Therefore, we tested the effect of USP39 knockdown on p53 activation and its downstream factors in A549 cells. USP39 knockdown significantly inhibited migration and invasion of A549 and HCC827 cells, also via activation of the p53 pathway, and downregulation of MMP2 and MMP9. Importantly, we verified these results in metastasis models in vivo. Collectively, these results not only establish that USP39 3PO functions as an oncogene in lung cancer, but reveal that USP39 has an essential role in regulating cell proliferation and metastasis via activation of the p53 pathway. = 0.0247) (Figure 1B). We also examined the relationship between the level of USP39 expression and the clinicopathological characteristics of patients from whom the tissue samples were derived. However, no correlations between the levels 3PO of USP39 expression with sex, Tumor-Node-Metastasis (TNM) stage, or lymph node invasion were present (Table S1). In addition, we analyzed the gene expression of in lung cancer samples using the Oncomine database (https://www.oncomine.org) and GEPIA database (http://gepia.cancer-pku.cn). The results showed that this USP39 mRNA level was significantly increased in lung cancer samples (Physique 1C,D). Next, we assessed USP39 expression in normal bronchial epithelial cells (BEAS-2B) and NSCLC cell lines (A549, NCI-H1299, NCI-H157 and NCI-H460). As depicted in Physique 1E,F, USP39 expression was significantly higher in NSCLC cell lines than in BEAS-2B cells (* < 0.05, ** < 0.01). These results suggest that USP39 may serve as a potential molecular target in lung cancer patients. Open in a separate windows Physique 1 USP39 expression in lung cancer tissues and lung cancer cell lines. (A) Representative images of USP39 immunohistochemical staining in normal lung tissues (left) and NSCLC tissues (right) were shown. Magnification 40 and 200. (B) Quantitative analysis of IHC results showed that USP39 3PO protein level was overexpressed in lung cancer tissues. (= 3 in normal group and = 77 in cancer group, * = 0.0247). (C,D) Gene expression data from Oncomine database and GEPIA database showed that mRNA level was overexpressed in human lung cancer. (E,F) The expression of UP39 was analyzed by Western blot and Real-time PCR in human normal lung cell BEAS-2B and various NSCLC cell lines: A549, NCI-H1299, NCI-H157 and NCI-H460 (* 0.05, ** 0.01). 2.2. Knocking Down USP39 Inhibits A549 Cell Growth in Vivo and In Vitro To investigate the functions of USP39 in lung cancer, we generated USP39 shRNAs (control, S1 and S2) lentiviruses and established A549 and HCC827 cell lines stably expressing these shRNAs. As shown in Physique 2A,B, Western blotting analysis revealed that this USP39 protein levels were significantly downregulated in both the shUSP39(S1) and shUSP39(S2) groups compared with the control sh group. Thus, it was exhibited that shRNAs targeting USP39 exerted significant knockdown effects on USP39 expression. To determine the role of USP39 expression on lung cancer cell viability, MTT assays and colony formation assays were performed on A549 and HCC827 cells. As shown in Physique 2CCF, knocking down USP39 significantly inhibited cell growth (** 0.01, *** 0.001, **** 0.0001). We then further examined the functional consequences of inhibiting USP39 around the growth of A549 cells in vivo. Xenograft tumors of the USP39 KD group exhibited smaller tumor volumes compared with tumors of control and control sh groups (Physique 2G,H). Together, these data indicate Mouse monoclonal to TEC that USP39 functions as a tumor promotor and positively regulates lung tumor growth. Open in a separate window Physique 2 Knocking down USP39 suppresses lung cancer cell proliferation in vivo and vitro. (A,B) Identification of knockdown efficiency in A549 and HCC827 cells by western blot assay. (C,D) Stable USP39 knockdown cell lines were plated into 96-well plates and cell 3PO viability was examined every 24 h by MTT assay, lasting for 5C6 days (**** 0.0001, = 4). (E,F) Meanwhile, Colonies (>50 M) were counted 10C12 days in A549 and HCC827 cells after transfected by lentivirus mediated USP39 shRNA or control sh groups (** 0.01, *** 0.001 and **** 3PO 0.0001). (G,H) Xenograft tumors were by injection of A549 cells stably suppressing USP39 compared with the control and control sh groups (= 4). Representative images of xenograft tumor were shown. Tumor mass volume was every 3 days after 9 days of injection (*** = 0.0003). 2.3. Knocking Down USP39 Inhibits the G2/M Cell Cycle Transition and Induces Apoptosis To elucidate the molecular.