USP14 promotes the malignant progression and ibrutinib resistance of mantle cell lymphoma by stabilizing XPO1

Abstract

Background: Mantle cell lymphoma (MCL) is a heterogeneous disease belonging to non-Hodgkin's lymphoma. In recent years, the morbidity rate of MCL is ascending, and the prognosis remains unfavorable. Ubiquitin-specific proteases14 (USP14) has been evidenced to be engaged in the process of malignant tumors. In this article, the role of USP14 in the malignant process of MCL and the mechanism of ibrutinib resistance were discussed. Methods: Through qRT-PCR and western blot, the mRNA and protein expressions of USP14 in MCL cells were tested. USP14 interference plasmid was constructed by cell transfection technology, and then CCK8 and EdU assays were applied to appraise cell proliferation. Cell cycle and cell apoptosis were estimated by flow cytometry and western blot. The sensitivity of MCL cells to ibrutinib was also investigated. Next, western blot, co-IP, Cycloheximide (CHX) assay and other techniques were used to detect the relationship between USP14 and XPO1. Finally, by simultaneously inhibiting USP14 and overexpressing XPO1, the impacts of USP14 on the malignant process of MCL and the regulatory mechanism of ibrutinib sensitivity in MCL were discussed. Results: USP14 expression was markedly fortified in MCL cell lines. Interference of USP14 suppressed MCL cell viability, potentiated cell cycle arrest, apoptosis, and ibrutinib sensitivity. This process might be achieved by USP14 deubiquitination through enhancing XPO1 stability. Conclusion: USP14 can promote the malignant progression and ibrutinib sensitivity of MCL by stabilizing XPO1.

Keywords: MCL; USP14; XPO1; ibrutinib resistance; ubiquitination.

Figure 1

USP14 interference impeded MCL cell proliferation. qRT-PCR (A) and western blot (B) tested USP14 expression. ***P<0.001 vs Wil2-s. The transfection efficacy sh-USP14 was detected by qRT-PCR (C) and western blot (D). (E) CCK8 and (F) EdU assays judged the cell viability and proliferation respectively. ***P<0.001 vs sh-NC.

Figure 2

Interference with USP14 induced G2/M cycle arrest and apoptosis of MCL cells. Flow cytometry analysis measured cell cycle (A) as well as apoptosis (B). Analysis of the proteins associated with cycle and apoptosis by western blot. ***P<0.001 vs sh-NC.

Figure 3

Interference with USP14 increased the sensitivity of MCL cells to ibrutinib. A. CCK8 estimated the cell inhibition rate. B. CCK8 judged cell viability. C. Flow cytometry measured cell apoptosis. D. Western blot tested Bcl-2 and Bax expressions. ***P<0.001 vs Control; ##P<0.01, ###P<0.001 vs Ibrutinib + sh-NC.

Figure 4

USP14 stabilized XPO1 through deubiquitination of XPO1. A. Western blot detected the expression of XPO1 after the inhibition of USP14. B. Co-IP proved that USP14 could interact with XPO1. C. CHX assay detected ubiquitination. D. USP14 knockdown increased the ubiquitination of XPO1 in MCL cells. ***P<0.001 vs sh-NC.

Figure 5

XPO1 elevation reversed the impact of USP14 deficiency on MCL cell proliferation. qRT-PCR (A) and western blot (B) tested XPO1 expression. ***P<0.001 vs Oe-NC. (C) CCK8 and (D) EdU assays judged the cell viability and proliferation respectively. ***P<0.001 vs Control; ###P<0.001 vs sh-USP14 + Oe-NC.

Figure 6

XPO1 elevation countervailed the impact of USP14 silencing on MCL cell cycle and apoptosis. Flow cytometry analysis measured cell cycle (A) as well as apoptosis (B). C. Analysis of the expression of proteins associated with cycle and apoptosis by western blot. *P<0.05, ***P<0.001 vs Control; #P<0.05, ###P<0.001 vs sh-USP14 + Oe-NC.

Figure 7

Overexpression of XPO1 reversed the effect of USP14 downregulation on the sensitivity of MCL cells to ibrutinib. A. Flow cytometry measured cell apoptosis. B. Western blot tested Bcl-2 and Bax expressions. *P<0.05, ***P<0.001 vs Control; ###P<0.001 vs Ibrutinib; ++P<0.01, +++P<0.001 vs Ibrutinib + sh-USP14 + Oe-NC.

Figure 8

USP14 down-regulation inhibited MCL cell growth in vivo. A. The images and weights of mice in each group. B and C. Image of tumor volume and size. D. IHC assay examined Ki-67 expression. E. Tunel assay detected the apoptosis rate of tumor cells in vivo. F. Western blot detected the expressions of USP14 and XPO1 in the tumor tissues. ***P<0.001 vs sh-NC.