Cell culture and reagents. Cell lines such as SW 620, HCT-8, HCT-116, SW 480, A549, H460, Panc-1, SGC-7901 and CHO were purchased from the American Type Culture Collection (Manassas, VA) and cultured under the conditions recommended by the vendor. CBFB siRNA, the mimics, mimics control, agomir and agomir control were synthesized by GenePharma (Suzhou, China). CBFB/GV141 vectors and vector control were synthesized by GeneWiz (Suzhou, China). DMEM and RPMI 1640 cell culture media were purchased from Gibco (Thermo, Massachusetts, USA).
Cell Transfection. To investigate the regulatory role of mimics in gene expression, synthetic DNAs or RNAs (mimics or mimics control; Table S1) were transfected into CRC cells using lipofectamine 2000 (Invitrogen, California, USA) for 48 h (RNA analysis) or 72 h (protein analysis).
RNA sequencing (RNA-seq). This assay was performed by Shanghai Oebiotech Co., Ltd. Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Texas, USA). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent, California, USA). The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, California, USA). Then the libraries were sequenced on HiSeq™ 2500 sequencing platform (Illumina, California, USA) and 125bp/150bp paired-end reads were generated.
RNA quantification. For quantitative real-time PCR (qPCR), total RNA was isolated from cells using TRIzol reagent (Takara, Tokyo, Japan). The total RNA extracted was reverse transcribed into cDNA using NxGen M-MuLV reverse transcriptase (Invitrogen, California, USA). qPCR was performed using quantitative RT-PCR master mix (Bio-Rad, California, USA) and primers (Table S1) on CFX96 Touch™ real-time PCR system (Bio-Rad, California, USA). RNA expression levels were normalized to those of GAPDH.
Western blot. Total proteins from cells and tumor tissues were extracted using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo, Massachusetts, USA). Alliquots of 20 µg proteins were separated on a 10% SDS-PAGE gel and electro-transferred onto a PVDF membrane. After blocking using 5% skim milk, the membrane was incubated with primary antibody at 4°C overnight and subsequently with the corresponding secondary antibody (Santa Cruz Biotech, Texas, USA) for 1 h at room temperature. The membrane was then developed using Clarity Western ECL substrates (Merck Millipore, Darmstadt, Germany) and visualized with a ChemiDocTM MP Imaging System (Bio-Rad, California, USA). Protein expression was normalized with GAPDH expression. The antibodies against CBFB (#62184S) and β-Actin (#3700S) were purchased from Cell Signaling Technology (Massachusetts, USA).
Dual-luciferase reporter (DLR) assay. 3’-UTR of CBFB gene was cloned downstream of the pGL3-control vector (Promega, Utah, USA)) using XbaI and Hpal endonucleases (NEB, Massachusetts, USA ). CHO cells were co-transfected with 200 ng pGL3 constructs and 100 nM mimics (GenePharma, Suzhou, China) using lipofectamine 2000 (Invitrogen, California, USA). Luciferase activity was measured after 24 h using the DLR assay system (Promega, Utah, USA).
CCK-8 assay. Cell proliferation was measured using the CCK-8 assay kit (Dojindo, Kyushu, Japan). Approximately 3000 cells were plated into each well of a 96-well plate (Corning, New York, USA) and transfected with 50 nM miRNA mimics using lipofectamine 2000. After 72 hours, 10 µL CCK-8 was added into 90 µL culture medium. Cells were subsequently incubated for 15 min at 37°C and the optical density was measured at 450 nm using M3 SpectraMax microplate reader (Biotek,Vermont, USA).
Tumor xenograft model. Protocols for animal husbandry and experiments were approved by the Institutional Animal Care and Use Committee at Soochow University. All animal experiments were complied with the ARRIVE guidelines and were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Male Nude mice were purchased from SLAC int. (Shanghai, China). All animals were kept in specific-pathogen-free (SPF) conditions in the Animal Resource Center at Soochow University.
To investigate anti-tumor activities of miR-34a and AM22, HCT-116 cells (3×106) or SW620 cells (5×106) were subcutaneously inoculated in the lower right flank of mice. Tumor-bearing mice were randomly assigned to different groups after tumors reached a volume of 100–150 mm3. An intravenous dose (1.45 mg/kg) of miR-34a agomir, AM22 agomir or agomir NC was administered once every 2 days. The tumor volume was detected by using vernier caliper every 3 days after inoculation. Tumor tissues were collected at the end of the study for analysis of CBFB protein.
Data analysis. Statistical analyses were conducted using Student's t-test and Pearson Correlation Coefficient. The results were presented as mean ± SD, with P < 0.05 considered as statistically significant.