Cell culture and transfection The RAW 264.7 cells were obtained from the ATCC (Manassas, VA, USA). All these cells were cultured with the RPMI-1640 medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Gibco, USA). All these cells were cultured in 37℃ humid atmosphere with 5% CO2. The miR-320a, MYC and PTEN knockdown and miR-320a overexpression lentivirus were purchased from the Genechem (Shanghai, China). And the polybrene (Genechem, Shanghai, China) was used for the promotion of the transfection efficacy. All the operation was followed the instruction.
TRAP staining The Leukocyte Acid Phosphatase (TRAP) Kits was used for the detection of the differentiation of osteoclast. All the operation was followed the instruction.
Luciferase reporter assays The vector and lentivirus were obtained from the Genechem (Shanghai, China). Luciferase Kit (Thermo Fisher Scientific, USA) was used for the detection of the fluorescence. All the operation of this assay was followed the instruction.
CHIP assays RAW 264.7 cells were cross-linked with 10% formaldehyde at room temperature for 10 minutes and quenched with 125 mM glycine for 5 minutes. DNA fragments averaging 200–500 bp are produced from cross-linked chromatin using ultrasound. After that, these chromatin fragments were immunoprecipitated with Goat anti-rabbit IgG H&L (Abcam, ab150077) and MYC (Abcam, ab32072). At last, precipitated DNA were purified with the ChIP DNA Clean Kits (Zymo Research).
qRT-PCR Total RNA was collected with the Trizol (Thermo Fisher Scientific, USA) methods. Next, reverse transcription kits (Takara, Japan) were used to reverse transcribe the RNA into cDNA. After that, the ABI7500 system (Thermo Fisher Scientific, USA) was used for the amplified of the cDNA. And the results were analyzed with the 2−ΔΔCt method. The primers used in this research were miR-320a forward primer 5’-TGCATAAAATAGGCCTTGTGTGAG-3’ reverse primer 5’-AGGCAGATTCACAGCCTCACTC-3’ c-Fos forward primer 5’-ACGTCCCCAAGCCAGGCTC-3’ reverse primer 5’-CTATGTAGCTCAGGAATAA-3’ NFATc1 forward primer 5’-ATAGGCCTTGTGTGAGCCA-3’ reverse primer 5’-GCCTTGTGTGAGCCACCAT-3’ TRAP forward primer 5’-GGCCTTGTGTGAGCCACCA-3’ reverse primer 5’-CTCTTCTCCCTCAGATATA-3’ CTSK forward primer 5’-AAAAGCTGGGTT GAGAGGGCG-3’ reverse primer 5’-CTGCCCAGCATCTGACCCTAA-3’ PTEN forward primer 5’AAAAGC UGGGUUGAGAGGGCGA-3’ reverse primer 5’-UUCUCC GAACGUGUCACGUTT-3’ MYC forward primer 5’-UCGCCCUCUCAA CCCAGCUUUU-3’ reverse primer 5’-CAGUACUUUUGUGUAGUACAA3’ GAPDH forward primer 5’-CACCATTGGCAATGAGCGGTTC-3’ reverse primer 5’-AGGTCTTTGCGGATGTCCACGT-3’ U6 forward primer 5’-AACGCTTCACGAATTTGC GT-3’ reverse primer 5’-CTGCCCAGCATCTGACCC TAA-3’.
Western blotting Total proteins was extracted with the RIPA buffer (Beyotime, China). Then, the concentration of these samples was determined with the BCA method (Beyotime, China). After that, these proteins were separated with the 8%-12% SDS-PAGE gel (Beyotime, China). These proteins were transferred to the PVDF membranes (Millipore, USA). Then, these membranes were blocked with 5% skim milk powder. After that, these membranes were incubated with primary antibodies at 4℃ overnight. The primary antibodies used in this study were MYC (1:500; Abcam, ab32072), c-Fos (1:500; Abcam, ab208942), NFATc1 (1:400Abcam, ab183023), TRAP (Abcam, ab133238), CTSK (Abcam, ab207086), PTEN (Abcam, ab267787) and GAPDH (Abcam, ab8245). These membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:1000; Cell signaling technology, 7074) for 2 hours in the second day. Finally, the immunoreactive signals were detected by the Pierce Western blotting Substrate (Thermo Fisher Scientific, USA).
Statistical analysis All the data in this study was analyzed with the GraphpadPrism 6.0. All the experiments in this research were repeated for three times. All the data in this study was displayed as mean ± SD. The comparison between diverse groups was performed with the student’s t test. The difference was considered as statistical significantly when the values of P was less than 0.05.