去泛素化酶USP14参与多发性骨髓瘤细胞增殖和细胞周期调控的作用机制OA

Objective To explore the mechanism by which the deubiquitinating enzyme ubiquitin-specific protease 14(USP14)stabi-lizes the nuclear export protein 1(XPO1)and participates in the proliferation and cell cycle regulation of multiple myeloma(MM)cells.Methods The mRNA levels of USP14 and XPO1 were detected by quantitative real-time polymerase chain reaction(qRT-PCR)in bone marrow samples from MM patients and healthy controls,and in HS-27A,U266,RPMI 8226,NCI-H929,and MM.1S cells.The U266 cells were divided into control group,silencing control group(si-NC),USP14 silencing group(si-USP14),USP14 si-lencing+overexpression control group(si-USP14+OE-NC),and USP14 silencing+overexpression XPO1 group(si-USP14+OE-XPO1).The U266 cells were transfected with the corresponding sequence using Lipofectamine™ 2000 for 48 h.The cells in control group received no treatment.Western blot assay was used to detect the silencing efficiency of USP14.Cell counting kit-8(CCK-8)and clone formation assays were used to detect the proliferation activity of cells[expressed as both the number of cell clones and the optical denstity at 450 nm(OD450 nm)].Flow cytometry was used to detect the changes in the cell cycle(G0/G1 phase,S phase).The interaction between USP14 and XPO1 was analyzed by co-immunoprecipitation(Co-IP).Ubiquitination experiments were conducted to detect the changes in XPO1 ubiquitination levels.Immunofluorescence was used to detect the co-localization of XPO1 and CyclinD1.Western blot was used to detect the expressions of cell proliferation-related proteins(PCNA,CyclinD1,p21),USP14 and XPO1.The nude mice were subcutaneously injected with the U266 cell suspension transfected with si-NC and si-USP14 plasmids through the right shoulder,named as si-NC group and si-USP14 group,with 10 mice in each group.The effect of USP14 silencing on the growth of transplanted tumors was then examined.Results The mRNA levels of both USP14 and XPO1 were significantly higher in bone marrow samples from MM patients than in healthy controls(P<0.05),and also higher in multiple myeloma cell lines(U266,RPMI 8226,NCI-H929,MM.1S)than in HS-27A cell line(P<0.05).The expressions of USP14,XPO1,PCNA,CyclinD1,and XPO1/CyclinD1,colony num-bers,OD450 nm values,and the proportion of S-phase cells significantly decreased in si-USP14 group compared to si-NC group,while p21 expression,the proportion of G0/G1-phase cells,and XPO1 ubiquitination levels increased(P<0.05).Compared with si-USP14+OE-NC group,the expressions of XPO1,PCNA,CyclinD1,XPO1/CyclinD1,the number of cell clones,OD450 nm,and the proportion of S-phase cells increased in si-USP14+OE-XPO1 group,while the expression of p21 and the proportion of G0/G1-phase cells,and the level of XPO1 ubiquitination decreased(all P<0.05).The co-immunoprecipitation experiment revealed an interaction between USP14 and XPO1.The nude mouse xenograft experiment showed that tumor volume and weight were significantly lower in si-USP14 group than in si-NC group(P<0.05).Conclusion USP14 can modulate the cell cycle by promoting XPO1 expression,thereby inhibiting the malignant proliferation of MM cells.