MiR-20a-5p represses the multi-drug resistance of osteosarcoma by targeting the SDC2 gene

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.

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:

si-SDC2:

5′-GAAACCACGACGCTGAATA-3′

hsa-miR-20a-5p

antagomiR: 5′-CUACCUGCACUAUAAGCACUUUA-3′

mimic:

sense 5′-UAAAGUGCUUAUAGUGCAGGUAG-3′

antisense 5′-CUACCUGCACUAUAAGCACUUUA-3′

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].

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 [5]. 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 [22].

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.

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 [23].

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) [24].

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/​).

Animal experiments were performed as previously described [22]. 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.

Our previous result suggested that G-292 and SJSA-1 cell lines are the multi-chemosensitive and multi-chemoresistant OS cell lines, respectively [22]. To identify the mechanisms that govern the multi-chemoresistance of OS cells, we performed an RNA-seq-based miR-omic analysis of G-292 and SJSA-1 cells (GEO Accession Number: GSE89930). The results showed that a dozen of miRNAs were differentially expressed in the SJSA-1 and the G-292 cells and miR-20a-5p was selected as one of the studied target miRNAs. Here the expression of miR-20a-5p by miR-omic analysis was 10.50-fold higher in G-292 cells compared with SJSA-1 cells [25]. We further tested the level of miR-20a-5p in G-292 and SJSA-1 OS cell lines by qRT-PCR. The level of miR-20a-5p in G-292 was 8.27-fold higher than that in SJSA-1, indicating a higher expression of miR-20a-5p in the sensitive OS cells [25].

A given miRNA usually suppresses the expression of various target genes and thus regulates related pathways. We thus predicted the target genes of miR-20a-5p based on the following websites: TargetScan (http://​www.​targetscan.​org/​) and microRNA.org (http://​www.​microrna.​org/​microrna/​getMirnaForm.​do). We subsequently compared the expression pattern of shared predicted mRNAs between G-292 and SJSA-1 cells by the RNA-seq based miR-omic analysis (GEO Accession Number: GSE89930). Dozens of genes have been found that differentially expressed in the two cell lines. Among them, the SDC2 gene is one of the most significantly differentiated genes that negatively correlate with the expression of miR-20a-5p. Consequently, the expression level of SDC2 was higher in SJSA-1 than G-292 at both mRNA (RNA-seq based miR-omic: 11.07:1.00, and qRT-PCR analysis: 102.82:1.00) and protein level (western blot: 2.17:1.00) (Fig. 1a–c). The higher expression of SDC2 in multi-chemoresistant cells SJSA-1 suggests that SDC2 is a positive regulator of OS multi-drug resistance.

The miR-20a-5p level was significantly higher in G-292 cells than SJSA-1 cells. We found that SDC2 negatively correlates with the level of miR-20a-5p. To check whether SDC2 is one of the authentic targets of miR-20a-5p, we determined the SDC2 level in the miR-20a-5p mimic transfected SJSA-1 and the antagomiR transfected G-292 cells versus the NC (scramble sequence control) transfected. The transfection of miR-20a-5p mimic in SJSA-1 cells increased its expression to about 39-fold, whereas the transfection of miR-20a-5p antagomiR in G-292 cells significantly decreased its level to 19% [25]. Following the changes of the miR-20a-5p level, a miR-20a-5p mimic transfection brought down the SDC2 mRNA to 56% (Fig. 2a) and protein to nearly 20% (Fig. 2c) compared to that in the NC transfected SJSA-1 cells. As expected, miR-20a-5p antagomiR transfection increased the mRNA level of SDC2 by 2.59-folds (Fig. 2b) and the protein level by 3.78-folds in G-292 cells (Fig. 2c).

Sequence analysis revealed that 3′-UTR region of SDC2 contains two potential binding motifs for miR-20a-5p (termed sit1 and sit2, respectively) (Fig. 2d). To further conclude whether the SDC2 is a target of miR-20a-5p, the wild type 3′-UTR region of SDC2 was inserted downstream of the luciferase gene in the pmiR-RB-REPORT™-control vector to create pmiR-RB-REPORT™-SDC2 UTR WT1 and pmiR-RB-REPORT™-SDC2 UTR WT2 (Fig. 2e). The constructs pmiR-RB-REPORT™-SDC2 UTR WT and pmiR-RB-REPORT™ enhancer control were transfected into G-292 and SJSA-1 cells respectively, to determine whether the differentially expressed miR-20a-5p in OS cells of distinct chemoresistance is indeed functional. The pmiR-RB-REPORT™-SDC2-UTR WT1 gave the relative luciferase activities of 0.90 and 0.60 in SJSA-1 and G-292 cells, respectively, and the WT2 was 0.90 and 0.99 respectively (Fig. 2e). The transfection of miR-20a-5p-mimic into SJSA-1 cells significantly brought down the luciferase activity of pmiR-RB-REPORT™-SDC2-UTR WT constructs, whereas the control cells showed almost the same activity upon the transfection of miR-20a-5p-mimic (Fig. 2f). Meanwhile, the transfection of miR-34a-5p-antagomiR into G-292 cells raised the luciferase activity of pmiR-RB-REPORT™-SDC2-UTR WT constructs (Fig. 2g). It is worthy to notice that sit1 has a more profound effect against the changed level of miR-34a-5p in both G-292 and SJSA-1 cells, which indicated that miR-20a-5p binds more firmly to the sit1 UTR, compared to sit2 UTR. This is also in accordance with the sequence analysis that miR-20a-5p has seven base pairings for sit1 UTR whereas six base pairings for sit2 UTR (Fig. 2d). Getting together, SDC2 is indeed, a direct target of miR-20a-5p and may execute the miR-20a-5p’s repressing effect on the OS drug resistance.

To further demonstrate that miR-20a-5p modulates multi-drug resistance by repressing SDC2 expression in OS cells, we compared drug-triggered cell death in SJSA-1 cells transfected with miR-20a-5p mimic or SDC2 siRNA. The results showed that transfection with the miR-20a-5p mimic reduced the SDC2 level to 31% of that found in the NC transfected cells. In addition, SDC2 siRNA inhibited SDC2 protein expression to approximately 45% of the NC control (Fig. 3a). The transfection of miR-20a-5p mimic or si-SDC2 in SJSA-1 cells decreased the chemoresistance to some extent against the following five drugs: Dox, Etop, MTX, CDDP, Carb, except the mimic to MTX and CDDP (Fig. 3b). Consequently, the transfection of miR-20a-5p mimic or si-SDC2 in SJSA-1 cells showed lower invasion capacity compared to the control cells (Fig. 3c). Afterwards, we increased the level of SDC2 by transfection of miR-20a-5p antagomiR or overexpression of SDC2 in G-292 cells. In agreement with the elevated level of SDC2 (Fig. 4a), the cell survival rate was increased for all the five drugs, except the antagomiR to MTX and GFP-SDC2 to CDDP (Fig. 4b). This discrepancy suggested that SDC2 might not mediate OS chemoresistance in response to MTX and CDDP. Similarly, the invasion capacity was elevated with the transfection of either miR-20a-5p antagomiR or GFP-SDC2 in G-292 cells (Fig. 4c).

Additionally, the miR-20a-5p mimic mediated SDC2 downregulation in SJSA-1 cells and increased the percentage of apoptotic cells from 0.82 to 0.91%. Similarly, knockdown of SDC2 with siRNA also elevated the apoptotic ratio from 0.59 to 0.87% in SJSA-1 cells (Fig. 5a–c). This result indicated that low levels of miR-20a-5p promoted OS cell survival probably by increasing SDC2 expression. All of these observations suggest that SDC2 gene does contribute a great deal to the miR-20a-5p’s repressing effect on the OS drug resistance.

Recently, miR-20a-5p was shown to suppress Dox chemoresistance of OS in tumor xenografts of nude mice via its repression of its target gene KIF26B [25]. In the present study, we semi-quantified via immuno-histological analysis the levels of SDC2 protein in the same set of the tumor tissues in mice that were subjected to an injection of Dox or PBS. The intratumoral injection of miR-20a-5p’s agomiR into SJSA-1 decreased SDC2 expression. By contrast, the injection of miR-20a-5p’s antagomiR into G-292 increased SDC2 expression in Dox- or PBS-treated mice (Fig. 6). The results further confirmed that miR-20a-5p has a profound negative effect on both the growth and chemoresistance of OS cell-derived tumor xenografts in nude mice.