MiR-578 mimic transfection led to overexpression of miR-578 in both 143B and U2OS cells (Fig

MiR-578 mimic transfection led to overexpression of miR-578 in both 143B and U2OS cells (Fig. following the manufacturers instructions to extract total RNA, which was used to synthesize complementary DNA (cDNA) with Reverse Transcription System (Promega, Madison, WI, USA). For RNase R treatment, total RNA (2 g) from 143B and U2OS cells was incubated with 5 U RNase R (Solarbio, Beijing, China) for 30 min at HOI-07 37 C. The expression levels of circ-CNST, linear CNST, miR-578, LDHA, and PDK1 were quantified with GoTaq? qPCR and RT-qPCR systems (Promega) and normalized to -actin and U6 mRNA levels. The primers were summarized in Table ?Table2.2. The relative expression was calculated using the 2 2?Ct method. Table 2 The primers and siRNAs used in this study = 8): sh-NC and sh-circ-CNST. Caliper was used to measure tumor dimensions every 3 days after 7-day inoculation, and volume was calculated according to 0.5 length width2 formula. At the termination of the experiment, mice HOI-07 were sacrificed and tumors were excised. Xenograft tumors were weighted with electronic scales and photographed with a camera. Tumor tissues were stored in liquid nitrogen for further RNA isolation. All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals (GB/T 35892-2018; Standardization Administration of the People Republic of China). Dual-luciferase reporter assay Circinteractome HOI-07 (https://circinteractome/circular_rna_query=hsa_circ_0017311&mirna) and Targetscan (http://www.targetscan/vert_71/=hsa-miR-578) databases were utilized to predict miR-578-binding sites in circ-CNST, LDHA, and PDK1. To test the abovementioned target relationships, dual-luciferase reporter assays were carried out. The wild-type (WT) sequences (with a specific binding site for miR-578) of circ-CNST, LDHA 3UTR, and PDK1 3UTR were respectively cloned into luciferase reporter vector pMIR-REPORT (Promega, containing Firefly), as well as the mutated (MUT) sequences (with mutations of miR-578-binding sites). 143B and U2OS cells were co-transfected with WT/MUT vectors, pRL-SV40 (internal control vector, containing Renilla), and miR-578 mimic or miR-NC mimic. After transfection for 24 h, cell lysate was collected and luciferase activities of Firefly and Renilla were measured by the Dual-Luciferase Reporter Assay Kit (Promega) on GloMax Discover Microplate Reader (Promega). Western blotting Total protein in cultured cells was extracted by RIPA Lysis Buffer (Beyotime), and protein concentration was measured using BCA Protein Assay Kit (Beyotime). Proteins were denatured in loading buffer by boiling at 100 C for Mouse monoclonal to HSV Tag 10 min, and denatured protein samples were subjected to Western blotting assay [20]. The antibodies used were presented in Table ?Table3.3. ECL? Western blotting system (Merck, Darmstadt, Germany) was used to detect protein signals and densitometric quantification was performed using ImageJ software (NIH, Bethesda, MD, USA). Table 3 The antibodies used in this study HOI-07 0.05 was deemed as statistically significant: asterisk represented 0.05, double asterisks represented 0.01, triple asterisks represented 0.001, and quadruple asterisk represented 0.0001. Results Circ-CNST was an upregulated circRNA in OS patients and cells Even though expression of circ-CNST in OS had once been identified, it was further validated in this present study. A set of OS patients were enrolled, and RT-qPCR data indicated that relative circ-CNST expression was increased in tumor tissues than adjacent normal tissues (Fig. HOI-07 ?(Fig.1a).1a). Besides, high circ-CNST was found in OS tumors from patients with shorter overall survival, advanced TNM stages, and lymph node metastasis (Figure S1A-S1C). Moreover, Chi-square test confirmed that circ-CNST level was significantly correlated with clinicopathologic features of OS patients, including TNM stage, lymph node metastasis, and tumor size (Table ?(Table1).1). In vitro, its level was higher in several OS cell lines, and 143B and U2OS cells exhibited the highest level of circ-CNST (Fig. ?(Fig.1b).1b). The structure stability was examined by RNase R digestion. Linear CNST expression was significantly attenuated, but circ-CNST expression was little affected in RNase R-treated 143B and U2OS cells (Fig. ?(Fig.1c,1c, d). These results indicated that circ-CNST was a stably upregulated circRNA in OS and might predict heavy tumor burden and poor prognosis. Open in a separate window Fig. 1 Expression of circ-CNST in OS patients and cells. a, b RT-qPCR measured relative circ-CNST expression in tumor tissues from OS patients (= 29) comparing to adjacent normal tissues, and in human OS cell lines (HOS,.