Fig 2: MAP2K4 suppresses malignant phenotypes caused by miR-BART22 in NPC cells.(a)(b) QPCR(n = 3 independent experiments, Student's t-test) and Western blot was used to examine the MAP2K4 mRNA and protein expressions in miR-BART22-overexpressed NPC cells and their control cells. (c) Tumor sphere formation experiment generated by the miR-BART22 NPC cells transfected with MAP2K4 and their empty control cells are shown; three independent experiments were performed. Original magnification, ×100, scale bar, 200 µm; (d) Transwell and Boyden Chamber assays, (e) Dose-response curves of HONE1-EBV+ and 5-8F treated with miR-BART22 inhibitor or controls. Experiments were repeated three times with similar results, mean ± SD, *P < .05. (f) Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, c-Jun, c-Myc, GSK3ß, p-GSK3ß, ß-catenin, E-cadherin, N-cadherin, Vimentin, Nanog, OCT4, Sox2, Ubc and TRAF6 expression in BART22-overexpressed NPC cells with MAP2K4 transfection. ß-actin served as controls. (g) Co-immunoprecipitation analysis of ubiquitin in BART22 overexpressed HONE1-EBV+ cells with MAP2K4 transfected. (h–i) Chromatin immunoprecipitation analysis of c-Jun binding to the transcriptional regulatory region of MYH9 in BART22 overexpressed HONE1-EBV+ and 5-8F cells with MAP2K4 transfected. All data are presented as the mean ± SD. Experiments were repeated three times. (j)(k) QPCR was used to examine the Ubc and MYH9 mRNA levels in BART22 overexpressed NPC cells with MAP2K4 transfected (n = 3 independent experiments, Student's t-test).

Fig 3: UBC was upregulated in prostate cancer tissues and cells. In prostate cancer tissues and paracancerous tissue, (a) IHC was used to detect ubiquitin-protein (UBC) expression. *P < 0.05 vs. paracancerous tissues; scale bar = 25 µm, the magnification is 400 times; scale bar = 100 µm, the magnification is 100 times. (b, c) qRT-PCR and WB were performed to detect ubiquitin-encoding gene UBA52, UBA80, UBB, and UBC expression. P < 0.05 vs. paracancerous tissues. In prostate cell line RWPE-1 and prostate cancer cell lines PC3, CWR22, VCAP, and DU145, (d) ICC were used to detect ubiquitin-protein expression. *P < 0.05 vs. the control group (prostate cell line RWPE-1); scale bar = 25 µm, the magnification is 400 times; scale bar = 100 µm, the magnification is 100 times. (e, f) qRT-PCR and WB detected ubiquitin-encoding gene UBA52, UBA80, UBB, and UBC expression. *P < 0.05 vs. the control group (prostate cell line RWPE-1).

Fig 4: SEPT6 inhibited tumor growth in nude mice. (a) The tumor volume of each group was detected at the time points of 4, 7, 11, 14, 17, 21, 24, and 28 d. (b) Nude mice were sacrificed on day 28, and tumor mass was measured in each group. (c) UBC expressions were detected by qRT-PCR. (d) WB was performed to detect SEPT6, UBC, ubiquitin, PCNA, and Ki67 expressions. *P < 0.05 vs. the NC group.

Fig 5: SEPT6 mediated UBC expression. (a) The expression of SEPT6 was detected by qRT-PCR in prostate cancer tissues and paracancerous tissues. *P < 0.05 vs. paracancerous tissues. (b) Pearson correlation coefficient analyzed the relationship between SEPT6 and UBC in prostate cancer tissues. (c) IF detected the colocation of SEPT6 and UBC in prostate cancer cells. Scale bar = 25 µm, the magnification is 400 times. (d) The overexpression and knockdown efficiency of SEPT6 was detected by qRT-PCR. *P < 0.05 vs. the NC group. (e, f) qRT-PCR and WB detected UBC expression, respectively. *P < 0.05 vs. the NC group.