USP21 promotes cell proliferation and metastasis through suppressing EZH2 ubiquitination in bladder carcinoma

Figures & data

Figure 1 USP21 is highly expressed in BC tissues and cell lines, and it predicted poor prognosis of BC.

Notes: (A) The mRNA level of USP21 in adjacent normal tissue and tumor tissue was detected by qRT-PCR. These results revealed that USP21 has an obviously high expression in BC tissues. (B) According to Kaplan–Meier method of analysis, the correlation of USP21 with survival curves. (C) The mRNA level of USP21 in normal urothelial cell line (SV-HUC-1) and two BC cell lines (5637 and T24) was analyzed by qRT-PCR. *Compared with SV-HUC-1, P<0.05. (D) The protein level of USP21 in normal urothelial cell line (SV-HUC-1) and two BC cell lines (5637 and T24) was analyzed by Western blotting.
Abbreviations: BC, bladder cancer; HUC, human urothelial cell; USP, ubiquitin-specific protease.

Table 1 Clinicopathologic variables in 66 bladder cancer patients

Variables	N=66	USP21 protein expression		P-value
		Low (n=23)	High (n=43)
Age (years)				0.423
<60	30	12	18
≥60	36	11	25
Drinking				0.073
Yes	36	16	20
No	30	7	23
Tumor size (cm)				0.001
Small (≤1.5)	33	18	15
Large (≥1.5)	33	5	28
Metastasis				0.006
Yes	38	8	30
No	28	15	13
Histologic grades				0.037
I–II	26	13	13
III–IV	40	10	30
pT				0.007
1–2	31	16	15
3–4	35	7	28
pN				0.032
0	34	16	18
1	32	7	25
Differentiation				0.351
Well/moderate	31	9	22
Poor	35	14	21

Abbreviation: USP, ubiquitin-specific protease.

Figure 2 Overexpression of USP21 promotes proliferation of bladder cancer cells.

Notes: (A) After transfected vector or USP21 or SCR or USP21 siRNA in 5637 and T24 cells according to the manufacture’s protocol, after 48 h of transfection, we extracted total RNA and performed qRT-PCR to detect mRNA level of USP21. (B) After transfected vector or USP21 or SCR or USP21 siRNA in 5637 and T24 cells according to the manufacture’s protocol, after 48 h of transfection, we collected and lysate whole cell, and performed Western blotting to detect protein level of USP21. (C) After overexpressed or silenced USP21 in 5637 and T24 cells, after 48 h of transfection, we placed 3×10³ cells in six-well plates, incubated for 14 days, and the cell were stained with 0.1% crystal violet and counted. (D) After overexpressed or silenced USP21 in 5637 and T24 cells, we evaluated cell proliferation by CCK-8 assay on the indicated day. *Compared with vector, P<0.05; **Compared with vector, P<0.01.
Abbreviations: SCR, scramble siRNA; USP, ubiquitin-specific protease.

Figure 3 USP21 promotes migration and invasion ability of BC cells and facilitates EMT.

Notes: (A) After overexpressed or silenced USP21 in 5637 cells, 48 h after transfection, we performed wound healing analysis to assess the migration ability of BC cells and measured the wound closure distance at 0 and 24 h. Overexpression of USP21 promoted the migration ability of BC cells; however, the inhibition of USP21 decreased BC cells migration ability. (B) After overexpressed or silenced USP21 in 5637 cells, 48 h after transfection, we placed 2×10³ cells on top chambers with serum-free medium, whereas the lower chamber with complete medium. After incubation, we fixed and stained cells with 0.5% crystal violet. Overexpression of USP21 promoted the invasion ability of BC cells; however, the inhibition of USP21 decreased BC cells invasion ability. (C) We overexpressed USP21 in 5637 cells and performed Western blotting and qRT-PCR to determine the function of USP21 in EMT process. Overexpression of USP21 decreased the expression of E-cadherin, whereas increased the expression of N-cadherin and fibronectin. (D) We knockdown USP21 in 5637 cells and incubated with or without MG132, a proteasome-specific inhibitor, and performed Western blotting and qRT-PCR to determine the function of USP21 in EMT process. Although cells were incubated without MG132, knockdown of USP21 increased the expression of E-cadherin, whereas decreased the expression of N-cadherin and fibronectin. However, while cells were incubated with MG132, it rescued the effect of USP21-depleted group. The magnification is 10×. *P<0.05, **P<0.01.
Abbreviations: BC, bladder cancer; EMT, epithelial–mesenchymal transition; SCR, scramble siRNA; USP, ubiquitin-specific protease.

Figure 4 Identifying USP21 as a EZH2-interacting protein.

Notes: (A) Co-IP assay was performed to determine whether USP21 interacted with EZH2 in vivo. 5637 cell lysates were first immunoprecipitated with anti-USP21 antibody and then immunoblotted using USP21 and EZH2 antibodies. (B) Co-IP assay was performed to determine whether EZH2 interacted with USP21 in vivo. 5637 cell lysates were first immunoprecipitated with anti-EZH2 antibody and then immunoblotted using EZH2 and USP21 antibodies. (C) GST pull-down analysis was used to determine whether USP21 directly interacted with EZH2 in vitro. We expressed GST-USP21 in bacterially and then incubated with transcribed/translated FLAG-EZH2.
Abbreviations: co-IP, co-immunoprecipitation; USP, ubiquitin-specific protease.

Figure 5 USP21 stabilizes EZH2 through its ubiquitinase activity.

Notes: (A) After overexpressed USP21 in 5637 cells, 48 h after transfection, Western blotting was used to detect the expression of indicated protein. Overexpression of USP21 had no effect on the expression of snail, slug, and twist, but it increased the expression of EZH2. (B) After overexpressed USP21 in 5637 cells, 48 h after transfection, qRT-PCR was used to detect the mRNA level of indicated gene. Overexpression of USP21 had no effect on those genes. *P<0.05. (C) After overexpressed wide-type USP21 (wt-USP21) and mutant USP21 (mut-USP21/C221A) in 5637 cells, 48 h after transfection, Western blotting was used to detect the expression of EZH2. Overexpression of wt-USP21 increased the expression of EZH2, whereas the mut-USP21/C221A had no effect on the expression of EZH2. (D) After overexpressed mut-USP21/C221A in 5637 cells, co-IP analysis was used to assess whether mut-USP21/C221A could interact with EZH2. (E) We knockdown the USP21 in 5637 cells and incubated with or without MG132, and performed Western blotting to detect the protein level of EZH2. While cells were incubated without MG132, the expression of EZH2 was decreased in USP21-silenced cells. While cells were incubated with MG132, the expression of EZH2 had no change in cells in which USP21 was silenced.
Abbreviations: Co-IP, co-immunoprecipitation; USP, ubiquitin-specific protease.