Lymphoma cell-derived extracellular vesicles inhibit autophagy and apoptosis to promote lymphoma cell growth via the microRNA-106a/Beclin1 axis

Abstract

Lymphoma is a common malignant tumor globally. Tumor-derived extracellular vesicles (Evs) participate in genetic information exchange between tumor cells. We investigated the role and mechanism of human Burkitt lymphoma cells Raji-derived Evs (Raji-Evs) in lymphoma cells. Effects of Evs on lymphoma cell proliferation, invasion, autophagy, and apoptosis were assessed using Cell Counting Kit-8 method, Transwell assay, laser confocal microscopy, Western blotting, and flow cytometry. microRNA (miR)-106a expression in lymphoma cells was determined using reverse transcription-quantitative polymerase chain reaction and then downregulated in Raji cells and then Evs were isolated (Evs-in-miR-106a) to evaluate its role in lymphoma cell growth. The binding relationship between miR-106a and Beclin1 was verified using RNA pull-down and dual-luciferase assays. Beclin1 was overexpressed in SU-DHL-4 and Farage cells and SU-DHL-4 cell autophagy and apoptosis were detected. The levels of miR-106a and Beclin1 in SU-DHL-4 cells were detected after adding autophagy inhibitors. The tumorigenicity assay in nude mice was performed to validate the effects of Raji-Evs in vivo. Raji-Evs promoted lymphoma cell proliferation and invasion and increased miR-106a. miR-106a knockdown reversed Evs-promoted lymphoma cell proliferation and invasion. miR-106a carried by Raji-Evs targeted Beclin1 expression. Beclin1 overexpression or miR-106a inhibitor reversed the effects of Evs on lymphoma cell autophagy and apoptosis. Autophagy inhibitors elevated miR-106a expression and lowered Beclin1 expression. Raji-Evs-carried miR-106a inhibited Beclin1-dependent autophagy and apoptosis in lymphoma cells, which were further verified in vivo, together with promoted tumor growth. We proved that Raji-Evs inhibited lymphoma cell autophagy and apoptosis and promoted cell growth via the miR-106a/Beclin1 axis.

Keywords: Beclin1; Lymphoma cells; apoptosis; autophagy; extracellular vesicles; microRNA-106a.

Figure 1.

Raji-Evs promoted human lymphoma cell proliferation and invasion. A. The morphology of the Evs was observed using TEM; B. the size distribution of the Evs was detected using qNano system; C. Expressions of positive markers (CD63 and CD9) and negative marker Calnexin of Evs were detected using WB, with the supernatant of Raji cell culture medium after GW4869 intervention as NC; D. Laser confocal microscopy observed the absorption of Dil-labeled Evs by SU-DHL-4 and Farage cells; E. CCK-8 assay was used to detect the proliferation of SU-DHL-4 and Farage cells; F. Transwell assay was used to detect the invasion of SU-DHL-4 and Farage cells in different groups. Three repeated experiments were conducted independently, and the data were expressed as mean ± standard deviation. Data in panels E/F were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01.

Figure 2.

Raji-Evs promoted human lymphoma cell proliferation and invasion via carrying miR-106a. A. RT-qPCR was used to detect the expression of miR-106a in SU-DHL-4 and Farage cells after Evs treatment; B. RT-qPCR was used to detect miR-106a expression in Raji cells and Raji-Evs after transfection of miR-106a inhibitor and its NC; C. RT-qPCR was used to detect the expression of miR-106a in SU-DHL-4 and Farage cells treated with Evs-in-miR-106a; D. CCK-8 assay was used to detect the proliferation of SU-DHL-4 and Farage cells; E. Transwell assay was used to detect the invasion of SU-DHL-4 and Farage cells. Three repeated experiments were conducted independently, and the data were expressed as mean ± standard deviation. Data in panels A/C/D/E were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01.

Figure 3.

Raji-Evs carried miR-106a into lymphoma cells to inhibit Beclin1 expression. A/B. The binding relationship between miR-106a and Beclin1 was verified using dual luciferase reporter gene assay and RNA pull-down assay; C. Beclin1 expression in SU-DHL-4 and Farage cells was detected using RT-qPCR; D. Beclin1 protein level in SU-DHL-4 and Farage cells was detected using WB. Three repeated experiments were conducted independently, and the data were expressed as mean ± standard deviation. Data in panels A-D were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01.

Figure 4.

Beclin1 overexpression reversed Raji-Evs-promoted proliferation and invasion of lymphoma cells. A. RT-qPCR was used to detect Beclin1 mRNA expression; B. WB was used to detect Beclin1 protein level; C. CCK-8 assay was used to detect the proliferation ability of lymphoma cells; D. Transwell assay was used to detect the invasion ability of lymphoma cells. Three repeated experiments were conducted independently, and the data were expressed as mean ± standard deviation. Data were analyzed using the t test. *p < 0.05; **p < 0.01.

Figure 5.

Raji-Evs inhibited autophagy and apoptosis of lymphoma cells via the miR-106a/Beclin1 axis. A. The autophagosomes and autolysosomes in SU-DHL-4 cells in each group were observed using TEM; B. The autophagy flow of SU-DHL-4 cells was detected using GFP-mRFP-LC3; C. Levels of autophagy-related proteins LC3-II, LC3-I and p62 in SU-DHL-4 cells were detected using WB; D. Apoptosis of SU-DHL-4 cells was detected using flow cytometry; E. Protein levels of Bcl-2 and Cleaved-caspase-3 in SU-DHL-4 cells were detected using WB; Autophagy inhibitors 1 nM Baf-A1 and 20 μM CQ were added to the cells. F. RT-qPCR was used to detect the expression of miR-106a and Beclin1 in SU-DHL-4 cells; (g) WB was used to detect the protein expression of Beclin1 in SU-DHL-4 cells. Three repeated experiments were conducted independently, and the data were expressed as mean ± standard deviation. Data in panels B-G were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01.

Figure 6.

Raji-Evs promoted mouse tumor growth and inhibited autophagy and apoptosis of lymphoma cells. A. The changes of tumor volume in each group were measured; B. The changes of tumor weight in each group were measured; C. mRNA expressions of miR-106a and Beclin1 in each group were detected using RT-qPCR; D. Levels of LC3-II, LC3-I, p62 and Cleaved-caspase-3 in mouse tumor of each group was detected using WB. N = 5. Data were expressed as mean ± standard deviation. Data in panels A-D were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01; ^#p < 0.05; ^##p < 0.01. In panel A, *comparison between the Evs group and the NC group; ^#comparison between the Evs-in-miR-106a group and the Evs group.