Human patient tissue specimens
Freshly breast tissue specimens obtained from breast cancer patients who underwent curative surgical treatment between April 2010 and March 2012 were immediately frozen in liquid nitrogen for further RNA extraction. All of patients were selected at Department of Oncology, Peking University Shenzhen Hospital. The study was approved by Peking University Shenzhen Hospital and written informed consent was obtained from all of the patients.
Cell lines culture
Three human breast cancer cell lines (MDA-MB-231, MCF-7, and SKBR-3) and one normal mammary epithelial cell line (MCF-10A) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). All of breast cancer cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) added with 10% fetal bovine serum at 37 °C with 5% CO2.
Quantitative real-time polymerase chain reaction (QRT-PCR)
The TRIzol® (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction from tissues and cells according to the manufacturer’s instructions. RNA was subjected to cDNA synthesis using a Prime Script RT Regent kit (Takara, Dalian, China). The SYBR Green PCR Master Mix (Takara, Dalian, China) was used for qRT-PCR assay on ABI PRISM 7900 Real-time PCR system (Applied Biosystems, Foster City, CA). GAPDH were used for normalization. The primer sequences were as follows: SNORD78(Forward:5’-GTGTAATGATGTTGATCAAATGTCTGAC-3’;Reverse: 5’-CACATTACTACAACTAGTTTACAGACTGG-3’),GAPDH(Forward:5’-CTCAAGGGCATCCTGGGCTAC-3’;Reverse:5’-CAGCCCCAGCGTCAAAGGT-3’).QRT-PCR results were analyzed using 2△△CT Methods.
RNA interference and cell transfection
Two siRNAs against SNORD78 was designed to knockdown SNORD78 expression and one negative control (si-NC) was purchased from Ribobio (Guangzhou, China). Cell transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The siRNAs targeted SNORD78 sequences were as follow: si-SNORD78-1(5’-GTTGATCAAATGTCTGACCTG-3’), si-SNORD78-2 (5’-GACCTGAAATGAGCATGTAGA-3’).
MTT assay
Cell growth rate was investigated using a MTT assay. 3 × 103 transfected cells/well were seeded into 96-well plates. At indicated time 0, 24, 48, and 72 h, cells were incubated with 20 µl MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) and cultured for 4 h at 37 °C with 5% CO2. The cell proliferation was analyzed using an ELISA reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the absorbance at 490 nm.
Cell clonogenic assay
300 transfected cells were placed in 12-well plates cultured in medium added with 10% fetal bovine serum at 37 °C with 5% CO2 for 14 days. Colonies were fixed with 100% methanol, stained with 0.1% crystal violet, and then were counted under a microscope.
Cell invasion assays
Transwell chambers with an 8 µm pore polycarbonate membrane (Costar; Corning Incorporated, Corning, NY, USA) were used to assess cell invasion ability. 1 × 105 cells were added on the upper chamber supplemented with 300 µl culture medium without FBS and 500 µl culture medium supplemented with 20% FBS was added on the lower chamber. After cell cultured for 24 h, cells were fixed with 100% methanol, and then stained with 0.1% crystal violet. Cells on the lower chamber were counted in five random fields under a light microscope.
Western blot analysis
Cells were lysed using radioimmunoassay precipitation assay (RAPA) lysis buffer with a proteinase inhibitor. The quantity of approximately 40 µg proteins in the lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were incubated with anti-WNT1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), GSK3β (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), β-catenin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and GAPDH (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 12 h at 4 °C and then washed three times for 5 min. Following horseradish peroxidase-conjugated secondary antibodies were incubated. The blot was visualized by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA).
Statistical analysis
All of results are presented as means ± SD from at least three or more independent experiments. The association between SNORD78 expression and clinical characteristics was evaluated using the chi-square test. Differences between groups were compared using Student’s t-test or an analysis of variance (ANOVA). A p < 0.05 were considered statistically significant.