Ubiquitin-specific protease 21 promotes tumorigenicity and stemness of colorectal cancer by deubiquitinating and stabilizing ZEB1

Figure 1 Ubiquitin-specific protease 21 and ZEB1 exhibited higher expression, and resulted into poor prognosis. A: The mRNA expressions of ubiquitin-specific protease 21 (USP21) and ZEB1 were confirmed in normal tissues and colorectal cancer (CRC) tissues through real time-quantitative polymerase chain reaction (RT-qPCR); B: The correlation between USP21 and ZEB1 was verified; C: The prognosis of CRC patients with low or high USP21 (or ZEB1) was determined; D: The mRNA expressions of USP21 and ZEB1 were assessed in normal colonic epithelial cells (NCM460) and CRC cells (HCT-116, SW480, SW620, LoVo) through RT-qPCR; E: The protein expressions of USP21 and ZEB1 were assessed in normal colonic epithelial cells (NCM460) and CRC cells (HCT-116, SW480, SW620, LoVo) through western blot. ^bP < 0.01, ^cP < 0.001. USP21: Ubiquitin-specific protease 21.

Figure 2 Ubiquitin-specific protease 21 remained the stability of ZEB1. A: The interaction between USP21 and ZEB1 was evaluated through Co-IP assay; B: The interaction between USP21 and ZEB1 was detected through GST pull down assay; C: The mRNA and protein expressions of USP21 were examined after USP21 knockdown through real time-quantitative polymerase chain reaction (RT-qPCR) and western blot; D: The mRNA expression of ZEB1 was tested after suppressing USP21 through RT-qPCR; E: The protein expression of ZEB1 was examined after silencing USP21 through western blot; F: The protein expression of ZEB1 was assessed after cyclohexane treatment through western blot; G: The protein expression of ZEB1 was measured after MG132 treatment through western blot; H: The ubiquitination level of ZEB1 was evaluated through western blot. ^bP < 0.01, ^cP < 0.001. NC: Negative control; CHX: Cyclohexane; USP21: Ubiquitin-specific protease 21.

Figure 3 Ubiquitin-specific protease 21 strengthened cell proliferation, migration and stemness. Groups were separated into the sh-NC, sh-USP21 and sh-USP21+pcDNA3.1/ZEB1 group. A: The cell proliferation was detected through colony formation assay; B and C: The cell migration and invasion abilities were determined through Transwell assay; D: The stemness was tested through sphere formation assay. ^cP < 0.001. NC: Negative control; USP21: Ubiquitin-specific protease 21.

Figure 4 Ubiquitin-specific protease 21 aggravated tumor growth in vivo. Groups were separated into the sh-NC, sh-USP21 and sh-USP21+pcDNA3.1/ZEB1 group. A and B: The tumor size, volume and weight were assessed through in vivo assays; C: The protein expressions of ki67, USP21 and ZEB1 in tumors were measured through immunohistonchemistry assay. ^cP < 0.001. NC: Negative control; USP21: Ubiquitin-specific protease 21.