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MicroRNA-155 targets MAP3K10 and regulates osteosarcoma cell growth
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Original article
MicroRNA-155 targets MAP3K10 and regulates osteosarcoma cell growth Chuangjian Wang a,1 , Xiaobo Zhang b,1 , Chunlin Zhang a , Fuying Zhai a , Ying Li a , Zongqiang Huang a,∗ a b
Department of Orthopaedics, The First Affiliated Hospital of Zhengzhou University, No. 1 JianShe Road, Zhengzhou, Henan 450000, China Central sterile supply Department, The First Affiliated Hospital of Zhengzhou University, No. 1 JianShe Road, Zhengzhou, Henan 450000, China
a r t i c l e
i n f o
Article history: Received 23 May 2016 Keywords: Osteosarcoma MicroRNA-155 Cell lines MAP4K10
a b s t r a c t Osteosarcoma is the most common type of bone cancer prevalent in young adults. Recent studies suggested that aberrant microRNA expression plays an essential role in osteosarcoma pathogenesis. In this study, we found miR-155 up-regulation in different osteosarcoma cell lines U2OS, Saos-2 and MG-63. Through bioinformatic prediction and analysis, we identified its target MAP3K10 that involved in cell proliferation and apoptosis. This work demonstrates novel interaction between microRNA, intercellular MAPK signaling and apoptosis in osteosarcoma, which will provide targets for therapeutic development. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Osteosarcoma is the most common form of primary malignant bone cancer, especially prevalent in children and adolescents. It is reported that approximately 4–5 cases per million [1]. This type of aggressive sarcoma is characterized by a high level of chromosomal instability and diverse karyotypes [2]. While a number of amplifications of chromosomal region 6p12 and 8q24 have been observed in osteosarcoma, oncogenes such as RB and cMYC mutations also have been associated in osteosarcomagenesis [3,4], the detailed molecular pathways involved in osteosarcoma remain unclear. Interestingly, recent studies have shown that microRNAs (miRNAs) deregulation occurs in osteosarcoma [5,6]. miRNAs is a family of small non protein coding RNAs (20–25 nucleotides) that play a critical role in the posttranscriptional gene regulation by targeting mRNAs for translational repression and/or degradation. MiR155 host gene BIC is abundantly expressed in most tissues and cell types [7], therefore, may play a critical role in a wide range of biological processes, such as hematopoiesis, inflammation. Moreover, miR155 levels are elevated in a broad array of cancers including lymphomas, leukemia and solid tumors. Deregulation of this miRNA may also significantly participate in the development of carcinogenesis.
∗ Corresponding author. E-mail address: [email protected] (Z. Huang). 1 These authors contributed equally to this work.
In this study, we investigated the role of miR155 in human osteosarcoma. We found miR155 is upregulated in a number of osteosarcoma cell lines. Inhibition of this miRNA leads to cancer cell death. We identified the target of miR155 as MAP3K10. More importantly, inhibition of this miRNA in cancer cell reduced the tumor growth in vivo. Our results revealed a novel role of miR155 in cancer cell growth and provided a new possibility of therapeutic intervention in treatment of osteosarcoma. 2. Materials and methods 2.1. Cell lines U2OS, Saos-2 and MG-63 cells were provided by Institute of Biochemistry and Cell Biology of Chinese Academy of Science and originated from American Type Culture Collection (ATCC). 2.2. RNA extraction Total RNA of cultured cells was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. 2.3. RT-PCR One g of total RNA per sample was synthesized using the RT reagent Kit (Formentas, USA) following the manufacturer’s protocol. And then, real-time PCR was performed using the FastStart Universal SYBR Green Master kit (Roche, Switzerland) on the
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ABI 7500 thermocycler (Applied Biosystems, USA). Primers were designed and provided by Ruibo Biotech (Guangzhou, China). 2.4. MiRNA target prediction MicroRNA target genes were predicted using the website program (www.microRNA.org). 2.5. Luciferase assay 293T cells were transfected with MAP3K10 3 UTR plasmid, Renilla luciferase pRL-TK vector (Promega USA), and miR-155 mimics(miR10004658-1-5, Ribobio) or miR-NC using lipofectamine 2000 mimics(miR01201-1-5,Ribobio) reagent (Invitrogen, USA). After 48 h, luminescence was examine using the Dual-Luciferase Reporter Assay System (Promega, USA). Luminescence results were normalized to the Renilla luminescence.
Fig. 1. Expressions of miR-155 are upregulated in human osteosarcoma cell lines. Mature miR-155 expressions were analyzed by real-time PCR and standardized to the endogenous control U6. Human osteosarcoma cell lines U2OS, Saos-2 and MG-63 were examined as well as the control cell hFOB1.19. (**p < 0.01).
2.6. Western blot analysis Cell protein extracts were immunoblotted with a primary antibody and then a secondary antibody. Primary antibodies, MAP3K10 antibody(867–880, SIGMA),were used at a diluted concentration of 1:2000 while the secondary antibody(A0545, SIGMA) was used at 1:10,000. 2.7. SiRNA transfection The human MAP3K10 siRNA(anti-MAP3K10) and negative control oligonucleotide (anti-NC) were designed and provided by Ribobio (Guangzhou, Guangdong, China). MG-63 cells were transfected with 40 nM of anti-MAP3K10 or anti-NC by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2.8. Apoptosis detection and analysis Cell apoptosis was detected using the Annexin V/PI apoptosis detection kit(BMS500FI, eBiosicence) using the manufacturer’s protocol. Briefly, cells were washed by ice-cold PBS and binding buffer and were then incubated for 15 min in 100 l of binding buffer containing 5 l of fluorochrome-conjugated Annexin V. Cells were washed using binding buffer and 5 l of PI. Signals were detected using flow cytometry. 2.9. Animal experiments MG-63 cells were transfected with anti-miR-155(miR20004658-1-5,Ribobio) or anti-miRNC(miR02201-1-5,Ribobio). After 6 h, cells were injected subcutaneously into nude mouse. Tumor sizes are measured once per week. We got the approval of using these mice from the Bioethics committee of the Zhengzhou University. The raising of the mice accorded with the “Guide for the Care and Use of Laboratory Animals”. 2.10. Statistical analysis Data were expressed as Mean ± SEM of three independent experiments. For all statistical tests, PRISM 5.0 (GraphPad Software Inc., USA) was used. P values less than 0.05 were identified as statistically significant.
3. Results 3.1. Human osteosarcoma cell lines overexpress miR-155 Osteosarcoma cells share some osteoblastic features, but they differ in many aspects, in particular the proliferation kinetics and osteoid production [8]. Thus, we compared three osteosarcoma cell lines of different origin, U2OS, Saos-2 and MG-63 [9], with normal human osteoblasts, hFOB1.19. We found that the expression of miR-155 is upregulated in all three osteosarcoma cell lines compared to normal osteoblasts (Fig. 1). This miRNA showed five-fold increase in U2OS, and about 10-fold increase in MG-63 cells. We focused on MG-63 cells for our following study.
3.2. MiR-155 deficiency leads to activation of apoptosis in MG-63 cells To elucidate the function of miR155 in osteosarcoma cell line, we used anti-miRNA oligonucleotides, anti-miR-155. Unlike the transitional antisense techniques that suppress expression of target RNAs, anti-miRNA oligonucleotides appear to work primarily through a steric blocking mechanism of action. These compounds are synthetic reverse complements that tightly bind and inactivate the miRNA instead of decreasing its expression. An anti-scramble was used as negative control (anti-miR-NC). Interestingly, when MG-63 cells were transfected with anti-miR-155, their proliferation was significantly reduced (Fig. 2A) compared to U2OS and Saos-2 cells (Supplemental Fig. 1). We next examined whether the reduced proliferation is due to apoptosis using Annexin V/PI staining. As shown in Fig. 2B, treatment of anti-miR-155 increased both doubled Annexin V positive population, and increased Annexin V and PI double positive cells about five fold. This suggests that miR155 deficiency leads to enhanced apoptosis and cell death. To confirm the activation of apoptosis upon miR155 inhibition, we tested Caspase-3/7 activity, which are the effector caspases that execute apoptosis by cleaving cellular proteins on specific Asp residues. Consistent with the Annexin V staining results, the Caspase-3/7 activity was increased when cells were treated with anti-miR-155, compared to cells treated with control anti-miR-NC (Fig. 2C). These findings demonstrate that miR-155 is important to inhibit the activation of apoptosis in osteosarcoma.
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Fig. 2. Inhibition of miR-155 suppressed cell proliferation and induced apoptosis of human osteosarcoma MG-63 cells in vitro. MG-63 cells were transfected with 100 nM of miR-155 inhibitor (anti-miR-155) or negative control inhibitor (anti-miR-NC). (A) After 48 h, cell proliferation was measured by CCK-8 assay. (B)Cell apoptosis was analyzed by Annexin V/PI assay. (C) Caspase-3/7 activities were examined by caspase 3/7 assay. (**p < 0.01).
Fig. 3. MiR-155 targets MAP3K10. (A) Bioinformatic analyses (www.microrna.org) were performed to search for miR-155 target genes and the binding sites of MAP3K10 within its 3 UTR. (B and C) 293 T cells were co-transfected with miR-155 along with reporter plasmid containing the 3 UTR of MAP3K10 or the mutated one. Relative luciferase activity was analyzed after 48 h (D) MG-63 cells were transfected either with miR-155 or miR-NC for 48 h. MAP3K10 protein levels were examined by western blots using GAPDH as a loading control (one of two similar blots is shown). Results are shown as Mean + SEM. (*p < 0.05).
3.3. MAP3K10 is the target of miR-155
3.4. MAP3K10 expressions were lower in osteosarcoma cell lines
A quick bioinformatics search revealed that miR-155 complement with 3 UTR region of MAP3K10 (Fig. 3A). To experimentally confirm that miR-155 could target MAP3K10, we made a luciferase construct with MAP3K10 3 UTR, and tested the luciferase activity in 293T cells during treatment of miR-155. As shown in Fig. 3B and C, we co-transfected MAP3K10 along with reporter plasmid containing the 3 UTR of MAP3K10 or the mutated one into 293T cells. MiR-155 repressed the activity of luciferase fused to the WT MAP3K10 3 UTR, while it failed to repress the mutated one. These data reveal that miR-155 directly targets the MAP3K10 3 UTR leading to repressed expression. We next tested if miR-155 targets MAP3K10 in osteosarcoma cells. Western blot analysis showed that, when MG-63 cells treated with miR-155, it reduced MAP3K10 protein level compared to cells treated with control miR-NC (Fig. 3D). In addition, depletion of MAP3K10 promoted the cell proliferation in MG-63 cells (Fig. 4). Taken together, these results confirmed MAP3K10 as a direct target of miR-155.
Expressions of MAP3K10 in osteosarcoma cell lines U2OS, Saos2 and MG-63 were examined by RT-PCR. As shown in Fig. 5, MAP3K10 levels were decreased in U2OS, Saos-2 and MG-63 cells compared with hFOB1.19 cells, among which it was the lowest in MG-63 cells.
4. Discussion Sarcomas are well known for their heterogeneity, and the heterogeneous and chaotic nature of osteosarcoma has confounded accurate molecular classification, prognosis, and prediction for this tumor [10]. Through examining three different osteosarcomaderived cells lines U2OS, Saso-2 and MG-63, we found they have common upreguated expression of miR-155. These results were consistent with several previous reports [11,12]. These reports and our findings unveiled a role of miR-155 in the proliferation of osteosarcoma cells. It will be interesting to know how the miR-
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Fig. 4. Inhibition of MAP3K10 increased cell proliferation of human osteosarcoma MG-63 cells in vitro. MG-63 cells were transfected with 40 nM of MAP3K10 siRNA (anti-MAP3K10) or negative control (anti-NC). (A) MAP3K10 expression was examined at 72 h. (A) After 48 h and 72 h, cell proliferation was measured by CCK-8 assay (*p < 0.05).
Fig. 5. Expressions of MAP3K10 are downregulated in human osteosarcoma cell lines. MAP3K10 expressions were analyzed by real-time PCR and standardized to the endogenous control GAPDH. Human osteosarcoma cell lines U2OS, Saos-2 and MG-63 were examined as well as the control cell hFOB1.19. (**p < 0.01).
155 host gene is regulated in osteoblast and what happens during cancerogenesis. We identified a direct target of miR-155 as MAP3K10 in osteosarcoma, one member of MAPK pathway directly involved in cell proliferation, differentiation, and apoptosis [13]. MiR-155MAP3K10 interaction was also previous reported in THP-1 cells[14]. Interestingly, a recent study found deregulation of a number of miR-
NAs that target MAPK pathway in osteosarcoma [5]. It is known that MAP3K10 functions preferentially on JNK pathway, which belongs to superfamily of MAP-kinases and is upstream of mitochondria dependent apoptosis pathway [15]. Thus, our results indicate that miR-155 is directly targets MAP3K10-JNK-apoptosis pathway. Further investigation is required to unravel a detailed molecular network of this pathway. But it will be important to know the specific regulation in osteosarcoma cells. In addition, microRNAs functions through multiple targets. Although the present study is focused on MAP3K10, there are several other potential functional targets involved in the molecular pathway of miR-155 in osteosarcoma, such as receptor interacting serine-threonine kinase 1 (RIPK1) and HMG-box transcription factor 1 (HBP1) [12,16]. RIPK1 has an essential role in the molecular pathway and signaling triggered by death receptors and pattern recognition receptors, which drives NF-B-mediated cell survival and caspase-8- dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The interaction between RIPK1 and miR155 and their function have been demonstrated in osteosarcoma cells [16]. HBP1, is a member of the sequence-specific high-mobility group (HMG) family, characterized by the DNA-binding domain HMG box. It’s a suppressor of Wnt pathway, participating in multiple cell progressions. And it was also found to be a target of miR-155 in osteosarcoma [12]. However, the molecular network among all the targets still remains to be further explored and the key target of the network to be identified. The significant reduction of cancer cell proliferation and increased apoptosis upon anti-miR-155 treatment prompted us to test cancer cell growth in vivo. MG-63 cells transfected with antimiR-155 or anti-miR-NC was injected into nude mice, and tumor size was measured once a week. In the preliminary result, we found starting MG-63 control induced tumor was growing significantly faster than MG-63 cells transfected with miR-155 (Supplemental Fig. 2). Three weeks post-injection, the control tumor was twice the size of miR-155 deficient tumor. However, transient transfection effect of anti-miR-155 could only maintain for around 5 days. Thus the effect of anti-miR-155 is on the early stage of the tumorigenesis in this animal model. This in vivo data suggests miR-155 is critical for oncogenesis of osteosarcoma. However, the present study has a number of limitations. First, it is based on in vitro cell and animal model. These results are waiting to be further validated in human samples. Second, transient transfection for the in vitro and in vivo studies was applied, while stable transfection using lenti-virus or gene-knock mice will achieve more promising results. Third, rescue effects of individual or combination of targets such MAP3K10, RIPK1 and HBP1 will further illuminate the key microRNA-protein pathway of miR-155 in osteosarcoma. Due to the complex genetic origin, the identifying diagnostic marker and drug targets have been challenging for osteosarcoma. In recent years, miRNAs have shown promising results as potential diagnostic and prognostic biomarkers and also provide target based therapeutic approaches. For instance, miR-199a inhibits mTOR and STAT expression and proliferation [17], miR-21 targets RECK and MMPs [18] Our finding of common upregulation of miR-155 in different osteosarcoma cell lines affords novel target for the biomarker development. Our in vivo study is also very encouraging, which suggests a possible approach of targeting miR-155 and MAPK-JNK pathway in the treatment of osteosarcoma.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2016.12.028.
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Original article
MicroRNA-155 targets MAP3K10 and regulates osteosarcoma cell growth Chuangjian Wang a,1 , Xiaobo Zhang b,1 , Chunlin Zhang a , Fuying Zhai a , Ying Li a , Zongqiang Huang a,∗ a b
Department of Orthopaedics, The First Affiliated Hospital of Zhengzhou University, No. 1 JianShe Road, Zhengzhou, Henan 450000, China Central sterile supply Department, The First Affiliated Hospital of Zhengzhou University, No. 1 JianShe Road, Zhengzhou, Henan 450000, China
a r t i c l e
i n f o
Article history: Received 23 May 2016 Keywords: Osteosarcoma MicroRNA-155 Cell lines MAP4K10
a b s t r a c t Osteosarcoma is the most common type of bone cancer prevalent in young adults. Recent studies suggested that aberrant microRNA expression plays an essential role in osteosarcoma pathogenesis. In this study, we found miR-155 up-regulation in different osteosarcoma cell lines U2OS, Saos-2 and MG-63. Through bioinformatic prediction and analysis, we identified its target MAP3K10 that involved in cell proliferation and apoptosis. This work demonstrates novel interaction between microRNA, intercellular MAPK signaling and apoptosis in osteosarcoma, which will provide targets for therapeutic development. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Osteosarcoma is the most common form of primary malignant bone cancer, especially prevalent in children and adolescents. It is reported that approximately 4–5 cases per million [1]. This type of aggressive sarcoma is characterized by a high level of chromosomal instability and diverse karyotypes [2]. While a number of amplifications of chromosomal region 6p12 and 8q24 have been observed in osteosarcoma, oncogenes such as RB and cMYC mutations also have been associated in osteosarcomagenesis [3,4], the detailed molecular pathways involved in osteosarcoma remain unclear. Interestingly, recent studies have shown that microRNAs (miRNAs) deregulation occurs in osteosarcoma [5,6]. miRNAs is a family of small non protein coding RNAs (20–25 nucleotides) that play a critical role in the posttranscriptional gene regulation by targeting mRNAs for translational repression and/or degradation. MiR155 host gene BIC is abundantly expressed in most tissues and cell types [7], therefore, may play a critical role in a wide range of biological processes, such as hematopoiesis, inflammation. Moreover, miR155 levels are elevated in a broad array of cancers including lymphomas, leukemia and solid tumors. Deregulation of this miRNA may also significantly participate in the development of carcinogenesis.
∗ Corresponding author. E-mail address: [email protected] (Z. Huang). 1 These authors contributed equally to this work.
In this study, we investigated the role of miR155 in human osteosarcoma. We found miR155 is upregulated in a number of osteosarcoma cell lines. Inhibition of this miRNA leads to cancer cell death. We identified the target of miR155 as MAP3K10. More importantly, inhibition of this miRNA in cancer cell reduced the tumor growth in vivo. Our results revealed a novel role of miR155 in cancer cell growth and provided a new possibility of therapeutic intervention in treatment of osteosarcoma. 2. Materials and methods 2.1. Cell lines U2OS, Saos-2 and MG-63 cells were provided by Institute of Biochemistry and Cell Biology of Chinese Academy of Science and originated from American Type Culture Collection (ATCC). 2.2. RNA extraction Total RNA of cultured cells was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. 2.3. RT-PCR One g of total RNA per sample was synthesized using the RT reagent Kit (Formentas, USA) following the manufacturer’s protocol. And then, real-time PCR was performed using the FastStart Universal SYBR Green Master kit (Roche, Switzerland) on the
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ABI 7500 thermocycler (Applied Biosystems, USA). Primers were designed and provided by Ruibo Biotech (Guangzhou, China). 2.4. MiRNA target prediction MicroRNA target genes were predicted using the website program (www.microRNA.org). 2.5. Luciferase assay 293T cells were transfected with MAP3K10 3 UTR plasmid, Renilla luciferase pRL-TK vector (Promega USA), and miR-155 mimics(miR10004658-1-5, Ribobio) or miR-NC using lipofectamine 2000 mimics(miR01201-1-5,Ribobio) reagent (Invitrogen, USA). After 48 h, luminescence was examine using the Dual-Luciferase Reporter Assay System (Promega, USA). Luminescence results were normalized to the Renilla luminescence.
Fig. 1. Expressions of miR-155 are upregulated in human osteosarcoma cell lines. Mature miR-155 expressions were analyzed by real-time PCR and standardized to the endogenous control U6. Human osteosarcoma cell lines U2OS, Saos-2 and MG-63 were examined as well as the control cell hFOB1.19. (**p < 0.01).
2.6. Western blot analysis Cell protein extracts were immunoblotted with a primary antibody and then a secondary antibody. Primary antibodies, MAP3K10 antibody(867–880, SIGMA),were used at a diluted concentration of 1:2000 while the secondary antibody(A0545, SIGMA) was used at 1:10,000. 2.7. SiRNA transfection The human MAP3K10 siRNA(anti-MAP3K10) and negative control oligonucleotide (anti-NC) were designed and provided by Ribobio (Guangzhou, Guangdong, China). MG-63 cells were transfected with 40 nM of anti-MAP3K10 or anti-NC by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. 2.8. Apoptosis detection and analysis Cell apoptosis was detected using the Annexin V/PI apoptosis detection kit(BMS500FI, eBiosicence) using the manufacturer’s protocol. Briefly, cells were washed by ice-cold PBS and binding buffer and were then incubated for 15 min in 100 l of binding buffer containing 5 l of fluorochrome-conjugated Annexin V. Cells were washed using binding buffer and 5 l of PI. Signals were detected using flow cytometry. 2.9. Animal experiments MG-63 cells were transfected with anti-miR-155(miR20004658-1-5,Ribobio) or anti-miRNC(miR02201-1-5,Ribobio). After 6 h, cells were injected subcutaneously into nude mouse. Tumor sizes are measured once per week. We got the approval of using these mice from the Bioethics committee of the Zhengzhou University. The raising of the mice accorded with the “Guide for the Care and Use of Laboratory Animals”. 2.10. Statistical analysis Data were expressed as Mean ± SEM of three independent experiments. For all statistical tests, PRISM 5.0 (GraphPad Software Inc., USA) was used. P values less than 0.05 were identified as statistically significant.
3. Results 3.1. Human osteosarcoma cell lines overexpress miR-155 Osteosarcoma cells share some osteoblastic features, but they differ in many aspects, in particular the proliferation kinetics and osteoid production [8]. Thus, we compared three osteosarcoma cell lines of different origin, U2OS, Saos-2 and MG-63 [9], with normal human osteoblasts, hFOB1.19. We found that the expression of miR-155 is upregulated in all three osteosarcoma cell lines compared to normal osteoblasts (Fig. 1). This miRNA showed five-fold increase in U2OS, and about 10-fold increase in MG-63 cells. We focused on MG-63 cells for our following study.
3.2. MiR-155 deficiency leads to activation of apoptosis in MG-63 cells To elucidate the function of miR155 in osteosarcoma cell line, we used anti-miRNA oligonucleotides, anti-miR-155. Unlike the transitional antisense techniques that suppress expression of target RNAs, anti-miRNA oligonucleotides appear to work primarily through a steric blocking mechanism of action. These compounds are synthetic reverse complements that tightly bind and inactivate the miRNA instead of decreasing its expression. An anti-scramble was used as negative control (anti-miR-NC). Interestingly, when MG-63 cells were transfected with anti-miR-155, their proliferation was significantly reduced (Fig. 2A) compared to U2OS and Saos-2 cells (Supplemental Fig. 1). We next examined whether the reduced proliferation is due to apoptosis using Annexin V/PI staining. As shown in Fig. 2B, treatment of anti-miR-155 increased both doubled Annexin V positive population, and increased Annexin V and PI double positive cells about five fold. This suggests that miR155 deficiency leads to enhanced apoptosis and cell death. To confirm the activation of apoptosis upon miR155 inhibition, we tested Caspase-3/7 activity, which are the effector caspases that execute apoptosis by cleaving cellular proteins on specific Asp residues. Consistent with the Annexin V staining results, the Caspase-3/7 activity was increased when cells were treated with anti-miR-155, compared to cells treated with control anti-miR-NC (Fig. 2C). These findings demonstrate that miR-155 is important to inhibit the activation of apoptosis in osteosarcoma.
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Fig. 2. Inhibition of miR-155 suppressed cell proliferation and induced apoptosis of human osteosarcoma MG-63 cells in vitro. MG-63 cells were transfected with 100 nM of miR-155 inhibitor (anti-miR-155) or negative control inhibitor (anti-miR-NC). (A) After 48 h, cell proliferation was measured by CCK-8 assay. (B)Cell apoptosis was analyzed by Annexin V/PI assay. (C) Caspase-3/7 activities were examined by caspase 3/7 assay. (**p < 0.01).
Fig. 3. MiR-155 targets MAP3K10. (A) Bioinformatic analyses (www.microrna.org) were performed to search for miR-155 target genes and the binding sites of MAP3K10 within its 3 UTR. (B and C) 293 T cells were co-transfected with miR-155 along with reporter plasmid containing the 3 UTR of MAP3K10 or the mutated one. Relative luciferase activity was analyzed after 48 h (D) MG-63 cells were transfected either with miR-155 or miR-NC for 48 h. MAP3K10 protein levels were examined by western blots using GAPDH as a loading control (one of two similar blots is shown). Results are shown as Mean + SEM. (*p < 0.05).
3.3. MAP3K10 is the target of miR-155
3.4. MAP3K10 expressions were lower in osteosarcoma cell lines
A quick bioinformatics search revealed that miR-155 complement with 3 UTR region of MAP3K10 (Fig. 3A). To experimentally confirm that miR-155 could target MAP3K10, we made a luciferase construct with MAP3K10 3 UTR, and tested the luciferase activity in 293T cells during treatment of miR-155. As shown in Fig. 3B and C, we co-transfected MAP3K10 along with reporter plasmid containing the 3 UTR of MAP3K10 or the mutated one into 293T cells. MiR-155 repressed the activity of luciferase fused to the WT MAP3K10 3 UTR, while it failed to repress the mutated one. These data reveal that miR-155 directly targets the MAP3K10 3 UTR leading to repressed expression. We next tested if miR-155 targets MAP3K10 in osteosarcoma cells. Western blot analysis showed that, when MG-63 cells treated with miR-155, it reduced MAP3K10 protein level compared to cells treated with control miR-NC (Fig. 3D). In addition, depletion of MAP3K10 promoted the cell proliferation in MG-63 cells (Fig. 4). Taken together, these results confirmed MAP3K10 as a direct target of miR-155.
Expressions of MAP3K10 in osteosarcoma cell lines U2OS, Saos2 and MG-63 were examined by RT-PCR. As shown in Fig. 5, MAP3K10 levels were decreased in U2OS, Saos-2 and MG-63 cells compared with hFOB1.19 cells, among which it was the lowest in MG-63 cells.
4. Discussion Sarcomas are well known for their heterogeneity, and the heterogeneous and chaotic nature of osteosarcoma has confounded accurate molecular classification, prognosis, and prediction for this tumor [10]. Through examining three different osteosarcomaderived cells lines U2OS, Saso-2 and MG-63, we found they have common upreguated expression of miR-155. These results were consistent with several previous reports [11,12]. These reports and our findings unveiled a role of miR-155 in the proliferation of osteosarcoma cells. It will be interesting to know how the miR-
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Fig. 4. Inhibition of MAP3K10 increased cell proliferation of human osteosarcoma MG-63 cells in vitro. MG-63 cells were transfected with 40 nM of MAP3K10 siRNA (anti-MAP3K10) or negative control (anti-NC). (A) MAP3K10 expression was examined at 72 h. (A) After 48 h and 72 h, cell proliferation was measured by CCK-8 assay (*p < 0.05).
Fig. 5. Expressions of MAP3K10 are downregulated in human osteosarcoma cell lines. MAP3K10 expressions were analyzed by real-time PCR and standardized to the endogenous control GAPDH. Human osteosarcoma cell lines U2OS, Saos-2 and MG-63 were examined as well as the control cell hFOB1.19. (**p < 0.01).
155 host gene is regulated in osteoblast and what happens during cancerogenesis. We identified a direct target of miR-155 as MAP3K10 in osteosarcoma, one member of MAPK pathway directly involved in cell proliferation, differentiation, and apoptosis [13]. MiR-155MAP3K10 interaction was also previous reported in THP-1 cells[14]. Interestingly, a recent study found deregulation of a number of miR-
NAs that target MAPK pathway in osteosarcoma [5]. It is known that MAP3K10 functions preferentially on JNK pathway, which belongs to superfamily of MAP-kinases and is upstream of mitochondria dependent apoptosis pathway [15]. Thus, our results indicate that miR-155 is directly targets MAP3K10-JNK-apoptosis pathway. Further investigation is required to unravel a detailed molecular network of this pathway. But it will be important to know the specific regulation in osteosarcoma cells. In addition, microRNAs functions through multiple targets. Although the present study is focused on MAP3K10, there are several other potential functional targets involved in the molecular pathway of miR-155 in osteosarcoma, such as receptor interacting serine-threonine kinase 1 (RIPK1) and HMG-box transcription factor 1 (HBP1) [12,16]. RIPK1 has an essential role in the molecular pathway and signaling triggered by death receptors and pattern recognition receptors, which drives NF-B-mediated cell survival and caspase-8- dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The interaction between RIPK1 and miR155 and their function have been demonstrated in osteosarcoma cells [16]. HBP1, is a member of the sequence-specific high-mobility group (HMG) family, characterized by the DNA-binding domain HMG box. It’s a suppressor of Wnt pathway, participating in multiple cell progressions. And it was also found to be a target of miR-155 in osteosarcoma [12]. However, the molecular network among all the targets still remains to be further explored and the key target of the network to be identified. The significant reduction of cancer cell proliferation and increased apoptosis upon anti-miR-155 treatment prompted us to test cancer cell growth in vivo. MG-63 cells transfected with antimiR-155 or anti-miR-NC was injected into nude mice, and tumor size was measured once a week. In the preliminary result, we found starting MG-63 control induced tumor was growing significantly faster than MG-63 cells transfected with miR-155 (Supplemental Fig. 2). Three weeks post-injection, the control tumor was twice the size of miR-155 deficient tumor. However, transient transfection effect of anti-miR-155 could only maintain for around 5 days. Thus the effect of anti-miR-155 is on the early stage of the tumorigenesis in this animal model. This in vivo data suggests miR-155 is critical for oncogenesis of osteosarcoma. However, the present study has a number of limitations. First, it is based on in vitro cell and animal model. These results are waiting to be further validated in human samples. Second, transient transfection for the in vitro and in vivo studies was applied, while stable transfection using lenti-virus or gene-knock mice will achieve more promising results. Third, rescue effects of individual or combination of targets such MAP3K10, RIPK1 and HBP1 will further illuminate the key microRNA-protein pathway of miR-155 in osteosarcoma. Due to the complex genetic origin, the identifying diagnostic marker and drug targets have been challenging for osteosarcoma. In recent years, miRNAs have shown promising results as potential diagnostic and prognostic biomarkers and also provide target based therapeutic approaches. For instance, miR-199a inhibits mTOR and STAT expression and proliferation [17], miR-21 targets RECK and MMPs [18] Our finding of common upregulation of miR-155 in different osteosarcoma cell lines affords novel target for the biomarker development. Our in vivo study is also very encouraging, which suggests a possible approach of targeting miR-155 and MAPK-JNK pathway in the treatment of osteosarcoma.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.prp.2016.12.028.
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