of functional domain deletion of KIFC1 on the biological functions of iCC cell lines.(9)Stable iCC cell lines overexpressing both TRIM58 and KIFC1 were constructed to identify whether TRIM58 affects the biological effects of KIFC1 in iCC cell lines.Results:(1)Co-IP and Western blot experiments showed that TRIM58 can bind to KIFC1 and downregulate its protein expression level without affecting its mRNA level.(2)Using the inhibitor CHX,MG 132,and CQ to treat intrahepatic cholangiocarcinoma cells,Western blot results showed that the decrease in KIFC1 protein expression level after CHX treatment could be restored by adding MG132 but not CQ.(3)CHX tracing and protein ubiquitination detection experiments showed that TRIM58 can reduce the stability of KIFC1 and increase its ubiquitination modification level in intrahepatic cholangiocarcinoma cells.(4)Successfully constructed expression vectors of TRIM58-ΔN(RING),TRIM58-ΔC(PRY/SPRY),KIFC1-ΔSMC,and KIFC1-ΔKISc.Co-IP results showed that TRIM58 can recognize and bind to the SMC domain of KIFC1 through the PRY/SPRY domain.(5)Western blot experiments showed that TRIM58 has a dose-dependent effect on the degradation of KIFC1 protein,and the ability of the TRIM58-ΔN(RING)and TRIM58-ΔC(PRY/SPRY)deletion mutants to degrade KIFC1 protein is significantly reduced compared with full-length TRIM58.According to the results of KIFC1 protein ubiquitination level detection,the deletion of the RING domain of TRIM58 leads to the most significant decrease in KIFC1 ubiquitination level.(6)When lysine 48 and 63 of Ub protein were mutated,the ubiquitination modification level of TRIM58 on the substrate KIFC1 was significantly reduced.(7)Knockdown of KIFC1 inhibited the proliferation,colony formation,scratch healing,and migration ability of iCC cells.(8)The deletion of the functional domain KISc weakened the promoting effect of KIFC1 on the scratch healing and migration ability of iCC cells.(9)TRIM58 can weaken the pro-cancer effect of KIFC1 on iCC by reducing the protein level of KIFC1.Conclusion:(1)In iCC cells,TRIM58 can bind to KIFC1 and mediate its degradation through the ubiquitin-proteasome pathway via ubiquitination modification.(2)During TRIM58-mediated ubiquitination of KIFC1,its RING domain exhibits E3 ubiquitin ligase activity,the PRY/SPRY domain recognizes and binds to the SMC domain of KIFC1,and the mediated polyubiquitin chain types are K48 and K63.(3)KIFC1 acts as an oncogene in iCC,and its oncogenic function requires the KISc domain.TRIM5 8 can significantly reduce the protein level of KIFC1 and weaken its oncogenic effect in iCC.更多还原