FEBS LettersVolume 587, Issue 9 p. 1366-1372 Short communication Free Access miR-16 inhibits cell proliferation by targeting IGF1R and the Raf1–MEK1/2–ERK1/2 pathway in osteosarcoma Lei Chen, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaThese authors contributed equally to this work.Search for more papers by this authorQing Wang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaThese authors contributed equally to this work.Search for more papers by this authorGuo-dong Wang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaSearch for more papers by this authorHua-song Wang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaSearch for more papers by this authorYong Huang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaSearch for more papers by this authorXi-ming Liu, Corresponding Author gklxm@163.com Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaCorresponding authors.Search for more papers by this authorXian-hua Cai, Corresponding Author wgcaixh@163.com Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaCorresponding authors.Search for more papers by this author Lei Chen, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaThese authors contributed equally to this work.Search for more papers by this authorQing Wang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaThese authors contributed equally to this work.Search for more papers by this authorGuo-dong Wang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaSearch for more papers by this authorHua-song Wang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaSearch for more papers by this authorYong Huang, Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaSearch for more papers by this authorXi-ming Liu, Corresponding Author gklxm@163.com Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaCorresponding authors.Search for more papers by this authorXian-hua Cai, Corresponding Author wgcaixh@163.com Department of Orthopaedics Surgery, Wuhan General Hospital of Guangzhou Command, Wuhan, ChinaCorresponding authors.Search for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Several miRNAs have been implicated in the development and progression of osteosarcoma (OS). In this study, we found that miR-16 is downregulated in OS cell lines and tissues. Overexpression of miR-16 suppresses OS cell proliferation and tumor growth in nude mice. Furthermore, we confirmed that IGF1R is a direct target of miR-16. Mechanistic investigation revealed that miR-16 overexpression inhibits the Raf1–MEK1/2–ERK1/2 pathway. In clinical specimens, IGF1R levels inversely correlate with miR-16 expression. Our results provide significant clues regarding the role of miR-16 as a tumor suppressor by targeting IGF1R in OS. 1 Introduction Osteosarcoma (OS) is one of the most common primary sarcoma of bone and a leading cause of cancer death due to its rapid proliferation among adolescents [1]. Despite the rapid development in treatment strategies, the cure rate of patients carrying osteosarcoma is still very poor. Therefore, there is an urgent need to develop novel strategies for the diagnosis, treatment and prognosis of OS. miRNAs (microRNAs) are a class of small endogenous non-coding RNA molecules, which regulate target-gene expression at post-transcriptional levels [2, 3]. Recent evidence has shown that about half of human miRNAs are located in cancer-associated genomic regions that are frequently amplified, deleted, or rearranged in cancer, suggesting that some miRNAs may act as oncogenes or tumor suppressors [4, 5]. Oncogenic and tumor suppressor miRNAs have been observed in OS, such as high expression of miR-20a, miR-19a, miR-93 [6-8], as well as reduced expression of miR-34a, miR-183 and miR-145 [9, 10], which contribute to the development and progression of OS. A recent study has shown that miR-16 is also downregulated in OS tissues compared to healthy bone tissue by miRNA microarray analysis [11]. Function and mechanism of miR-16 as a tumor suppressor have been studied in chronic lymphocytic lymphoma [12], pituitary adenomas [13], breast and ovarian cancer [14, 15]. However, the biological function and molecular mechanism of miR-16 in OS remain to be unknown. In the present study, we identified significant downregulation of miR-16 in OS cell lines and tissues, and found that overexpression of miR-16 potently inhibited OS cell proliferation in vitro and in vivo. In addition, we confirmed IGF1R as a direct target of miR-16 and explored the underlying mechanism of miR-16 in OS. Our findings demonstrate a novel role of miR-16 in human OS. Four osteosarcoma cell lines, including HOS, KHOS, U2OS, and MG-63, were obtained from the ATCC. Human osteoblast cell line HOB was purchased from PromoCell (Heidelberg, Germany). The OS cell lines were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. HOB cells were cultured in osteoblast growth medium (PromoCell). Cells were incubated at 37 °C in humidified incubator containing 5% CO2. Eighteen OS and the adjacent normal tissues were obtained from patients who underwent surgery at Wuhan General Hospital of Guangzhou Military Region, Wuhan, China. The study was approved by the Ethics Committee of Wuhan General Hospital of Guangzhou Command, People\'s Liberation Army, and written informed consent was obtained from each patient. For evaluating IGF1R expression, qRT-PCR was performed with an ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The primers for IGF1R were: forward 5′-GAGAAGGAGGAGGCTGAATACCG-3′ and reverse 5′-GTGATGTTGTAGGTGTCTGCGGC-3′. The relative level of miR-16 was determined using a mirVana™ qRT-PCR miRNA Detection Kit (Ambion, Foster City, CA, USA) as described previously [16]. All samples were normalized to the internal control (β-actin or U6) and fold changes were calculated through 2−ΔΔCt analysis method. The IGF1R shRNA was purchased from GeneChem (Shanghai, China), and cloned into the pLVTHM vector. The oligonucleotides used were 5′-GATCCGGCCAGAAATGGAGAATAATTCAAGAGATTATTCTCCATTTCTGGCCTCA-3′ and 5′-AGCTTGAGGCCAGAAATGGAGAATAATCTCTTGAATTATTCTCCATTTCTGGCCG-3′. The pre-miR-16 sequence was cloned into the pCDH-CMV-MCS-EF1-coGFP vector. The primers for pre-miR-16 were: 5′-CCTGAATTCATAATACTGAAAAGACTATCAATAAAACTG-3′ (forward) and 5′-CTTGGATCCGTAAAGTAGCAGCACATAATGGTTTGTGGA-3′ (reverse). Lentivirus particles were harvested 48 h after pCDH-CMV-miR-16 or pLVTHM-shIGF1R transfection with the corresponding packaging plasmids into 293T cells. Then KHOS and U2OS cells were infected with recombinant lentivirus-transducing units plus 10 mg/ml polybrene (Sigma, St. Louis, Missouri, USA). The miR-16 antisense inhibitor (anti-miR16) and negative control were purchased from Ambion (Austin, TX). MG63 cells were transfected with anti-miR16 and negative control using Lipofectamine 2000. Cells were collected 48 h after transfection. The wild-type and mutant 3′-UTR fragments were cloned into the downstream of the luciferase reporter gene in pGL3-control vector. The primers were as the following: forward 5′-TCCACGCGTCCACAACAGCAGTAAGAAGAAAAGC-3′ and reverse 5′-TTGCTCGAGCCTAAGCAAAGGCAAGGGAAAGAGA-3′ for IGF1R; forward 5′-AGAACGCGTCCATTCTCG TTTTAGGACTCTTCTT-3′, reverse 5′-CGTCTCGAGCAGGTTTGAAAAATCCTACTGTCGC-3′ for Kras; forward 5′-CACACGCGTCACCACTTTTCTGCTCCCTTTCTCC-3′, reverse 5′-GTCCTCGAGCAAAGGGATAGAAAAGAAGGCAACA-3′ for Raf1. For luciferase reporter assays, KHOS and U2OS cells were transiently cotransfected with appropriate reporter plasmid and a control Renilla luciferase vector in the presence of either miR-16 or miR-control. 48 h after transfection, cells were harvested and lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The transfected cells were plated in 24-well plates at 5 × 103 per well and cultured for 1, 2, 3, 4 and 5 days. Cell counts were estimated by trypsinizing the cells and performing analysis with a Coulter counter (Beckman Coulter, Fullerton, CA, USA). For analysis of cell cycle, 2 × 105 transfected cells were suspended in 1 ml solution containing 0.4 mM sodium citrate, 25 μg/ml propidium iodide (PI), and 50 μg/ml RNase. The stained cells were analyzed in a FACScan flow cytometer (BD Biosciences, USA) using the ModFit LT program (BD Biosciences, USA). Proteins were separated on 10% SDS–PAGE gel and transferred to nitrocellulose membrane (Bio-RAD, Richmond, CA, USA). After blocked with 5% non-fat milk, the membrane was incubated with primary antibody against Kras, Raf1, p-Raf1, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2 or β-actin (Santa Cruz Biotechnology, CA, USA). The membranes were then incubated with antimouse or antirabbit IgG conjugated to horseradish peroxidase. Protein bands were visualized using ECL reagents (Pierce, Rockford, IL, USA). Five-week-old male athymic BALB/c nude mice were purchased from the Experimental Animal Center of the Third Military Medical University. All animal studies complied with the Third Military Medical University animal use guidelines and the protocols approved by the Third Military Medical University Animal Care Committee. 5 × 106 U2OS cells stably expressing miR-16 or miR-control were injected subcutaneously to the skin under the front legs of the mouse. The mice were observed over 4 weeks for tumor formation. The tumor volume (V) was examined twice weekly with a caliper and calculated by using the formula: V 2)/2, where a is the larger and b is the smaller dimension of the tumor. After the mice were sacrificed, the tumors were recovered and the wet weights of each tumor were determined. The experiments were performed using five mice per group. The SPSS 13.0 program was used for statistical analysis. Data were expressed as mean ± S.D. of at least 3 independent experiments. The differences between groups were analyzed using Student′s t-test when comparing only two groups or assessed by one-way analysis of variance when more than two groups were compared. The relationship between IGF1R and miR-16 expression was explored using Pearson χ 2 test. Data were considered to be statistically significant when P 0.05 (∗) and P 0.01 (∗∗). To investigate the significance of miR-16 in OS carcinogenesis, we analyzed the expression of miR-16 in OS cell lines (HOS, KHOS, U2OS, MG-63) and clinical specimens by qRT-PCR. The results showed that miR-16 expression was significantly decreased in all OS cell lines compared to HOB (Fig. 1A). Consistent with these observations, OS clinical specimens expressed lower levels of miR-16 compared to the adjacent normal bone tissues (Fig. 1B). These results suggest that reduced expression of miR-16 is correlated with OS carcinogenesis and that miR-16 might act as a tumor suppressor in OS. Figure 1Open in figure viewerPowerPoint The expression of miR-16 in OS cell lines and clinical specimens. (A) The relative expression of miR-16 in normal osteoblast HOB and four OS cell lines was measured by qRT-PCR. (B) The relative expression of miR-16 in 18 paired OS and adjacent normal bone tissues. Results represent the mean ratio between miR-16 and U6 from 3 experiments. To explore the effect of miR-16 on OS cell proliferation, we first constructed miR-16-overexpressed OS cells by lentiviral vectors carrying the pre-miR-16 gene. Significant increase of miR-16 expression in KHOS and U2OS cells was confirmed by qRT-PCR (Fig. 2A). As shown in Fig. 2B, cell growth was significantly decreased in miR-16-overexpressed cells compared to their corresponding controls. Moreover, cell cycle analysis of miR-16-overexpressed cells showed significant increase in G0/G1 phase and reduction in S phase (Fig. 2C). In contrast, anti-miR16 promoted cell growth and cell cycle G1/S transition in MG63 cells (Fig. 2B and C). These results suggest that miR-16 can suppress OS cell proliferation in vitro. Figure 2Open in figure viewerPowerPoint miR-16 overexpression suppressed OS cell proliferation in vitro. (A) qRT-PCR analysis of miR-16 in KHOS and U2OS cells infected with Lv-miR16 or Lv-control. (B) Cell proliferation assay of KHOS and U2OS cells infected with Lv-miR16 or Lv-control, and MG63 cells transfected with anti-miR16 or anti-control. (C) Cell cycle analysis of KHOS and U2OS cells infected with Lv-miR16 or Lv-control, and MG63 cells transfected with anti-miR16 or anti-control. To understand the underlying mechanism of growth inhibition induced by miR-16, we searched for the potential targets of miR-16 using TargetScan and Pictar. Among the predicted genes, we were particularly interested in IGF1R because it was closely correlated with cancer cell proliferation. To verify IGF1R is a direct target of miR-16, IGF1R 3′-UTR containing wild-type or mutant miR-16-binding site was cloned downstream of the luciferase reporter gene (Fig. 3A). Luciferase reporter assays showed that miR-16 overexpression significantly repressed the relative luciferase activity in KHOS and U2OS cells when the plasmid contains wild-type IGF1R 3′-UTR; however, the luciferase activity was unaffected when the 3′-UTR contained mutant binding site (Fig. 3B). Consistently, qRT-PCR and Western blotting analysis showed that miR-16 overexpression decreased the levels of IGF1R in KHOS and U2OS cells (Fig. 3C and D). In contrast, anti-miR16 transfection increased IGF1R levels in MG63 cells (Fig. 3C and D). Figure 3Open in figure viewerPowerPoint IGF1R is a direct miR-16 target in OS cells. (A) Diagram of IGF1R 3′UTR-containing reportor construct. Mutations were generated at the predited miR-16-binding site located in the IGF1R 3′-UTR. (B) Luciferase reporter assays in KHOS and U2OS cells. The wild-type or mutant reporter plasmids were co-transfected into KHOS and U2OS cells which were infected by Lv-control or Lv-miR16. (C) qRT-PCR analysis of the expression of IGF1R. (D) Western blotting analysis of the expression of IGF1R. (E) Silencing of IGF1R in U2OS cells was verified by Western blotting. Cell proliferation assay (F) and cell cycle analysis (G) in U2OS cells infected with Lv-miR16, Lv-shIGF1R or Lv-control. Based on the results above, we hypothesized that miR-16 might reduce OS cell proliferation by repressing IGF1R expression. To test this hypothesis, we performed loss-of-function studies. As shown in Fig. 3E, significant inhibition of IGF1R expression in U2OS cells was verified by Western blotting. Silencing of IGF1R led to cell growth inhibition (Fig. 3F) and cell-cycle arrest (Fig. 3G), which was similar to those induced by miR-16 overexpression. These results demonstrate that IGF1R is a direct target of miR-16 in OS cells. Given that IGF1R can activate many downstream genes closely associated with malignant cell proliferation, such as Kras, Raf1, MEK1/2 and ERK1/2, which were key components of the Raf1-MEK1/2-ERK1/2 pathway, we further evaluated the potential effect of miR-16 on these molecules in OS cells. As illustrated in Fig. 4A, the levels of these molecules and corresponding phosphorylated molecules were down-regulated in miR-16-overexpressed U2OS cells. These findings were also confirmed by using shIGF1R. We also observed that knockdown of miR-16 MG63 cells increased the levels of all these moleculars above (Fig. 4A, right). These results demonstrate that miR-16 may be an important regulator in the Raf1–MEK1/2–ERK1/2 pathway. Figure 4Open in figure viewerPowerPoint miR-16 regulates the expression of major molecules of the Raf1–MEK1/2–ERK1/2 pathway in OS cells. (A) The protein levels of Kras, Raf1, p-Raf1, MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 were reduced by miR-16 overexpression or IGF1R silencing (left) in U2OS cells, and the levels of these moleculars were increased by anti-miR16 transfection in MG63 cells (right). (B) Diagram of Kras 3′UTR-containing reportor construct. Mutations were generated at the predited miR-16-binding site located in the Kras 3′-UTR. (C) Diagram of Raf1 3′UTR-containing reportor construct. Mutations were generated at the predited miR-16-binding site located in the Raf1 3′-UTR. (D) Luciferase reporter assay. U2OS cells were co-transfected with luciferase reporter plasmids containing the WT or Mut miR-16 target sites in the Kras 3′UTR with miR-16 or miR-control precursors. (E) Luciferase reporter assay. U2OS cells were co-transfected with luciferase reporter plasmids containing the WT or Mut miR-16 target sites in the Raf1 3′UTR with miR-16 or miR-control precursors. Given the above observations, we wondered whether there is other targets of miR-16 in the Raf1–MEK1/2–ERK1/2 pathway. Using TargetScan, another two miR-16 target sites were found in Kras and Raf1 3′-UTR segments respectively (Fig. 4B and C). The relative luciferase activity of these reporters contained the miR-16 binding sites but not the corresponding mutant counterpart was significantly reduced in miR-16-overexpressed U2OS cells (Fig. 4D and E). These results indicate that miR-16 regulates the expression of IGF1R, Kras and Raf1 by directly binding to the 3′-UTR target sites, supporting our conclusion that miR-16 can suppress OS cell proliferation by regulating Raf1–MEK1/2–ERK1/2 pathway. To further determine whether miR-16 could suppress OS cell growth in vivo, U2OS cells stably expressing miR-16 or miR-control were injected subcutaneously to the skin under the front legs of the mice. The tumor growth was closely monitored. As shown in Fig. 5A and B, the tumor size and volume were markedly reduced in miR-16-overexpressed tumors compared to control tumors. More than 2-fold decrease in tumor weight was observed in miR-16-overexpressed tumors compared to controls (Fig. 5C). Moreover, the levels of IGF1R, Kras, Raf1, p-Raf1, MEK1/2, p-MEK1/2, ERK1/2, and p-ERK1/2 were reduced compared to controls (Fig. 5D). Figure 5Open in figure viewerPowerPoint miR-16 overexpression suppresses OS tumorigenesis in vivo. (A) Representative picture of tumors formed in nude mice. U2OS cells infected with Lv-miR16 produced smaller tumors than control cells. (B) Growth curve of tumor volumes. The tumor volume was significantly reduced by the overexpression of miR-16. Each data point represents the mean ± S.D. of 5 mice. (C) The weight of tumors. The average tumor weight was significantly reduced by the overexpression of miR-16. (D) The protein levels of IGF1R, Kras, Raf1, p-Raf1, MEK1/2, p-MEK1/2, ERK1/2 and p-ERK1/2 were decreased in miR-16-overexpressed tumors. We further examined the levels of miR-16 and IGF1R in 18 OS tissues and the adjacent normal tissues. As shown in Fig. 6A, the average expression of IGF1R was significantly higher in OS tissues compared to the adjacent normal tissues. Correlation analysis indicated that IGF1R expression was reduced along with miR-16 upregulation in these 18 pairs of OS tissues (R= 0.794, Pearson χ 2 test, Fig. 6B). Figure 6Open in figure viewerPowerPoint Expression of IGF1R was upregulated in OS tissues and negatively correlated with miR-16 levels. (A) Relative expression of IGF1R in 18 OS tissues and the adjacent normal tissues using qRT-PCR analysis. (B) Correlation analysis of miR-16 and IGF1R expression was performed by Pearson χ 2 test in 18 OS tissues. miR-16 is a member of the miR-16 family, which is composed of miR-15a, miR-15b, and miR-16. This family is known to act as tumor suppressors and has been shown to play important roles in inhibiting cell proliferation, promoting apoptosis, and suppressing tumorigenicity [16, 17]. For instance, Bonci et al [18] identified that miR-15a and miR-16 overexpression resulted in growth arrest, apoptosis and marked regression of prostate tumor xenografts. In osteosarcoma, a preliminary study has shown that miR-16 is downregulated by microarray analysis [11]. Our results varified the decreased expression of miR-16 in OS cell lines and OS tissues. Moreover, we found that miR-16 could inhibit cell growth and induce a G0/G1 arrest in OS cells, and suppress tumorigenesis in a murine model of OS xenograft, suggesting its potential tumor suppressor role in OS. Similar to classical transcription factors, miRNAs exert their functionality through regulating specific target genes. Of note, one miRNA can regulate hundreds of target genes concomitantly. miR-16 family can induce apoptosis and cell cycle arrest in G1 by repressing specific target genes, including Bcl-2 and CCNE1 [14, 19]; miR-16 also inhibited cell proliferation via downregulating ARL2 and Bmi-1 [15, 20]. In this study, potential targets of miR-16 were analyzed using different prediction algorithms. Among a panel of candidate targets, IGF1R was further experimentally validated as a miR-16 target in OS. Insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase that mediates IGF1-induced signaling events and has pivotal roles in cellular processes such as proliferation, cell migration and differentiation [21]. In recent years, mounting evidence indicates that IGF1R may be involved in tumorigenesis in many cancers, including gastric and breast cancer [22, 23]. In our studies, we demonstrated that miR-16 inhibited IGF1R expression, and confirmed that IGF1R was a direct target of miR-16 in OS cells. Moreover, we found that silencing of IGF1R induced cell growth inhibition and a G0/G1-phase arrest similar to the phenotypes induced by miR-16 overexpression. These results suggested that the growth suppressive effect of miR-16 was at least partly mediated by repressing IGF1R expression. Since IGF1R is a major component in the activation of Raf1–MEK1/2–ERK1/2 pathway, which is related to tumorigenesis, metastasis and apoptosis in many cancers [24, 25], to further explore the molecular mechanisms of growth inhibition induced by miR-16, we examined the expression of major molecules of the Raf1–MEK1/2–ERK1/2 pathway. Our results demonstrated that the levels of Kras, Raf1, MEK1/2, ERK1/2 and corresponding phosphorylated molecules were downregulated in miR-16-overexpressed OS cells and xenograft tumors. 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