2016; 17:47C62

2016; 17:47C62. that an increased output of the miR-493-5p/ITGB1 axis could neutralize the regulatory impact of NR2F1-AS1 knockdown on the malignant phenotype of NSCLC cells. In summary, the NR2F1-AS1/miR-493-5p/ITGB1 pathway initiates pro-oncogenic behavior in NSCLC tumor progression, and the NR2F1-AS1/miR-493-5p/ITGB1 axis may provide new molecular targets for anticancer therapy against NSCLC. and and decreased tumor growth [24]. Additionally, the interference of NR2F1-AS1 expression reportedly increases cell apoptosis and induces cell cycle arrest (S)-(-)-Citronellal in osteosarcoma [24]. Further, NR2F1-AS1 exerts tumor-promoting roles in esophageal squamous cell carcinoma progression and is implicated in the regulation of cellular viability, colony formation, proliferation, migration, invasion, and sphere-forming ability [25]. Our present study focused on investigating whether NR2F1-AS1 contributes to the aggressive phenotype of NSCLC cells and showed that the proliferation, migration, and invasion were attenuated and apoptosis induction was promoted in NSCLC cells following NR2F1-AS1 knockdown. Furthermore, the depletion of NR2F1-AS1 suppressed NSCLC tumor growth electrophoresis (10% gel) was performed for the separation of proteins with equivalent concentrations, following which they were transferred to polyvinylidene fluoride membranes. Blocking was performed by incubating the imprinted membranes for 2 h at room temperature with 5% nonfat dried milk diluted in Tris-buffered saline containing 0.1% of Tween 20. After overnight incubation with primary antibodies at 4C, a horseradish peroxidase-conjugated secondary antibody (cat. no. ab6721; Abcam, Cambridge, UK) was diluted at a concentration of 1 1:5000 and further used for incubating the membranes at room temperature for (S)-(-)-Citronellal 1 h. The protein bands were developed using ECL Western Blotting Substrate kit (Abcam). The primary antibodies and their sources are as follows: ITGB1 (cat. no. ab52971; Abcam) and GAPDH (cat. no. ab181602; Abcam). GAPDH was used as the loading control. Statistical analysis All experiments were performed in triplicate and repeated at least three times. All results were expressed as mean standard deviation. Students t-test was used to examine the differences between two groups. Comparisons among 3 groups were performed using one-way analysis of variance along with Dunnetts post-hoc test. Correlations between NR2F1-AS1 and miR-493-5p expressions in 73 NSCLC tissues were tested using Pearson’s correlation coefficient. Chi-squared test was used to analyze the association between the NR2F1-AS1 expression and clinical characteristics of 73 patients with NSCLC. The 5-year overall survival of these 73 patients with NSCLC was determined using the KaplanCMeier method, which was further analyzed with log-rank test. P < 0.05 was considered statistically significant, and P < 0.01 indicated a highly significant difference. Ethics Statement All experimental protocols were approved by the Clinical Research Ethics Committee of The Second Xiangya Hospital of Central South University. Moreover, written informed consent was collected from all participants in the study. Xenograft tumor assay was conducted in accordance with the guidelines of Animal Protection Law of the Peoples Republic of China (2009) for experimental animals and with the approval of Ethics Committee of Animal Experiments of The Second Xiangya Hospital of Central South University. Footnotes Contributed by AUTHOR CONTRIBUTIONS: Chan Zhang and Shangjie Wu conceived and supervised this study. Chan Zhang, Shangjie Wu , Rong Song, and Changming Liu implemented all experiments. 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