As shown in Physique ?Determine7,7, the USP9X-shRNA group formed much smaller tumors and displayed reduced tumorigenicity, showing that downregulation of USP9Xreduced primary glioma cell tumorigenesis in vivo. 3.862, = 0.049). Importantly, there were no significant differences between the two groups in terms of patient age, gender, KPS, histologic grade, medical procedures and chemotherapy (Supplementary Table S1). USP9X expression was a significantly independent prognostic factor (= 0.002) with a relative risk of 0.365 (95% confidence interval, 0.193 C 0.688) in a Cox multivariate 2′,5-Difluoro-2′-deoxycytidine analysis, which was performed with the following variables for each case: USP9X expression, radiotherapy and tumor size (Supplementary Table S2). These results suggested that USP9X was indeed an independent unfavorable prognostic factor for patients with high grade glioma and that USP9X-targeted studies could help explore new therapeutic strategies for this tumor. Table 1 The correlation of USP9X expression and clinicopathological features in high grade glioma patients 0.05) (Figure 5a, 5b). We also explored the mechanism through which USP9X affected U251 and A172 cell proliferation abilities. After USP9X expression was suppressed by the siRNA, the expression of c-Myc mRNA and protein decreased. Subsequently, we detected whether USP9X affected the cell cycle of glioma cells. Compared with the control group and si-NC group, the G1 phase of the siRNA 1-3 groups was significantly longer ( 0.05) (Supplementary Figure S2a, S2b). In contrast, the duration of the S phase of the siRNA 1-3 groups decreased ( 0.05) (Supplementary Figure S2c,d). We also detected whether USP9X affected the apoptosis of glioma cells. Compared with the control group and si-NC group, the apoptosis rates of the siRNA 2′,5-Difluoro-2′-deoxycytidine 1-3 groups were significantly higher ( 0.05) (Supplementary Figure S2g, S2h). The results HNRNPA1L2 of the western blots suggested that cleaved caspase 3 and cleaved caspase 8 proteins emerged in the siRNA 1-3 groups (Physique 5c, 5d). However, there was 2′,5-Difluoro-2′-deoxycytidine no expression of cleaved caspase 3 and caspase 8 in the control group and si-NC group. Open in a separate windows Physique 5 Cell proliferation decreased after USP9X knockdowna and b. Cell proliferation curves of U251 and A172 cells after USP9X knockdown. c and d. USP9X siRNAs were transfected into U251 and A172 cells for 72 h, cells were harvested and lysed for western blot to detect caspase 3 and caspase 8. The expression of USP9X was significantly correlated with -catenin, c-Myc and cyclinD1 in high grade glioma tissues Among 54 glioma cases, there were 26 USP9X positive cases, representing a positive rate of 48.1% (Supplementary Table S3). Using immunohistochemistry, we found that USP9X positive reactions primarily occurred in the cytoplasm of high grade glioma cells and produced brownish yellow granules (Physique ?(Figure6a).6a). A total of 28 patients were -catenin positive, representing a positive rate of 51.9%. -catenin was primarily expressed in the cell membrane and cytoplasm of tumor cells, and a few cell nuclei were -catenin positive. The expression of USP9X and -catenin was significantly correlated (Spearman’s rank test, 0.001) in high grade glioma tissues. The positive rates of c-Myc and cyclinD1 were 48.1% and 40.7 %, respectively. We found that c-Myc and cyclinD1 positive reactions primarily occurred in the cell nuclei and produced brownish yellow granules (Physique ?(Figure6a).6a). The expression of USP9X was also significantly correlated with the expression of c-Myc and cyclinD1 in high grade glioma tissues (Spearman’s rank test, 0.001). Moreover, the rate of apoptosis in USP9X positive cases was 5.2 0.8%, and the rate of apoptosis in USP9X negative cases was 28.7 1.2%, as detected by TUNEL method. As shown in Physique 6a,b, apoptotic cells were rarely observed in USP9X positive patients (left) and quite a few apoptotic cells were observed in USP9X unfavorable patients (right). Open in a separate window Physique 6 Representative immunohistochemical staining for USP9X, -catenin, c-Myc and cyclinD1 in high grade glioma tissuesa. Positive USP9X, -catenin, c-Myc, cyclinD1 expression in patient a (Original magnification, 400). b. Unfavorable USP9X, -catenin, c-Myc, cyclinD1 expression in patient b (Original magnification, 400). Downregulation of USP9X decreased tumorigenesis of glioma cells in vivo To determine the role of USP9X in modulating glioma tumorigenesis in vivo, we implanted primary glioma cells and lentivirus infected primary glioma cells into the dorsal side of the.