猪重组NK-lysin抑制肝细胞性肝癌的转移:基于下调FKBP3表达、抑制氧化磷酸化和糖酵解

Procine recombinant NK-lysin inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 and inhibiting oxidative phosphorylation and glycolysis:a proteomic analysis

目的应用生物信息学方法探究猪重组NK-lysin(prNK-lysin)抑制肝癌细胞转移潜在的作用靶点和通路。方法将肝癌细胞设置为空白对照组,PBS处理组和prNK-lysin处理组,37℃作用6 h后,应用高效液相色谱串联质谱对肽段进行鉴定,按照Foldchange=1.2倍且P<0.05筛选出差异表达蛋白。基于GO、KEGG等数据库对差异表达蛋白进行GO功能分析和KEGG通路分析。运用RT-qPCR验证细胞中多肽-N-乙酰半乳糖胺基转移酶13(GALNT13)、跨膜蛋白51(TMEM51)和FKBP脯酰异构酶3(FKBP3)的mRNA相对表达量。Western blot验证FKBP3的蛋白表达量。结果蛋白组学表明,与空白对照组相比,prNK-lysin处理组中有1989个差异表达蛋白;与PBS处理组相比,prNK-lysin处理组中有2753个差异表达蛋白;PBS处理组和空白对照组相比,有15个差异表达蛋白。相对于PBS处理组和空白对照组,prNK-lysin处理组中共有1909个差异表达蛋白。GO和KEGG分析表明差异表达蛋白主要参与Viral process、translational initiation、RNAbinding等过程,主要富集于Ribosome、Protein process in endoplasmic reticulum、RNA transport等通路。RT-qPCR表明,与空白对照组相比,prNK-lysin处理组显著升高了细胞内GALNT13(1.54±0.06 vs 1.02±0.17,P<0.05)和TMEM51(1.27±0.07 vs 1.00±0.04,P<0.01)的mRNA相对表达量,显著降低了FKBP3(0.43±0.06 vs 1.02±0.24,P<0.05)的mRNA相对表达量。Western blotting表明prNK-lysin处理组与空白对照组相比显著降低了细胞内FKBP3(0.68±0.02 vs 1.02±0.03,P<0.001)的蛋白表达量。结论prNK-lysin处理后肝癌细胞SMMOL/LC-7721的蛋白质组与空白组相比发生显著变化,prNK-lysin作用于FKBP3蛋白,并能通过影响细胞内氧化磷酸化和糖酵解等通路发挥其抑制作用。

Objective To investigate the potential mechanisms that mediate the inhibitory effect of porcine recombinant NK lysin(prNK-lysin)against liver cancer cell metastasis.Methods HPLC-tandem mass spectrometry was used to identify the differentially expressed proteins in prNK-lysin-treated hepatocellular carcinoma SMMOL/LC-7721 cells in comparison with the control and PBS-treated cells.GO functional annotation and KEGG pathway analysis of the differentially expressed proteins were performed using GO and KEGG databases.RT-qPCR was used to determine the mRNA expression levels of polypeptide-N-acetylgalactosaminotransferase 13(GALNT13),transmembrane protein 51(TMEM51)and FKBP prolyl isomerase 3(FKBP3)in the cells,and the protein expression of FKBP3 was verified using Western blotting.Results Proteomic analysis identified 1989 differentially expressed proteins in prNK-lysin-treated cells compared with the control cells,and 2753 compared with PBS-treated cells.Fifteen proteins were differentially expressed between PBS-treated and the control cells,and 1909 were differentially expressed in prNK-lysin group compared with both PBS and control groups.These differentially expressed proteins were involved mainly in the viral process,translational initiation and RNA binding and were enriched mainly in ribosome,protein process in endoplasmic reticulum,and RNA transport pathways.RT-qPCR showed that compared with the control group,prNK-lysin treatment significantly increased the mRNA expressions of GALNT13(P<0.05)and TMEM51(P<0.01)and lowered FKBP3 mRNA expression(P<0.05).Western blotting also showed a significantly decreased expression of FKBP3 protein in prNK-lysin-treated cells(P<0.001).Conclusion Treatment with prNK-lysin causes significant changes in protein expression profile of SMMOL/LC-7721 cells and inhibits hepatocellular carcinoma metastasis by downregulating FKBP3 protein and affecting the cellular oxidative phosphorylation and glycolysis pathways.