泛素连接酶三基序蛋白21对结直肠癌鞘氨醇激酶2的调控作用及其机制

REGULATORY EFFECTS AND MECHANISM OF UBIQUITIN LIGASE TRIPARTITE MOTIF CONTAINING-21 ON SPHIGOSINE KINASE 2 IN COLORECTAL CANCER

目的探讨泛素连接酶三基序蛋白21(TRIM21)对结直肠癌(colorectal cancer,CRC)中鞘氨醇激酶2(SphK2)的调控作用及机制。方法将转染Flag-SphK2的PLC/RPF/5细胞分为a、b、c、d、e组,分别采用表皮生长因子刺激、葡萄糖饥饿处理、过氧化氢刺激、缺氧处理、转化生长因子-β刺激,通过质谱鉴定与SphK2互作的蛋白。将HEK-293T细胞分为f、g、h、i、j、k、l、m、n、o组,f~k组分别转染Flag-SphK2、Flag-SphK2+HA-TRIM21质粒、Flag-SphK2、Flag-SphK2+His-Ubquitin、Flag-SphK2+His-Ubquitin+1μg HA-TRIM21质粒、Flag-SphK2+His-Ubquitin+4μg HA-TRIM21质粒,l~o组分别分别转染HA-TRIM21质粒0、0.3、0.6、0.9μg,f~g组进行免疫共沉淀实验鉴定SphK2与TRIM21蛋白的相互作用情况,h~k组进行体内泛素化实验鉴定TRIM21对SphK2泛素化水平的影响,l~o组进行Western-blot实验检测TRIM21过表达对SphK2蛋白水平的影响。将His-TRIM21、GST-SphK2蛋白混合液分为p、q组,分别加入琼脂糖珠、谷胱甘肽琼脂糖珠,进行蛋白体外结合实验鉴定SphK2与TRIM21蛋白的相互作用情况。将EP管分为r、s、t三组,每组分别加入GST-SphK2+E1/E2/Ub、GST-SphK2+His-TRIM21、GST-SphK2+His-TRIM21+E1/E2/Ub蛋白,进行体外泛素化实验鉴定TRIM21对SphK2泛素化水平的影响。将RKO细胞分为u、v、w、x四组,各组分别感染LV-sgTRIM21-1、LV-sgTRIM21-2、LV-sgTRIM21-3、LV-sgControl慢病毒,采用Western-blot实验检测TRIM21敲除对RKO细胞SphK2蛋白水平及半衰期的影响,采用CCK-8实验检测TRIM21敲除对RKO细胞5-氟尿嘧啶(5-Fu)敏感性的影响。结果质谱分析、免疫共沉淀和蛋白质体外结合实验显示TRIM21与SphK2存在蛋白互作。体内、体外泛素化实验确定TRIM21介导SphK2发生多聚泛素化。在HEK-293T细胞中,TRIM21的外源过表达导致SphK2蛋白水平上调(F=196.22,P<0.05)。与x组相比,TRIM21敲除的RKO细胞系中SphK2蛋白表达水平降低、SphK2蛋白半衰期缩短、对5-Fu的敏感性增加(F=24.83~890.51,P<0.05)。结论在CRC中,TRIM21通过催化SphK2泛素化,提高了SphK2蛋白的表达水平。敲除TRIM21能提高CRC细胞对5-FU的敏感性。

Objective To investigate the regulatory effects and mechanism of the ubiquitin ligase tripartite motif containing-21(TRIM21)on sphingosine kinase 2(SphK2)in colorectal cancer(CRC).Methods PLC/RPF/5 cells transfected with Flag-SphK2 were divided into groups a,b,c,d,and e to receive epidermal growth factor stimulation,glucose starvation,hydrogen peroxide stimulation,hypoxia stimulation,and transforming growth factor-βstimulation,respectively.Mass spectrometry was used to identify proteins interacting with SphK2.HEK-293T cells were divided into groups f,g,h,i,j,k,l,m,n,and o.Groups f to k were transfected with Flag-SphK2,Flag-SphK2+HA-TRIM21,Flag-SphK2,Flag-SphK2+His-Ubquitin,Flag-SphK2+His-Ubquitin+1μg HA-TRIM21 plasmids,and Flag-SphK2+His-Ubquitin+4μg HA-TRIM21 plasmids,respectively.Groups l to o were transfected with 0,0.3,0.6,and 0.9μg of HA-TRIM21 plasmids,respectively.Co-immunoprecipitation assay was used to test protein interaction between SphK2 and TRIM21 in groups f and g.In vivo ubiquitination assay was performed to analyze the effects of TRIM21 on SphK2 ubiquitination in groups h to k.Western-blot was used to analyze the effects of TRIM21 overexpression on the SphK2 protein level in groups l to o.Using a mixed His-TRIM21 and GST-SphK2 protein solution with either agarose beads(group p)or glutathione-agarose beads(group q),GST-pull down assay was conducted to identify the interaction between SphK2 and TRIM21.With GST-SphK2+E1/E2/Ub,GST-SphK2+His-TRIM21,and GST-SphK2+His-TRIM21+E1/E2/Ub separately added into EP tubes(groups r,s,and t,respectively),in vitro ubiquitination assay was performed to study the effects of TRIM21 on SphK2 ubiquitination.RKO cells were divided into groups u,v,w,and x to be infected with LV-sgTRIM21-1,LV-sgTRIM21-2,LV-sgTRIM21-3,and LV-sgControl lentiviruses,respectively.Western-blot was performed to measure the effects of TRIM21 knockout on the level and half-life of SphK2 protein in RKO cells.Cell Counting Kit-8 was used to analyze the effect of TRIM21 knockout on the sensitivity of RKO cells to 5-fuorouracil(5-Fu).Results Protein interaction between TRIM21 and SphK2 was demonstrated by mass spectrometry,co-immunoprecipitation assay,and GST-pull down.The in vivo and in vitro ubi-quitination assays showed that TRIM21 mediated the polyubiquitination of SphK2.The exogenous overexpression of TRIM21 signi-ficantly increased the protein level of SphK2 in HEK-293T cells(F=196.22,P<0.05).Compared with the x group,RKO cells with TRIM21 knockout showed a significantly decreased SphK2 protein level,a significantly shorter half-life of SphK2 protein,and significantly increased sensitivity to 5-Fu(F=24.83-890.51,P<0.05).Conclusion TRIM21 increases the expression level of SphK2 protein by catalyzing SphK2 ubiquitination in CRC.Knocking out the TRIM21 gene can improve the sensitivity of CRC cells to 5-Fu.