Thirty microliters of proteins were put through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane (EMD Millipore, Billerica MA, USA)

Thirty microliters of proteins were put through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane (EMD Millipore, Billerica MA, USA). co-cultured with NFs or CAFs, or after added exosomes. qPCR was used to detect the expression of miR-382-5p. Western blot analysis was used to measure the expression of migration and invasion-associated proteins. In the present study, the CAF density in tumor tissues was found to be relevant to OSCC lymph node metastasis and TNM stage. Furthermore, we revealed that miR-382-5p was overexpressed GW 542573X in CAFs compared with that in fibroblasts of adjacent normal tissue and miR-382-5p overexpression was responsible for OSCC cell migration and invasion. Finally, we demonstrated that CAF-derived exosomes transported miR-382-5p to OSCC cells. The present study confirmed a new mechanism of CAF-facilitated OSCC progression and may be beneficial for identifying new cancer therapeutic targets. detected the distinguishable miRNAs in CAFs and normal fibroblasts (NFs), and the expression of several miRNAs, including miR-382-5p, was significantly higher in CAFs than in NFs (31). In OSCC, it has been reported that a newly identified circRNA, hsa-circ-0008309, could sponge miR-382-5p and regulate ATXN1 expression (32); however, the function of GW 542573X miR-382-5p in OSCC migration and invasion remains unknown. In the present study, the role of CAFs in mediating OSCC cell migration and invasion was investigated, and the participation of exosomal miR-382-5p in this process was elucidated. Materials and methods Patients and tissue samples Forty-seven OSCC patients, 27 male and 20 females, ranged from 39 to 72 years old, who underwent tumor resection at the Department of Oral Maxillofacial Surgery of Liaocheng People’s Hospital from 1 January 2014 to 31 December 2017 GW 542573X were enrolled in this study. Patient, clinical and pathologic characteristics were retrieved from the Medical Records Room. All patients signed informed consent prior to participating in this study, and this study was approved by the Ethics Committee of Shandong University (Shandong, China). Antibodies and reagents Anti–SMA (cat. no. 19245), anti-MMP-3 (cat. no. 14351), anti-MMP-9 (cat. no. 13667), anti–catenin (cat. no. 8480) and anti-N-cadherin (cat. no. 13116) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and anti–actin (SC-70319) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). GW4869 was purchased from Selleck Chemicals (Houston, TX, USA). Cell culture Tongue squamous cell carcinoma CAL-27 cells GW 542573X were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and incubated in Gibco DMEM (Thermo Fisher Scientific, Inc.) with 10% GW 542573X fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.) at 37C in a 5% CO2 incubator. Isolation of fibroblasts Fibroblasts were isolated according to a previous study (33). Briefly, fibroblasts were isolated from freshly resected OSCC tissue and adjacent normal tissue from the OSCC patients treated at the Department of Stomatology, Liaocheng People’s Hospital. The adjacent normal tissues were excised at Rabbit Polyclonal to p18 INK least 3 cm distant from the margin of the tumor, and were verified by two senior pathologists. Tissues were minced into small pieces of about 1 mm3 and seeded onto 10-cm dishes in DMEM with 10% FBS at 37C in a 5% CO2 incubator. Approximately 10 days later, homogeneous groups of fibroblasts formatted around the pieces in the dishes. The fibroblasts were passaged for more than 10 times and were then used for subsequent experiments. Co-culture of the OSCC cells with CAFs or NFs To elucidate the biological function of CAFs in OSCC metastasis, we employed a co-culture system (Corning Inc., Corning, NY, USA) (Fig. 3A). Briefly, CAFs or NFs were seeded in the upper chamber of the system, and the OSCC cells in the lower chamber. The upper chamber and lower chamber were separated with a semipermeable membrane with pores ~0.4-m, which allowed the passage.