MicroRNA-142-3p, a novel target of tumor suppressor menin, inhibits osteosarcoma cell proliferation by down-regulation of FASN
Yao-Qi Yang . Jin Qi . Jian-Qiang Xu . Ping Hao
Received: 13 June 2014 / Accepted: 4 July 2014 /Published online: 18 July 2014 C International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Menin, encoded by MEN1 gene, has been viewed as a tumor suppressor in several types of tu- mors, such as insulinoma, parathyroid tumor, and adre- nocortical and lung carcinoma. However, its expression and molecular regulation mechanism in osteosarcoma has not been elucidated. Here, our results show menin expression is significantly down-regulated in osteosarco- ma tissues, compared with adjacent normal tissues. Be- sides, we report that MicroRNA-142-3p as a novel target of menin. Up-regulation of MicroRNA-142-3p by menin overexpression inhibits cell proliferation in U2OS and MG63 cells. At the molecular level, MicroRNA-142-3p inhibits the protein expression of FASN through binding to its 3’-untranslated region. Therefore, we elucidate a novel regulation pathway in osteosarcoma cells and suggest a potential therapeutic approach for the tumor therapy.
Keywords Osteosarcoma . Menin . MicroRNA . MicroRNA-142-3p . FASN
Yao-Qi Yang and Jin Qi contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s13277-014-2316-z) contains supplementary material, which is available to authorized users.
Y .- Q. Yang . J .- Q. Xu · P. Hao
Department of Orthopaedics, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, No. 197, Ruijin 2nd Road, 200025 Shanghai, China
e-mail: jianqiang_xu@yeah.net
J. Qi
Shanghai Institute of Traumatology and Orthopaedics, School of Medicine, Shanghai Jiao Tong University, No. 197, Ruijin 2nd Road, 200025 Shanghai, China
Introduction
Menin, the product of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene, plays important roles in gene transcription, cell cycle progression and apoptosis, and DNA damage and repair [1, 2]. Its protein mutations have been tightly associated with multiple endocrine neoplasia type 1 (MEN1), including insulinoma, parathyroid tumor, and pituitary tumors [3, 4]. Besides, menin is shown to inhibit cell proliferation in lung and hepatocellular carcinoma [5, 6], suggesting that menin is a critical checkpoint governing tumor suppression in the develop- ment of human cancers. At the molecular level, menin up- regulates expression of cell cycle regulators, including p18 and p27, through recruitment of several histone and DNA methyl- transferase [7]. Besides, menin represses Wnt/ß-catenin and hedgehog signaling in pancreatic tumor cells [8, 9]. However, a recent study revealed that menin might promote hepatocellular carcinogenesis through up-regulation of Yap1 transcription [10]. Therefore, understanding the mechanisms underlying menin- mediated regulation of cell proliferation may provide a potential target for gene therapy for human cancers.
Osteosarcoma, the most common type of bone tumor, occurs most often in teenagers [11]. Previous studies have shown that aberrant expression of many oncogenes and (or) tumor suppressors, such as C-myc, vascular endothelial growth factor (VEGF) and P53, usually contributes to the cell proliferation, apoptosis, metastasis, and radiosensitization in osteosarcoma [12, 13]. However, the specific role of menin in osteosarcoma remains unexplored until now.
Materials and methods
Cell culture and tissue samples
Osteosarcoma cell lines (U2OS and MG63) were obtained from American Type Culture Collection (Rockville, MD).
Cells were culture in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Beijing) supplemented with 10 % fetal bo- vine serum (Gibco, Beijing). Cultures were maintained at 37 ℃ in a humidified atmosphere with 5 % CO2. Tumor tissues and adjacent non-tumor normal tissues were collected from routine therapeutic surgery at our department. All sam- ples were obtained with informed consent and approved by the hospital institutional review board.
Analysis of miRNA expression using TaqMan RT-PCR
Total RNA from tissue samples and cell lines was harvested using the microRNA (miRNA) Isolation Kit (Ambion, USA). Expression of mature miRNAs was assayed using Taqman MicroRNA Assay (Applied Biosystems) specific for hsa- miR-142-3p. Briefly, 10 ng of total RNA were reverse tran- scribed to complementary DNA (cDNA) with specific stem- loop RT primers. Quantitative real-time PCR was performed by using an Applied Biosystems 7900 Real-time PCR System and a TaqMan Universal PCR Master Mix. All the primers were obtained from the TaqMan miRNA Assays. Small nu- clear U6 small nuclear RNA (snRNA) (Applied Biosystems) was used as an internal control.
Plasmid construction and transfection
For miR-142-3p expression plasmid, human miR-142-3p pre- cursor was cloned into pSilencer 4.1 (Ambion, Austin, TX). The negative control plasmid consists of a scrambled se- quence (Ambion). To inhibit miR-142-3p function, an Ambion miRNA antisense for miR-142-3p was used, along with the negative control. For transfection, a complex of Lipofectamine 2000 (Invitrogen, CA, USA) and 25-nM miRs mentioned above was prepared following the manufacturer’s instructions.
BrdU assays
A cell proliferation enzyme-linked immunosorbent assay (5-bromo-2’-deoxyuridine, BrdU; Beyotime) was used to analyze the incorporation of BrdU during DNA synthe- sis following the manufacturer’s protocols. All experi- ments were performed in triplicate. Absorbance was measured at 450 nm in the Spectra Max 190 ELISA reader (Molecular Devices, Sunnyvale, CA).
Western blot
Cells or tissues were harvested and lysed with ice-cold lysis buffer (50-mM Tris-HC1, pH 6.8, 100-mM 2-ME, 2 % w/v SDS, 10 % glycerol). After centrifugation at 20,000×g for 10 min at 4 ℃, proteins in the supernatants were quantified and separated by 10 % SDS PAGE, transferred to
nitrocellulose membrane (Amersham Bioscience, Bucking- hamshire, UK). After blocking with 10 % nonfat milk in PBS, membranes were immunoblotted with antibodies as indicated, followed by HRP-linked secondary antibodies (Cell Signaling). The signals were detected by SuperSignal West Pico Chemiluminescent Substrate kit (Pierce, Rockford, IL) according to manufacturer’s instructions. Anti-menin, p18, and p27 antibodies were purchased from Cell signaling (USA). Protein levels were normalized to total glyceraldehyde 3-phosphate dehydrogenase (GAPDH), using a mouse anti- GAPDH antibody (Santa Cruz, USA).
Luciferase reporter assay
Total cDNA from MG63 cells was used to amplify the 3’UTR of fatty acid synthesis (FASN) by PCR. The FASN 3’UTR was cloned into pMir-Report (Ambion), yielding pMir-Report- FASN. Mutations were introduced in potential miR-100 binding sites using the QuikChange site-directed muta- genesis Kit (Stratagene). Cells were transfected with the pMir-Report vectors containing the 3’-UTR variants, and miR-100 precursor, control plasmids for 36 h. The pRL- TK vector (Promega, USA) carrying the Renilla lucifer- ase gene was used as an internal control to normalize the transfection efficiency. Luciferase values were deter- mined using the Dual-Luciferase Reporter Assay System (Promega).
Statistical analysis
Data are expressed as the mean±SEM from at least four separate experiments. Differences between groups were ana- lyzed using Student’s t test or one-way ANOVA analysis. A value of p<0.05 was considered statistically significant.
Results
Down-regulation of menin in human osteosarcoma tissues
First, we analyzed menin expression in 20 paired osteosarco- ma tissues and adjacent non-tumor tissues by way of real-time PCR. As shown in Fig. 1a, we observed a significant decrease of menin mRNA expression in tumor tissues. Besides, its down-regulation was further confirmed by western blot (Fig. 1b).
To address whether down-regulation of menin could affect osteosarcoma progression, U2OS cells were transfected with small interfering RNA (siRNA) oligos to inhibit endogenous expression of menin. Compared with scramble negative con- trols (NC), siRNA targeting menin dramatically reduced its mRNA and protein levels (Fig. 1c, d). As a result, we
A
Menin
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1.5-
Relative mRNA level
Patient 1
Patient 2
Patient 3
1.0
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N
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N
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GAPDH
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Menin (U2OS Cells)
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U2OS Cells
Relative mRNA level
1.0
SİRNA NC Menin
Menin
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GAPDH
0.0
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siRNA
E
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U20S Cellis
U2OS Cells
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U2OS cells
200
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Cell Numbers (x 104)
SIRNA-NC
15-
siRNA-Menin
5
MTT Assay (%)
150
BrdU Assay (A450 absorption)
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SİRNA
observed that the growth rate of U2OS cells was dramat- ically increased upon menin inhibition, as assayed by cell numbers, MTT, and BrdU incorporation assays (Fig. le- g). Similar results were also obtained in MG63 cells (Supplementary Fig. 1a-e), suggesting that osteosarcoma development is characterized by down-regulation of menin expression.
Menin overexpression inhibits osteosarcoma cell proliferation
We then tested our proposed involvement of menin in osteo- sarcoma and delivered its expression plasmids into U2OS and MG63 cells (Fig. 2a, b). pCDNA3.1, an empty vector, was used as the control. The cell growth rate displayed a typical pattern, which indicated that menin overexpression inhibited U2OS cell proliferation (Fig. 2c, d). Besides, the results from MTT and BrdU assays also supported our notion (Fig. 2e, h). Taken together, these data demonstrate that menin is a tumor suppressor in osteosarcoma.
Regulation of MicroRNA-142-3p by menin
At described above, menin modulates the transcription of p18 and p27 in several human cancers [7]. In agreement with these reports, we also observed that expression of p18 and p27 was increased by menin overexpression in U2OS and MG63 cells (Supplementary Fig. 2a, b). However, whether additional mechanisms involved in the roles of menin remain poorly understood. Here, to explore the specific target of menin in osteosarcoma, microRNA (miR) arrays prepared from U2OS cells were performed. As suggested by our array data, miR-142-3p was dramatically elevated in U2OS cells overexpressing menin (data not shown). Besides, our real-time PCR analysis also confirmed the up- regulation of miR-142-3p by transfection of menin in U2OS and MG63 cells (Fig. 3a, b). In agreement, targeted knockdown of menin also reduced miR-142-3p expres- sion (Fig. 3c, d). Moreover, the expression level of miR- 142-3p was also reduced in osteosarcoma tissues, com- pared with adjacent non-tumor tissues (Fig. 3e), further
A
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U2OS Cells EV Menin
MG63 Cells EV Menin
Menin
Menin
GAPDH
GAPDH
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U2OS cells
MG63 cells
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Cell Numbers (x 104)
EV
Cell Numbers (x 104)
EV
POH
6-
Menin
6-
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Menin
4.
4-
2.
2-
ME
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*
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Day
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U2OS Cells
MG63 Cells
150-
150-
MTT Assay (%)
MTT Assay (%)
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100
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50-
50
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EV
Menin
EV
Menin
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H
U2OS Cells
MG63 Cells
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BrdU Assay (A450 absorption)
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BrdU Assay (A450 absorption)
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**
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Menin
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Menin
supporting the notion that menin is an upstream regulator for the induction of miR-142-3p.
Next, to determine whether menin might be a transcriptional activator of miR-142-3p, a reporter vector encoding luciferase under control of the miR-142-3p promoter was constructed into
PGL3 vectors. Concurrent expression of menin increased miR- 142-3p promoter activity (Fig. 3f). Moreover, chromatin im- munoprecipitation (ChIP) assays also confirmed the bound of menin protein onto the proximal promoter region of miR-142- 3p, but not the distal region (Fig. 3g). Therefore, our results.
A
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miR-142-3p U2OS Cells
miR-142-3p MG63 Cells
miR-142-3p U2OS Cells
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25-
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Relative miRNA Level
Relative miRNA Level
Relative miRNA Level
15-
20-
15-
1.0-
10-
10-
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5-
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Menin
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Menin
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NC
Menin
SİRNA
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miR-142-3p MG63 Cells
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miR-142-3p Promoter
miR-142-3p Promoter
1.5-
1.5-
miR-142-3p
Relative Luciferase Activity
80-
1.5-
Relative miRNA Level
Relative miRNA Level
Percentage of Input
**
60-
1.0-
1.0-
1.0-
40-
0.5-
**
0.5-
0.5-
20-
0.0
NC
Menin
0.0
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N
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EV
Menin
0.0
lgG
Menin
lgG Menin Distal Promoter
SİRNA
Proximal Promoter
miR-142-3p regulates proliferation and inhibits tumorigenicity
In order to investigate the roles of miR-142-3p in osteosarco- ma, we transfected a has-miR-142-3p mimic into U2OS cells and measured cell proliferation. Using MTT and BrdU assays, we observed that the growth rate of miR-142-3p overexpress- ing cells was dramatically decreased, compared to negative control (NC)-transfected cells (Fig. 4a, b). In addition, inhibi- tion of miR-142-3p by its antisense markedly enhanced the growth of U2OS cells, compared to NC-transfected cells (Fig. 4c, d). Similar results were also obtained in MG63 cells (data not shown). Taken together, these results support an anti- proliferative role for miR-142-3p in osteosarcoma cells.
miR-142-3p directly targets FASN in osteosarcoma cells
To predict the possible target gene of miR-142-3p, we carried out a computational analysis on TargetScan (http://www. targetscan.org/), which suggested that miR-142-3p may di- rectly target the 3’-untranslated region of fatty acid synthesis (FASN). To confirm FASN as the target gene of miR-142-3p, we engineered the fragment of FASN gene containing the binding sites for miR-142-3p into luciferase reporter vector and carried out luciferase reporter activity assay. As illustrated in Fig. 5a, transfection of miR-142-3p caused a substantial
reduction of luciferase activity on the luciferase expression constructs carrying the target fragment. Besides, the repressive effect of miR-142-3p on the FASN 3’-UTR was abrogated by point mutations in the miR-142-3p-binding seed region of the FASN 3’-UTR (Fig. 5a). Moreover, ectopic expression of miR-142-3p decreased the protein expression levels of FASN in U2OS and MG63 cells (Fig. 5b, c), while transfection of miR-142-3p antisense up-regulated FASN protein abundance (Fig. 5d, e). Together, our results confirm that FASN gene is a target of miR-142-3p. Consistent with this, menin overexpres- sion also led to a reduction of FASN protein levels in U2OS cells, which was reversed by miR-142-3p inhibition (Fig. 5f), indicating that miR-142-3p was a mediator in the regulation of FASN by menin.
Discussion
In this study, we demonstrated that menin expression was reduced in osteosarcoma tissues, compared to adjacent normal tissues. At the molecular level, we found that miR-142-3p was a transcriptional target of menin. Overexpression of miR-142- 3p inhibited cell proliferation while its inhibition enhanced tumor cell proliferation. Furthermore, we showed that miR- 142-3p could directly target the FASN 3’-UTR, suggesting
A
U2OS Cells
B
U2OS Cells
150
0.8
MTT Assay (%)
BrdU Assay (A450 absorption)
100
0.6
*
*
0.4
50
0.2
0
0.0
NC
miR-142-3p
NC
miR-142-3p
C
U2OS Cells
D
U2OS Cells
200
1.0
*
MTT Assay (%)
150
BrdU Assay (A450 absorption)
0.8
0.6
100
0.4
50
0.2
0
0.0
NC
miR-142-3p AS
NC
miR-142-3p AS
that FASN is negatively regulated by miR-142-3p in osteosar- coma cells. Collectively, our findings reveal a previous un- known menin/miR-142-3p/FASN regulatory network in the progression of osteosarcoma. In agreement with these results,
Fig. 5 miR-142-3p down- regulates FASN expression by targeting its 3’-UTR. (a) Relative luciferase activity of wild-type (WT) or mutant (Mut) FASN 3’- UTR in U2OS cells transfected with negative control (NC) or miR-142-3p. (b, c) Western blot analysis of FASN expression levels in U2OS (B) and MG63 (C) cells transfected with negative control (NC) or miR-142-3p. (d, e) Western blot analysis of FASN expression levels in U2OS (d) and MG63 (e) cells transfected with negative control (NC) or miR-142-3p antisense (AS)
a recent study demonstrated that menin is required for optimal processing of the microRNA let-7a, resulting in suppression of Irs2 expression and insulin signaling in pancreatic beta cells [14]. Therefore, miRNAs may participate in the precise mech- anism by which menin suppresses gene expression, which needs to be determined in the future.
MicroRNAs are small non-coding RNAs regulating gene expression at the post-transcriptional and/or translational levels [15, 16]. MicroRNAs play important roles in various biological processes, including development, cell prolifera- tion, differentiation, metabolisms [17]. Recent studies have proved that expression of certain MicroRNAs is dysregulated in many types of cancer, suggesting microRNAs exert key roles that lead to carcinogenesis [18, 19]. It has been docu- mented that miR-142-3p plays important roles in several kinds of cancer cells. MicroRNA-142-3p was shown to suppress the migration and invasion of hepatocellular carcinoma cells through regulation of RAC1 [20]. MiR-142-3p also inhibited the growth of colon cancer cells by targeting CD133, ABCG2, and Lgr5 [21]. Therefore, together with these studies, our data indicate that miR-142-3p as a tumor suppressor.
In addition, our results reveal that miR-142-3p could reg- ulate FASN expression in osteosarcoma cells. FASN is a key lipogenic enzyme catalyzing the terminal steps in the de novo biogenesis of fatty acids in cancer pathogenesis [22]. Previous experimental evidence has suggested that FASN is involved in cancer proliferation and metastasis in breast, colon, and hepa- tocellular carcinoma [22, 23]. Besides, a positive correlation between FASN and Ki-67 protein, a proliferation marker, was identified in osteosarcoma [24]. Indeed, inhibition of fatty acid synthase suppresses osteosarcoma cell invasion and mi- gration [25]. Therefore, FASN may be a promising target in the treatment of osteosarcoma.
In summary, the key finding of the current study is that menin regulate osteosarcoma cell proliferation, at least in
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FASN
FASN
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NC miR-142-3p AS
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FASN
FASN
FASN
GAPDH
GAPDH
GAPDH
part, through up-regulation of miR-142-3p, which in turn, down-regulates the oncogene FASN. Understanding the precise roles played by menin/miR-142-3p/FASN regula- tory axis will not only advance our knowledge of osteo- sarcoma biology, but also will help determine if menin and miR-142-3p has potential as a novel therapeutic target for the treatment of osteosarcoma.
Acknowledgements This work was supported by the Shanghai Jiao Tong University School of Medicine Project in China, No.12XJ10064
Conflict of interest None
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