microRNA-362-3p targets USP22 to retard retinoblastoma growth via reducing deubiquitination of LSD1

Abstract

Accumulating evidence has reported the role of microRNA (miR) in retinoblastoma (RB). Therefore, the objective was to discuss how miR-362-3p exerted its function in RB cell progression via regulating ubiquitin-specific protease 2 (USP22) and lysine-specific histone demethylase 1 (LSD1). MiR-362-3p, USP22 and LSD1 expression in RB cells and tissues were tested. The biological functions of RB cells were detected via over-expressing miR-362-3p and down-regulating USP22. The target relationship of USP22 and miR-362-3p as well as the interaction of USP22 and LSD1 in RB was verified. Down-regulated miR-362-3p and up-regulated USP22 and LSD1 were demonstrated in RB tissues and cells. Restoring miR-362-3p and depleting USP22 attenuated invasion, proliferation and migration, and facilitated apoptosis of RB cells. USP22 was a target gene of miR-362-3p. USP22 deubiquitinated LSD1 in RB. It is revealed that miR-362-3p targets USP22 and then restrains invasion, proliferation and migration while promotes apoptosis of RB via reducing LSD1 modified by deubiquitination.

Keywords: Retinoblastoma; apoptosis; deubiquitination; lysine-specific histone demethylase 1; microRNA-362-3p; proliferation; ubiquitin-specific protease 22.

Figure 1.

MiR-362-3p and miR-362-5p expression is decreased while USP22 and LSD1 expression is increased in RB tissues and cells. (a), Expression of miR-362-3p and miR-362-5p in RB tissues and cell lines tested by RT-qPCR. (b), Expression of USP22 and LSD1 in RB tissues tested by RT-qPCR. (c), Expression of USP22 and LSD1 in RB cell lines WERI-Rb1 and Y79 tested by western blot assay. * P < 0.05 vs. ARPE-19 cells. + P < 0.05 vs. normal group. Normal (n = 11), RB (n = 46), WERI-Rb1, Y79, ARPE-19 (N = 3). Measurement data were depicted as mean ± standard deviation, comparison between two groups was conducted by t test, comparisons among multiple groups were assessed by one-way ANOVA followed with Tukey’s post hoc test

Figure 2.

Up-regulating miR-362-3p restrains cell progression in RB. (a), Comparison of proliferative capacity of WERI-Rb1 and Y79 cells by CCK-8 assay. (b), Comparison of invasive ability of WRI-Rb1 and Y79 cells by Transwell assay. (c), Comparison of migration ability of WERI-Rb1 and Y79 cells by scratch test. (d), Analysis of apoptosis rate of WRI-Rb1 and Y79 cells by flow cytometry. * P < 0.05 vs. mimic NC group. + P < 0.05 vs. inhibitor NC group. N = 3. Measurement data were depicted as mean ± standard deviation, comparisons among multiple groups were assessed by one-way ANOVA followed with Tukey’s post hoc test

Figure 3.

USP22 is targeted by miR-362-3p. (a), The binding site of miR-362-3p on USP22 3’UTR and the luciferase activity of WERI-Rb1 and Y79 cells. (b), miR-362-3p and USP22 expression in WERI-Rb1 cells by RT-qPCR and Western blot assay. (c), miR-362-3p and USP22 expression in Y79 cells by RT-qPCR and Western blot assay. * P < 0.05 vs. mimic NC group. + P < 0.05 vs. inhibitor NC group. N = 3. Measurement data were depicted as mean ± standard deviation, comparison between two groups was conducted by t test, comparisons among multiple groups were assessed by one-way ANOVA followed with Tukey’s post hoc test

Figure 4.

Down-regulating USP22 inhibits cell progression in RB. (a), Comparison of the proliferative capacity of WERI-Rb1 and Y79 cells by CCK-8 assay. (b), Comparison of the invasive ability of WRI-Rb1 and Y79 cells by Transwell assay. (c), Comparison of migration ability of WERI-Rb1 and Y79 cells by scratch test. (d), Analysis of apoptosis rate of WRI-Rb1 and Y79 cells by flow cytometry. * P < 0.05 vs. sh-NC group. + P < 0.05 vs. oe-NC group. N = 3. Measurement data were depicted as mean ± standard deviation, comparisons among multiple groups were assessed by one-way ANOVA followed with Tukey’s post hoc test

Figure 5.

USP22 deubiquitinates LSD1 for protein stabilization in RB. (a), Immunoprecipitation of USP22 in WERI-Rb1 cells with USP22 antibody. (b), USP22 and LSD1 expression in WERI-Rb1 cells by RT-qPCR. (c), USP22 and LSD1 expression in Y79 cells by RT-qPCR.* P < 0.05 vs. sh-NC group. + P < 0.05 vs. oe-NC group. N = 3. Measurement data were depicted as mean ± standard deviation, comparisons among multiple groups were assessed by one-way ANOVA followed with Tukey’s post hoc test