miR-17-5p promotes the growth of osteosarcoma in a BRCC2-dependent mechanism
Retraction in: /10.3892/or.2024.8710
- Authors:
- Published online on: January 5, 2016 https://doi.org/10.3892/or.2016.4542
- Pages: 1473-1482
Abstract
Introduction
Osteosarcoma is a primary malignant bone tumor originated in bone mesenchymal cells, characterized by proliferating tumor cells directly forming immature bone or bone-like tissue. Osteosarcoma, which often occurs in adolescents with poor efficacy and survival, accounts for 20% of primary malignant bone tumors. Typical osteosarcoma is a highly malignant intramedullary tumor, with frequent metastasis, accounting for 85% of all bone sarcomas (1). Along with chemotherapy, surgical techniques, bone reconstruction and other treatment methods have been development, and osteosarcoma limb salvage therapy has gradually taken the place of amputation, thus, >80% of patients prefere limb salvage surgery, and the 5-year survival rate has increase from 20 to 55 to 75% (2). However, the molecular biological mechanisms of osteosarcoma have not been clarified and there are still patients who are not sensitive to chemotherapy and have poor prognosis. Therefore, it is necessary to search new diagnostic and therapeutic methods for osteosarcoma.
miRNAs are a class of single-stranded, 20–24 nucleotides (nt) in length small non-coding RNA molecules. Mature miRNAs have 5′ end with a phosphate group and 3′ end with a hydroxyl group. miRNAs play negative regulatory roles by fully or partially complementation fixation with target genes in 3′-UTR region to cause mRNA degradation or translation inhibition (3). Lee et al (4) found miRNA lin-4 in caenorhabditis elegans for the first time in 1993. Reinhart et al (5) found let-7 in study of nematode growth regulation. Since then the study of miRNA began and developed rapidly. Studies have shown that a variety of miRNAs play roles as oncogene or anti-oncogene in a variety of tumors including sarcoma, and participate in all stages of tumor growth and development (6).
miR-17-5p is one of the members of the miRNA cluster of miR-17-92 which contains seven members and is also one of the most studied miRNAs related to cancers in this cluster. miR-17-5p is also functionally involved in the regulation of the malignancies of multiple cancers (7–9). However, its expression and clinical significance in osteosarcoma is still unclear. In the present study, we showed that miR-17-5p was overexpressed in osteosarcoma cells and tissues compared with normal bone tissues using qRT-PCR. miR-17-5p upregulation in osteosarcoma tissues significantly associated with cell proliferation, invasion, advanced TNM stage and tumor growth. Both gain-of-function and loss-of-function studies showed that miR-17-5p increased the ability of osteosarcoma cells to proliferate and to invade through Matrigel in vitro and increased the tumor volume and weight in vivo. We further demonstrated that BRCC2 is a direct target of miR-17-5p. miR-17-5p appears to be a potential therapeutic target for use in osteosarcoma treatment.
Materials and methods
Patients and tissue samples
From 2009 to 2014, a total of 116 primary osteosarcoma and corresponding non-cancerous bone tissue samples were collected from Liaoning Provincial Tumor Hospital, for quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis. No patients had previously received blood transfusion, chemotherapy or radiotherapy. All patients underwent neoadjuvant chemotherapy and wide resection of tumor. Tumor biopsies were collected before neoadjuvant chemotherapy and were fresh-frozen, stored at −80°C. The patient information is summarized in Table I. Clinical tumor stage was classified according to the Enneking staging system. Tumor response to pre-operative chemotherapy was assessed using Huvos grading system. This study was approved by the Research Ethics Committee of Liaoning Provincial Tumor Hospital. Written informed consent was obtained from all the patients.
 | Table IAssociation of miR-17-5p expression in human osteosarcoma tissues with clinicopathological features. |
Cell culture
Human osteosarcoma cell lines Saos-2, 143B, MG63 and U2OS and human normal osteoblastic cell line hFOB 1.19 were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. These cells were cultured in Dulbecco's modified eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with heat-inactivated 10% fetal bovine serum (FBS; Invitrogen) at 37°C in a humidified incubator containing 5% CO2.
RNA extraction and quantitative real-time PCR
Total RNAs were extracted from cultured human tissue specimens and cells using TRizol reagent (Invitrogen) according to the manufacturer's instructions. For the detection of mature miR-17–5p and relative mRNA, RNA was reverse transcribed by using a TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). The reaction was incubated at 94°C for 4 min followed by 35 cycles of 20 sec at 94°C, 30 sec at 60°C and 30 sec at 72°C. All PCRs were done in triplicate on an ABi 7500 real-time PCR system (Applied Biosystems), and miRNA and mRNA expression was normalized to U6 snRNA and GAPDH, respectively, using the 2−ΔΔCt method, and at least 3 independent experiments were performed to generate each data set.
miR-17-5p transfection
miRNA-17-5p mimics and its inhibitor were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). For transfection, MG63 and U2OS cells were grown to 90% confluence, and transfected with miR-17-5p mimics or its inhibitor using Lipofectamine 2000 (Invitrogen) by incubation in Opti-MEM I media for 4 h according to the manufacturer's protocols. Cells were harvested 48 or 72 h post-transfection. The transfection efficiency was confirmed by quantitative real-time PCR analysis. For detecting miR-17-5p, cells were transfected again with miR-17-5p mRNA mimics or its silenced gene.
MTT assay
The transfected cells were seeded in 96-well plates at a density of 3×103 cells/well. MTT solution (20 μl of 5 mg/ml MTT) was added to each well (for a total volume of 250 μl), and the plates were incubated for 4 h at 37°C. Following the removal of the culture medium, the remaining crystals were dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 570 nm using a microplate reader. Cell proliferation was assessed daily for four consecutive days.
Immunocytochemistry
For immunocytochemistry staining, cells were fixed with 2% paraformaldehyde and incubated with a monoclonal antibody against Ki-67 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; at a final concentration of 4 μg/ml), followed by anti-rabbit FITC-conjugated secondary antibody (Invitrogen) and costaining 4′,6-diamidino-2-phenylindole (DAPI; 1.5 μg/ml) to stain cell nuclei. Labelled cells were assessed by fluorescence microscope.
In vivo tumor growth model
Male BALB/c nude mice aged 4–6 weeks were purchased from the Hunan Slac Jingda Laboratory Animal Co., Ltd. (Changsha, China). For tumor growth assay, cells stably overexpressing miR-17-5p or scramble miRNA were resuspended in PBS and 1×106 cells (200 μl) were subcutaneously injected in the dorsal flank of nude mice. Tumor size was measured every 3 days and tumor volumes were calculated with the following formula: Volume = (L × W2)/2, in which L meant the longest diameter and W meant the shortest diameter. Twenty-two days later, mice were sacrificed, and tumors were dissected and weighted. Animal handling and research protocols were approved by the Animal Care and Use Ethnics Committee.
mRNA expression arrays and data preprocessing
Total RNA quality and quantity were determined using Agilent 2100 Bioanalyzer and NanoDrop ND-1000. Affymetrix HU U133 plus 2.0 arrays were used according to the manufacturer's protocol. The data were initially normalized by robust multi-array average (RMA) normalization algorithms in expression console software (Affymetrix, Santa Clara, CA, USA). Significantly altered genes between miR-17-5p overexpression and its control cells were assessed by scatter plots and the genes upregulated and downregulated ≥5-fold. Clustering analysis was done using gene list by Gene Cluster v3.0 software, and heat maps were visualized using Java TreeView v1.1.4r3 software. Gene set enrichment analysis was carried out using Conceptgen (http://conceptgen.ncibi.org/core/conceptgen/index.jsp). Gene sets were either obtained from the ConceptGen or from published gene signatures.
Cell migration and invasion assays
The effects of miR-17-5p expression on cell migration and invasion were assessed using the wound-healing and Transwell assays as previously described (7).
Western blotting
Western blotting was used to detect the expression of miR-17-5p at the protein level as previous described. Anti-β-actin (Abcam) was used as the protein-loading control. The protein complex was detected with enhanced chemiluminescence reagents digital images were visualized using the electrochemiluminescence detection system (Invitrogen).
RNA interference for miR-17-5p
One siRNA lentivirus against miR-17-5p (Sigma-Aldrich) and non-targeting siRNA (Sigma-Aldrich) were transfected into U2OS-anti-miR-17-5p cells in 48-well plates according to the manufacturer's instructions. The multiplicity of infection (MOI, number of transducing lentiviral particles per cell) was 5. Puromycin selection was performed at a concentration of 0.5 μg/ml for 10 days.
Luciferase reporter assays
Cells cultured in 6-well plates were transfected with 0.05 μg of the pRL-TK vector (Promega, Madison, WI, USA) containing Renilla luciferase together with 30 nM miR-17-5p mimics or inhibitor. Cells cultured 24 h were then transfected with miR-17-5p-wild-type (WT) or miR-17-5p-mutant reporter plasmid containing firefly luciferase using Lipofectamine 2000. Luciferase activity was measured using Dual-luciferase assay system (Promega) 48 h post-transfection. Renilla activity was normalized to firefly activity to control for transfection efficiency.
Statistical analysis
The data are presented as mean ± standard error of mean (SEM). Statistical significance was determined using t-test or analysis of variance (ANOVA) using the SPSS 18.0 program. P<0.05 was considered as statistically signifi-cant difference.
Results
miR-17-5p is upregulated in human osteosarcoma tissues and cell lines
To explore the expression level of miR-17-5p in human osteosarcoma carcinoma development, 116 paired osteosarcoma carcinoma tissues and adjacent non-tumor tissues were detected. According to the qRT-PCR analysis, miR-17-5p was strongly upregulated in tumor tissues compared with that in the matched non-tumor tissues (Fig. 1A and B). We then analyzed miR-17-5p expression in osteosarcoma without or with metastasis property; we found that miR-17-5p higher expression was significantly correlated with metastasis property in osteosarcoma tissues (Fig. 1C). To investigate the clinical impact of elevated miR-17-5p expression in osteosarcoma, we assessed the association between miR-17-5p expression levels and clinical outcome. miR-17-5p expression levels were statistically increased in patients with >8 cm tumor size, distant metastasis and poor response to chemotherapy in comparison to <8 cm tumor size, non-distant metastasis and good response to chemotherapy. Patients in clinical stage IIB or III showed higher miR-17-5p expression levels than in clinical stage IIA (Table I). Then, we examined miR-17-5p expression levels in a panel of 4 widely used human osteosarcoma cell lines (143B, U2OS, MG63 and Saos-2) in comparison to levels in non-malignant cell lines hFOB 1.19. Correspondingly, miR-17-5p expression levels were consistently increased in osteosarcoma cell lines (Fig. 1D).
Establishment of stable miR-17-5p transfectants in osteosarcoma cell lines
We used MG63 and 143B cells to establish stable cell lines that constitutively overexpressed the miR-17-5p with the aim of revealing the role that miR-17-5p expression has in the development or progression of osteosarcoma. We also used an inhibitor to generate a stable miR-17-5p knockdown in the MG63 and 143B osteosarcoma cell lines. The transfection efficiency was confirmed using qRT-PCR analyses. As shown in Fig. 2A, the MG63 and 143B cells that had been transfected with the miR-17-5p expression plasmid displayed significantly increased miR-17-5p expression compared with the vector cell lines. In addition, as shown in Fig. 2B, the MG63 and 143B cells that had been transfected with the miR-17-5p inhibitor displayed significantly decreased miR-17-5p expression compared with the control cells.
miR-17-5p promotes osteosarcoma cells proliferation in vitro
We first explored the effects of miR-17-5p expression on cell growth using the MTT assay. As shown in Fig. 3, high expression of miR-17-5p resulted in more rapid proliferation compared with the vector in MG63 and 143B cells, whereas miR-17-5p inhibitor significantly inhibited the growth of these cells. As shown in Fig. 4B, we found that the overexpression of miR-17-5p in MG63 and 143B cells significantly upregulated Ki-67 as shown by the staining, and also the knockdown of miR-17-5p in these cells strongly downregulated the Ki-67. in total these above findings demonstrated that miR-17-5p induces a more aggressive phenotype of osteosarcoma and indicated that miR-17-5p is potentially oncogenic.
miR-17-5p promotes osteosarcoma cells migration and invasion
We next assessed whether miR-17-5p could affect the ability of osteosarcoma cells to migrate and invade using a Transwell assay. miR-17-5p overexpression promoted both migration and invasion in 143B cells (Fig. 5A and B), and also promoted both migration and invasion in MG63 cells (Fig. 5C and D). In addition, miR-17-5p knockdown in 143B cells significantly decrease cell migration and invasion (Fig. 5A and B), also inhibited both migration and invasion in MG63 cells (Fig. 5C and D). These results indicated that miR-17-5p significantly promoted the invasion and migration of osteosarcoma cells.
Increased miR-17-5p expression promotes xenograft tumor formation
To further evaluate the potential effect of miR-17-5p on osteosarcoma cell proliferation in vivo, 143B cells transfected with miR-17-5p, anti-miR-17-5p and NC were subcutaneously inoculated into nude mice. As shown in Fig. 6A, tumors formed by miR-17-5p overexpressing cells grew more quickly than those by vector control cells following inoculation, and the difference in average tumor volume between experimental and control animals continued to increase 2-fold at the experimental endpoint (22 days) (Fig. 6A) and the mean wet weight of tumors in control group was significantly lower than in the miR-17-5p overexpression group (Fig. 6B). In parallel, smaller size and lower weight tumors excised from animals of the miR-17-5p-silencing group were also observed as compared with those of the control group (Fig. 6C). The mean wet weight of tumors in the former group was significantly higher than in the latter group (Fig. 6D). Thus, these data indicate that miR-17-5p may promote xenograft tumor formation of osteosarcoma cells in vivo.
miR-17-5p directly targets BRCC2 and BRCC2 levels are inversely correlated with miR-17-5p levels in osteosarcoma tissues
To uncover mRNA targets of miR-17-5p in osteosarcoma, we used mRNA expression arrays and bioinformatics databases to identify potential targets. As shown in Fig. 7A and B, BRCC2 showed the most significant difference between miR-17-5p transfected 143B cells and control cell. To experimentally verify this potential target, 143B and MG63 cells were transfected with miR-17-5p and anti-miR-17-5p, and then mRNA target and protein levels were assessed by qRT-PCR and western blot analysis. As shown in Fig. 7C–F, miR-17-5p overexpression was reduced and miR-17-5p knockdown increased the expression of BRCC2 in osteosarcoma cells. To determine whether BRCC2 3′-UTR is direct target of miR-17-5p, BRCC2 3′-UTR reporter constructs or 3′-UTR mutant controls were transfected into osteosarcoma cells before transfection with miR-17-5p and luciferase activity was measured (Fig. 8A). As shown in Fig. 8B and 8C, the luciferase activity was significantly decreased in osteosarcoma cells overexpressing miR-17-5p co-transfected with 3′-UTR-BRCC2-wt vector and miR-17-5p mimic compared with those co-transfected with 3′-UTR-BRCC2-mut vector and miR-17-5p mimic. Furthermore, as shown in Fig. 8D and E, increasing activity was observed in osteosarcoma cells silencing miR-17-5p, suggesting that the fragment at the 3′-UTR of the BRCC2 was the complementary site for the miR-17-5p seed region, and thus, that BRCC2 was a direct target of miR-17-5p. Moreover, BRCC2 expression was inversely proportional to miR-17-5p expression in osteosarcoma tissues (Fig. 8F). Therefore, it was shown that miR-17-5p directly inhibits BRCC2 expression.
Disscussion
Osteosarcoma is a common occurrence in adolescents, with high degree of malignancy and high mortality. As the effectiveness of chemotherapy has increased together with the development of surgery techniques, the 5-year survival rate of patients with osteosarcoma has significantly improved (7). However, even after amputation and chemotherapy, there are still ~40% of patients who died with tumor and lung metastases (8). The development of gene therapy, immune therapy, molecular targeted therapeutics and other biological treatment technology has opened up new roads for the treatment of osteosarcoma. Study of the relationship between miRNA and osteosarcoma can provide a new direction for the diagnosis and treatment of osteosarcoma.
miR-17 gene, located in chromosome 13q31-32, is considered to be an oncogene. Expression of the gene can promote cell proliferation, inhibit apoptosis of tumor cells and leads to tumorigenesis. In B lymphocyte tumors, expession of miR-17 gene is higher and inhibits apoptosis of tumor cells. Oncogene c-Myc can induce the expression of miR-17, and miR-17 inhibits the expression of the transcription factor E2F1. Thereby promoting cell proliferation mediated by c-Myc, is related closely with tumor formation. In some other hematopoietic cells and solid tumors, high expression of miR-17 gene has also been shown to promote tumorigenesis (9). miR-17-5p, which is located in chromosome 13q31-32, is an important member of the miR-17-92 cluster. Previous studies have found that miR-17-5p is upregulated in certain types of human cancers. Li et al (10) found that miR-17-5p plays an important role in osteosarcoma cell invasion and migration by suppressing HBP1 and subsequent activation of Wnt/β-catenin. Shan et al (11) found that mature miR-17-5p and passenger strand miR-17-3p could synergistically induce the development of hepatocellular carcinoma. Ma et al (12) found that miR-17-5p is an oncogenic miRNA that regulates tumorigenesis and progression by targeting the gene encoding P130 and subsequently activating the Wnt/β-catenin pathway. However, the expression pattern, clinical significance, and biological role of miR-17-5p in osteosarcoma, remain largely undefined. In the present study, we found that miR-17-5p was upregulated in osteosarcoma cell lines and primary tumor samples. miR-17-5p was able to promote osteosarcoma cell proliferation and mobility.
Breast cancer cell 2 (BRCC2) was originally identified as a B1.2-kb transcript in the MDA-MB-231 human breast cancer cell line. The longest predicted open reading frame of BRCC2 is 862 bp, and its mRNA encodes a protein that is 108 amino acids in length. BRCC2 is an intronless gene that has been mapped to human chromosome 11q24.1 using fluorescence in situ hybridization (13). BRCC1 and BRCC2 are tumor suppressor genes involved in DNA repair and gene integrity (14). In the present study, we showed that miR-17-5p could promote the growth of the tumor by interacting with BRCC2. Previous studies had demonstrated that BRCC2 downregulation was associated with poor disease-free and overall survival in breast cancer. In clinical cancer samples, BRCC2 expression was significantly downregulated in cancer lesions compared with paired normal tissues. By silencing or overexpressing BRCC2 in cancer cells, BRCC2 could inhibit cell growth and metastasis in vitro (14). An in vivo assay showed that BRCC2 not only markedly inhibited cancer cell xenograft formation and growth, but also inhibited cancer cell metastasis in a lung metastasis model (14). Moreover, BRCC2 inhibited cancer metastasis via regulation of the Akt pathway (14). Thus, the potential mechanism of miR-17-5p promoting migration and invasion is via regulating BRCC2 and Akt pathways.
The present study shows that miR-17-5p is significantly upregulated in osteosarcoma. It demonstrates that miR-17-5p has powerful oncogenic, proliferation and invasion regulatory effects that are mediated by BRCC2. The study, therefore, presents that miR-17-5p may act as an oncogene and could be a promising therapeutic target in osteosarcoma.
References
|
Unni KK: Dahlin's Bone Tumors: General Aspects and Data on 11,087 Cases. 5th edition. Lippincott-Raven; Philadelphia: 1996 | |
|
Jaffe N: Osteosarcoma: review of the past, impact on the future. The American experience. Pediatric and Adolescent Osteosarcoma. Jaffe N, Bruland O and Bielack S: Springer; New York, NY: pp. 239–262. 2010 | |
|
Zeng Y and Cullen BR: Sequence requirements for microRNA processing and function in human cells. RNA. 9:112–123. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Lee RC, Feinbaum RL and Ambros V: The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 75:843–854. 1993. View Article : Google Scholar : PubMed/NCBI | |
|
Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, Horvitz HR and Ruvkun G: The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature. 403:901–906. 2000. View Article : Google Scholar : PubMed/NCBI | |
|
Kobayashi E, Hornicek FJ and Duan Z: MicroRNA involvement in osteosarcoma. Sarcoma 2012. Article ID 359739. 2012. | |
|
Bielack SS, Kempf-Bielack B, Delling G, Exner GU, Flege S, Helmke K, Kotz R, Salzer-Kuntschik M, Werner M, Winkelmann W, et al: Prognostic factors in high-grade osteosarcoma of the extremities or trunk: An analysis of 1,702 patients treated on neoadjuvant cooperative osteosarcoma study group protocols. J Clin Oncol. 20:776–790. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Marulanda GA, Henderson ER, Johnson DA, Letson GD and Cheong D: Orthopedic surgery options for the treatment of primary osteosarcoma. Cancer Control. 15:13–20. 2008. | |
|
O'Donnell KA, Wentzel EA, Zeller KI, Dang CV and Mendell JT: c-Myc-regulated microRNAs modulate E2F1 expression. Nature. 435:839–843. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Li H, Bian C, Liao L, Li J and Zhao RC: miR-17-5p promotes human osteosarcoma cancer cell migration and invasion through suppression of HBP1. Breast Cancer Res Treat. 126:565–575. 2011. View Article : Google Scholar | |
|
Shan SW, Fang L, Shatseva T, Rutnam ZJ, Yang X, Du W, Lu WY, Xuan JW, Deng Z and Yang BB: Mature miR-17-5p and passenger miR-17-3p induce hepatocellular carcinoma by targeting PTEN, GalNT7 and vimentin in different signal pathways. J Cell Sci. 126:1517–1530. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Ma Y, Zhang P, Wang F, Zhang H, Yang Y, Shi C, Xia Y, Peng J, Liu W, Yang Z, et al: Elevated oncofoetal miR-17-5p expression regulates colorectal cancer progression by repressing its target gene P130. Nat Commun. 3:12912012. View Article : Google Scholar : PubMed/NCBI | |
|
Li X, Kong X, Wang Y and Yang Q: BRCC2 inhibits breast cancer cell growth and metastasis in vitro and in vivo via down-regulating AKT pathway. Cell Death Dis. 4:e7572013. View Article : Google Scholar | |
|
Nathanson KL, Wooster R and Weber BL: Breast cancer genetics: What we know and what we need. Nat Med. 7:552–556. 2001. View Article : Google Scholar : PubMed/NCBI |
