*P<0.05 and **P<0.01 vs. consumption, -ketoglutarate (-KG) production and ATP production were significantly decreased by circ_0000003 knockdown in TSCC cells, and these effects were reversed by miR-330-3p inhibition. In conclusion, circ_0000003 facilitates TSCC cell proliferation, migration, invasion and glutamine catabolism by regulating the miR-330-3p/GLS pathway. luciferase activities. Pull-down assay with biotinylated miR-330-3p TSCC cell lysates were collected using RIPA buffer plus RNase inhibitor (Promega Corp.), followed by transfection with biotin-labeled wild-type (WT) miR-330-3p (Bio-miR-330-3p-WT), mutated (MUT) miR-330-3p (Bio-miR-330-3p-MUT) or antagonistic miR-330-3p probe (Bio-NC-probe), which were all designed and synthesized by GenePharma. Then, the TSCC cell lysates were mixed with M-280 streptavidin magnetic beads (Sigma-Aldrich; Merck KGaA) for 3 h at 4C. The pull-down products were subjected to RT-qPCR for circ_0000003 expression. RNA immunoprecipitation (RIP) assay RIP assay was carried out using the RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore Corp.). In brief, TSCC cells were lysed in RIP lysis buffer, followed by incubation with anti-Ago2 antibody (catalog no.ab57113, Abcam) and protein G magnetic beads. After 6 washes, the immunocomplexes bound by Ago2 were eluted, and then incubated with proteinase K at 55C for 30 min to digest the proteins, followed by RNA extraction and RT-qPCR analysis for the expression of circ_0000003 or GLS mRNA. Statistical analysis All data were statistically analyzed via SPSS 25.0 (IBM Corp.). Paired t-tests were used in the comparisons between TSCC tissues and their adjacent normal tissues. Unpaired t-tests were used in the comparisons between two groups of TSCC cells. Differences among 3 groups were assessed using one-way ANOVA followed by Tukeys post-hoc test. The overall survival curve was generated and assessed by Kaplan-Meier and log-rank assessments. The associations between circ_0000003 expression and clinicopathological parameters of TSCC patients were assessed using the 2 Rabbit Polyclonal to MAPK3 2 test (for analyzing sex and age) or Fishers exact test (for analyzing TNM stage and tumor size) in Table II. Among circ_0000003, miR-330-3p and GLS mRNA, their linear correlations were assessed using Pearsons correlation analysis RIPK1-IN-7 and two-tailed t-test. Table RIPK1-IN-7 II. Correlation between circ_0000003 and the clinicopathological features of the TSCC patients (N=40).
Age (years)??5517980.1020.749??>55231112Sex lover??Male2411130.4170.519??Female1697TNM stage??I+II181356.4650.011a??III+IV22715Tumor size (cm)??2211474.9120.027a??>219613 Open in a separate window The 2 2 RIPK1-IN-7 test was utilized for comparison between groups aP<0.05 indicates a statistically significant result. TSCC, tongue squamous cell carcinoma; TNM, Tumor-Node-Metastasis. Results Circ_0000003 is usually upregulated in TSCC and promotes TSCC cell proliferation To determine the role of circ_0000003 in TSCC, we tested circ_0000003 expression levels in 40 pairs of TSCC tissues and their paired adjacent normal tissues. As shown in Fig. 1A, circ_0000003 expression was significantly increased in the TSCC tissues compared with that noted in the paired adjacent normal tissues. We also examined circ_0000003 expression levels in HOKs and TSCC cell lines (SCC25, SCC4, Cal27 and SCC1), and found that circ_0000003 expression was significantly increased in the TSCC cells when compared with the HOKs cells (Fig. 1B). The above data revealed that circ_0000003 is usually highly expressed in TSCC tissues and cell lines. Furthermore, the clinicopathological characteristics of the 40 TSCC patients are shown in Table II. High expression of circ_0000003 was found to be significantly correlated with advanced TNM stage and increased tumor size. In addition, high expression of circ_0000003 predicted a poor patient prognosis (Fig. 1C). Open in a separate window Physique 1. Circ_0000003 is usually upregulated in TSCC tissues and cell lines and promotes TSCC cell proliferation in vitro. (A) Circ_0000003 level was RIPK1-IN-7 examined in TSCC tissues (n=40) and their paired adjacent nontumor tissues (n=40). **P<0.01 vs. the nontumor tissues. (B) Circ_0000003 level was measured in HOK and TSCC cells (SCC25, SCC4, Cal27 and SCC1). **P<0.01 vs. the HOK. (C) Survival rates of TSCC patients with high and low circ_0000003 expression were analyzed via Kaplan-Meier survival analysis. (D) Circ_0000003 shRNA (sh-circ_0000003) transfection silenced the circ_0000003 expression in Cal27 and SCC1 cells. (E) CCK-8 assay was conducted to detect the cell proliferative ability of Cal27 and SCC1 cells with circ_0000003 knockdown. (F) p-circ_0000003 transfection enforced the circ_0000003 expression in SCC25 and SCC4 cells. (G) CCK-8 assay was conducted to detect the cell proliferative ability of SCC25 and SCC4 cells with circ_0000003 overexpression. *P<0.05 and **P<0.01 vs. controls. circ_0000003, hsa_circ_0000003; TSCC, tongue squamous cell.