miR-543 expression was significantly upregulated in tumors from patients with OSCC and in OSCC cell lines

miR-543 expression was significantly upregulated in tumors from patients with OSCC and in OSCC cell lines. (CYP3A5) as a direct target of miR-543 using software analysis and dual-luciferase reporter assays. In conclusion, the results of the present study suggest that miR-543 acts as a tumor promoter and serves a vital role in OSCC proliferation and invasion. These results confirm that YK 4-279 miR-543 may serve as a potential novel target for the treatment of OSCC. luciferase plasmid with a ratio of 2:2:1 (31,32). Lysates were collected 72 h post-transfection. Firefly and luciferase activities were measured using a Dual-Luciferase Reporter System (Promega Corporation, Madison, WI, USA). Detection value ratio=luciferase detection value/firefly luciferase detection value. Immunohistochemistry (IHC) IHC staining of human OSCC tissues was performed on deparaffinized OSCC tissue sections using primary antibody against CYP3A5 (dilution, 1:100; cat. no. ab108624; Abcam) overnight at 4C. For the negative control for immunohistochemistry analysis, the primary antibody was replaced with normal IgG (dilution 1:100; cat. no. ab172730; Abcam) overnight at 4C. The slides were subsequently treated with biotinylated anti-rabbit YK 4-279 secondary antibody anti-rabbit IgG H&L (HRP) (dilution 1:4,000; cat. no. ab205718; Abcam) and counterstained with hematoxylin. Control experiments were performed using non-immune immunoglobulins as opposed to specific antibody. Immunostained images were captured using a digital camera; five images were captured at random. Statistical analysis SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis, and the data are presented as the mean standard error of the mean. Statistical analysis was performed using a paired t-test or one-way analysis of variance (ANOVA). The data of three or more groups were evaluated using analysis of variance and least significant difference (LSD) post hoc tests when the variance was normal. P 0.05 was considered to indicate a statistically significant difference. All experiments were conducted at least three times. Results High expression of miR-543 in OSCC cell lines and human tissues In order to investigate the potential mechanism of miR-543 in OSCC, the expression of miR-543 was detected by RT-qPCR in 20 pairs of OSCC tissues and adjacent non-tumor tissues obtained during clinical operations (Fig. 1A). The results of RT-qPCR demonstrated that miR-543 was significantly increased in cancerous tissues when compared with that noted in the adjacent non-tumor tissues (Fig. 1B; P 0.0001). In order to further determine the role of miR-543 in OSCC, the gene expression levels of miR-543 in the OSCC cell lines SCC9, SCC25 and CAL27 were detected. When compared with human normal oral keratinocytes (HOK) cells, miR-543 exhibited a higher expression in SCC9, SCC25 and CAL27 cells (Fig. 1C). Open in a separate window Figure 1. High expression of miR-543 in OSCC cell lines and human tissues. (A and B) Expression of miR-543 was detected in 20 paired clinical samples. Results of RT-qPCR revealed that miR-543 was increased in cancerous tissues when compared with that in adjacent non-tumor tissues. P 0.0001, cancer tissues vs. adjacent non-tumor tissues. (C) RT-qPCR was performed to analyze the expression of miR-543, which was significantly increased in 3 OSCC cell lines, SCC9, SCC25 and CAL27, when compared with that noted in HOK cells. *P 0.05, ***P 0.001. miR, microRNA; OSCC, oral squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. miR-543 promotes the growth of OSCC cell lines in vitro To investigate the mechanism of action underlying miR-543 in OSCC, the present study attempted to determine whether miR-543 affects OSCC cell line proliferation. SCC9, SCC25 and CAL27 cells were transfected with mimic NC, miR-543 mimic, inhibitor NC or miR-543 inhibitor; the results demonstrated a high transfection efficiency (Fig. 2A and B). Rabbit Polyclonal to ADCY8 The CCK-8 assay results indicated that when compared YK 4-279 with the.