Cell lines and culture
Two osteosarcoma cell lines used in this study-G-292 (ATCC NO. CRL-1423) and SJSA-1 (ATCC NO. CRL-2098) were purchased from the ATCC (https://www.atcc.org/). The two cell lines were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% glutamine at 37 °C in 5% CO2.
The mimic/antagomiR/siRNA/overexpression plasmids transfection
All the mimic, antagomiR, siRNA, and the scramble sequence control (NC) as well as riboFECT CP transfection kits were supplied by Guangzhou Ribobio (Guangzhou, China). The mammalian expression constructs for SDC2 with GFP tag (EX-W2418-M98) were supplied by GeneCopoeia™ (http://www.genecopoeia.com/). Transfection of both ribonucleic acid reagents or plasmids mentioned above and the reporter plasmids in a Cignal Finder Pathway Reporter package (Qiagen, Hilden, Germany) was performed according to the manufacturer’s instruction.
The sequences used in this study are as follows:
The luciferase reporter assay
Two partial sequences of the human SDC2 3′-untranslated region (276 bp, 1–276 and 533 bp, 1729–2261) with the miR-20a-5p targeting motif were cloned at the downstream of the firefly luciferase gene in pmiR-RB-REPORT™ to construct pmiR-RB-REPORT™-luc-SDC2-WT1 and pmiR-RB-REPORT™-luc-SDC2-WT2, respectively. Cells were seeded into 96-well plates at around 1 × 104 cells per well and transfected with a mixture of 50 ng pmiR-RB-REPORT™-luc-SDC2-WT1/WT2, 5 ng Renilla plus 5 pmol mimic or scramble control (NC) nucleotides, with the riboFECT CP transfection reagents according to the manufacturer’s instruction. Both firefly and Renilla luciferase activities were measured 24 h after transfection by the Dual-Luciferase Reporter Assay System (Promega) using a Promega GloMax 20/20 luminometer. The relative firefly luciferase activities were normalized with the Renilla luciferase activities as a for transfection efficiency.
Clinical grades of the following drugs were used (NCI dictionary of cancer terms, http://www.cancer.gov/dictionary), Dox (Haizheng, Zhejiang, China); Etop (Hengrui, Jiangsu, China); MTX (Lingnan, Guangdong, China) and CDDP (Haosen, Jiangsu, China) [5, 20, 21].
Chemoresistance profiling (IC50 measurements)
Cells in the logarithmic phase of growth were seeded in triplicate in 96-well plates at the density of 0.5 × 104/well and treated with fourfold serially diluted drugs for 72 h. Cell survival was then measured by a thiazolyl blue tetrazolium bromide (CCK8, 450 nm reading)-based cell proliferation assay . Both the linear regression parameters and the IC50 (the concentration of drug required for 50% of cells to be killed) with the no-drug control as the reference were calculated. The relative chemoresistance was presented as the fold for each of the cell line over the lowest IC50 .
Cells were harvested and rinsed with PBS twice. Then, 3 μl of FITC-labeled enhanced annexinV and 3 μl (20 μg/ml) of propidium iodide were added to 100 μl of cell suspension. After incubation in the dark for 15 min at room temperature, the samples were diluted with 100 μl of PBS. Flow cytometry was performed on a FACSCalibur instrument. The results were analyzed according to the manufacturer’s instructions. The experiments were performed independently three times, and a representative is shown.
In vitro invasion assays
Cell invasion assays were performed in a 24-well plate with 8 mm pore size chamber inserts (Corning, USA). For invasion assays, 1 × 104 cells stably expressing mimic, antagomiR or NC were placed into the upper chamber in each well with the matrigel-coated membrane, which was diluted in serum-free culture medium. In the assay, cells were suspended in 100 μl of DMEM without FBS when they were seeded into the upper chamber. In the lower chamber, 500 μl of DMEM supplemented with 10% FBS was added. After incubation for 30 h at 37 °C and 5% CO2, the membrane inserts were removed from the plate, and non-invading cells were removed from the upper surface of the membrane. Cells that moved to the bottom surface of the chamber were stained with 0.1% crystal violet for 30 min. The cells were then imaged and counted in at least five random fields using a CKX41 inverted microscope (Olympus, Tokyo, Japan). The assays were conducted three independent times .
RNA-seq analysis was performed by BGI-Tech of China, and RNA-seq library preparation and sequencing were performed by BGI (Shenzhen, China). Following purification, RNA was fragmented using divalent cations at an elevated temperature, and first-strand cDNA was synthesized using random hexamer primers and Superscript TMIII (Invitrogen™, Carlsbad, CA, USA). Second-strand cDNA was synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. Short fragments were purified with a QiaQuick PCR extraction kit (Qiagen) and resolved with EB buffer for end reparation and poly (A) addition. The short fragments were subsequently connected using sequencing adapters. After agarose gel electrophoresis, suitable fragments were used as templates for PCR amplification. During the QC steps, an Agilent 2100 Bioanaylzer and an ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library. Finally, the library (200-bp insert) was sequenced using Illumina HiSeq2000 (Illumina Inc., San Diego, CA, USA). The single-end library was prepared following the protocol of the IlluminaTruSeq RNA Sample Preparation Kit (Illumina) .
Total RNA was isolated from the cells during the logarithmic phase using Trizol (Tiangen Biotech Co., Ltd., Beijing, China). For the mRNA analysis, the cDNA primed by oligo-dT was made with a prime Script RT reagent kit (Tiangen Biotech Co., Ltd., Beijing, China), and the mRNA level of SDC2 was quantified by a duplex-qRT-PCR analysis where the TaqMan probes with a different fluorescence for β-actin (provided by Shing Gene, Shanghai, China) were used in the FTC-3000P PCR instrument (Funglyn Biotech Inc., Canada). Using the 2−ΔΔCt method, the normalization with the β-actin level was performed before the relative level of the target genes was compared. The sequences of primers and probes used for the qRT-PCR analysis are as follows:
HSDC2 F: 5′-CCTATTGATGACGATGACTACGC-3′
HSDC2 R: 5′-CCTATTGATGACGATGACTACGC-3′
HSDC2 probe: 5′-ROX-CCTATTGATGACGATGACTACGC-3′
hACTB F: 5′-GCCCATCTACGAGGGGTATG-3′
hACTB R: 5′-GAGGTAGTCAGTCAGGTCCCG-3′
hACTB probe: 5′-CY5-CCCCCATGCCATCCTGCGTC-3′
Cells were lysed with a lysis buffer (60 mM Tris–HCl, pH 6.8, 2% SDS, 20% glycerol, 0.25% bromophenol blue, 1.25% 2-mercaptoethanol) and heated at 95 °C for 10 min before electrophoresis. The protein was separated by 12% SDS-PAGE and then transferred to polyvinyl difluoride membranes (Millipore, Bedford, MA) by electroblotting. After blocking with 5% non-fat dry milk, the blots were incubated with primary antibodies (against SDC2 and GAPDH). The target bands were revealed by an enhanced chemiluminescence reaction (Pierce), and the relative density (level) of proteins over the GAPDH band was quantified with the Gel-Pro Analyzer (Media Cybernetics). Anti-SDC2 (YT4490) was purchased from ImmunoWay (http://www.immunoway.com/index.asp) and anti-rabbit IgG (SA00001-2) was purchased from San Ying Biotechnology, China (https://www.ptglab.com/).
In vivo studies
Animal experiments were performed as previously described . Expressions of SDC2 protein were measured using immunochemical analysis on 5-mm slices of formalin fixed paraffin-embedded tumor xenografts in nude mice. Antigens were retrieved by pretreating dewaxed sections in a microwave oven at 750 W for 5 min in a citrate buffer (pH 6) processed with the Super Sensitive Link-Labeled Detection System (Biogenex, Menarini, Florence, Italy). The enzymatic activities were developed using 3-amino-9-ethylcarbazole (Dako, Milan, Italy) as a chromogenic substrate. Following counterstaining with Mayer hematoxylin (Invitrogen), slides were mounted in aqueous mounting medium (glycergel, Dako). Pictures were taken using a LEICA DM 4000B microscope, while the relative level of each protein was calculated using LEICA software, percentage of the mock over the chemotherapeutic treated tumors was calculated and plotted.
The data are presented as the mean, and the error bars indicate the S.D. All statistical analyses were performed with GraphPad Prism 5. Two-tailed Student’s t test, a one-way analysis of variance or Mann–Whitney U test was used to calculate statistical significance. A P value of < 0.05 was considered significant.