| Survivin is the smallest member of the inhibitor of apoptosis proteins(IAP)family.Decreasing the expression of endogenous Survivin can inhibit tumor cell proliferation and promote apoptosis.There are currently many strategies for inhibiting Survivin expression,but there is a lack of a method to degrade Survivin based on the protein level targeted by antibody protein.Survivin exists in the cytoplasm(including mitochondria)and nucleus of cells.The main functions of Survivin in different subcells may be different.Therefore,the purpose of this subject is establishing a method for ubiquitination degradation endogenous Survivin targeted by nanobodies,to study the effects of Survivin degradation on apoptosis and cell growth,and then to explore major functions of Survivin in different subcellular locations.Based on the above objectives,this study has obtained the following results and conclusions:Firstly,we established a method for endogenous Survivin degradation mediated by ubiquitin ligase TRIM21.The Nb4A-Fc-T2A-TRIM21 system was constructed using Nb4A(Nanobodies screened for Survivin antigen by our laboratory)targeting Survivin and the ability of the antibody’s Fc region to specifically bind to the carboxy-terminal PRYSPRY domain of TRIM21.Western blot analysis showed that Nb4A-Fc-T2A-TRIM21 can degrade the endogenous Survivin protein in a large amount,and its ability to degrade Survivin was 3.72 times that of Nb4A and 2.84 times that of TRIM21.And the proteasome inhibitor MG132 was used to verify that the degradation of Survivin by this method is mediated by the ubiquitin-proteasome pathway.Secondly,flow cytometry showed that the apoptosis rate of Nb4A-Fc-T2A-TIRM21 on MCF-7 cells reached 58.52%.However,the apoptosis rate on L-02 cells with low-Survivin was only 14.56%.This indicates that Nb4A-Fc-T2A-TIRM21 can promote apoptosis by degrading Survivin in cancer cells.And Western blot analysis found that the expression of pro-apoptotic proteins Caspase-3 and Bax were inversely proportional to the expression of Survivin,while the expression of anti-apoptotic protein Bcl-2 had no significant correlation with the expression level of Survivin.It can be initially proved that Survivin exerts its anti-apoptotic function by directly or indirectly inhibiting the Caspase pathway,which is different from the anti-apoptotic pathway of the Bcl-2 anti-apoptotic protein family.Finally,Nb4A-Fc-T2A-TRIM21 was fused with nuclear localization signal NLS to target the degradation of Survivin in the nucleus.By comparing the apoptotic rate,it was found that the pro-apoptotic effect of Nb4A-Fc-T2A-TRIM21 on MCF-7 cells was about 2.37 times that of Nb4A-Fc-NLS-T2A-TRIM21-NLS.MTT experiments found that the cell viability rate was only 49.44% after Nb4A-Fc-NLS-T2A-TRIM21-NLS on MCF-7 cells for 48 h,which was lower than the cell viability rate of the Nb4A-Fc-T2A-TRIM21 group(60.45%).However,it had no significant effect on the proliferation activity of L-02 cells.By detecting the distribution of the cell cycle and the expression level of Cyclin D1,we found that the targeted degradation of Survivin in the nucleus accelerated the transition from G1 to S phase,and blocked the S and G2 phases,thereby inhibiting cell division.And the down-regulation of Survivin in the nucleus would largely down-regulate the expression of Cyclin D1.However,the degradation of Survivin in the cytoplasm did not significantly affect the process of the cell cycle.In summary,we confirmed that Survivin’s dual function is closely related to its localization;the cytoplasmic Survivin mainly plays an anti-apoptotic function,and the nuclear Survivin mainly promotes cell proliferation and participates in the regulation of the cell cycle.This study provides a new strategy and research basis for targeting Survivin tumor therapy accurately. |
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