角蛋白18与剪切因子PSF相互作用介导PSF的膜易位并维持髓系白血病细胞的化疗敏感性

Interaction between PSF and cytokeratin 18 mediates PSF relocation to cell membrane and maintains chemosensitivity of myeloid leukemia

目的:鉴定多嘧啶结合蛋白相关的剪切因子(polypyrimidine tract-binding protein-associated splicing factor,PSF)在髓系白血病细胞内的伴侣蛋白,探讨PSF在敏感HL60细胞和耐药的HL60/DOX细胞中易位细胞膜的模式和机制。方法:通过脂质体瞬时转染PSF真核表达载体过表达PSF,进一步结合流式细胞术在转染后24 h、48 h、72 h检测PSF在细胞膜上的表达水平。构建链亲和素结合肽(streptavidin binding peptide,SBP)和PSF的融合表达载体,转染载体,过表达SBP-PSF融合蛋白。通过链亲和素磁珠共沉淀法和质谱分析,鉴定PSF在细胞内的伴侣蛋白。在慢病毒载体中插入角蛋白18(cytokeratin 18,K18)干扰序列,转染293T细胞制备病毒液。用慢病毒感染HL60和HL60/DOX细胞,获得稳定干扰K18的细胞株。结合流式细胞术检测干扰K18的HL60和HL60/DOX细胞中细胞膜上PSF的表达水平,由此证实CK18在HL60和HL60/DOX细胞中协同转运PSF易位细胞膜机制的异同。结果:瞬时过表达PSF后的24 h、48 h、72 h连续3 d检测细胞膜PSF表达水平,HL60敏感株细胞膜上PSF表达率分别为22.4%±3.5%、37.9%±6.0%、58.3%±8.8%;耐药株HL60/DOX细胞膜上PSF的表达率分别为4.7%±0.5%、3.9%±0.6%、2.9%±0.6%;各时间点下敏感株和耐药株的差异有统计学意义,P<0.01。免疫共沉淀和质谱证实K18为PSF在细胞内的伴侣蛋白。干扰K18的表达后,再次分析细胞膜PSF表达,发现PSF在敏感株细胞膜上的表达水平由原来的48.9%±5.4%降至6.2%±1.0%;在耐药株细胞膜上的表达水平由9.11%±1.2%降至2.21%±0.51%。结论:K18是PSF在细胞内的伴侣蛋白,在敏感细胞中,K18与PSF相互作用可帮助PSF向细胞膜转运;而在耐药株中,由于该功能障碍导致PSF在胞内发生积累从而介导耐药性。

Objective:To identify the chaperone of polypyrimidine tractor-binding protein-associated splicing factor(PSF)in myeloid leukemia cells,and to explore the mechanism and redistributive pattern to cell surface of PSF in chemo-sensitive HL60 cells and resistant HL60/DOX cells.Methods:The eukaryotic expression vector of PSF was transfected with liposomes transiently,then flow cytometry was used to detect the expression level of PSF on the cell surface 24 h,48 h and 72 h after vector transfections.We constructed a chimeric expression vector,streptavidin binding peptide(SBP)-PSF,meanwhile this vector was transfected and made SBP-PSF fusion protein overexpress.In addition,we used streptavidin magnetic beads to precipitate the cellular chaperonin of PSF and then identified its chaperonin by mass spectrometry(MS).Lentiviral vectors containing cytokeratin18(K18)interference sequences were transfected into 293T cells to prepare lentivirus.HL60 and HL60/DOX cells were infected with lentivirus to obtain stable interfering K18 cell lines.Next,flow cytometry was used to test the membrane relocation level of PSF.Together,these methods confirmed the similar or different mechanisms of the PSF redistributing to membrane synergistically mediated by K18 in HL60 and HL60/DOX cells.Results:The expression of membrane relocated PSF was detected every day for three days(at the end of 24 h、48 h and 72 h)after transient overexpression.The expressing rate of PSF on the cell surface was 22.4%±3.5%,37.9%±6.0%,58.3%±8.8%,respectively in sensitive HL60 cells,while that was 4.7%±0.5%,3.9%±0.6%,2.9%±0.6%,respectively in resistant HL60/DOX cells.The difference of expressing rate on each day was significant,P<0.01.We identified K18 detected by co-immunoprecipitation and mass spectrum assay which was the cellular chaperone of PSF.We found that K18 knockdown decreased the PSF expression level which redistributed on cell surface from 48.9%±5.4%to 6.2%±1.0%in sensitive HL60 cells,and from 9.11%±1.2%to 2.21%±0.51%in resistant HL60/DOX cells,respectively.Conclusion:K18 is the intracellular chaperonin of PSF.The interaction of PSF and K18 mediates its redistribution to cell membrane in sensitive cells.While in resistant cells,PSF failed to relocate at the cell surface and accumulated in cells,which mediated resistance to chemotherapeutics.