Introduction
Oral cancer is one of the most common cancers worldwide with an annual estimated incidence of approximately 275 000. More than 90% of oral cancers are squamous cell carcinomas arising in the oral cavity [
1]. The initiation and progression of OSCC involve a multistep process of aberrant genetic events following the action of various carcinogens, which might be derived from the chronic use of tobacco, alcohol, and betel quid [
2]. Cigarette smoking and alcohol drinking are the major risk factors for OSCC in western countries. In southeast Asia, betel quid chewing and cigarette smoking are the predominant risk factors for OSCC [
2]. Currently, the treatment of OSCC is problematic. In most countries, the 5-year survival rate for oral cancer is approximately 50% [
1], with locoregional recurrence and/or distant metastasis the major causes of death in these patients [
3]. To improve the treatment outcome, elucidation of the molecular mechanisms involved in the carcinogenesis and progression of OSCC is, therefore, needed.
In recent years, biomedical investigation has increasingly focused on a relatively small number of microRNAs (miRNAs), noncoding RNAs approximately 17 to 25 nucleotides in length [
4]. These miRNAs are transcribed by RNA polymerase II in hairpin structures and processed by RNase III Drosha into long precursor miRNAs (pre-miRNAs) in the nucleus [
4]. The precursors are then transported into the cytoplasm and processed by RNase III Dicer to form mature miRNA, which regulates gene expression by controlling mRNA translation and stability. It does this through the translational repression of its target mRNA, or by increasing its degradation using an RNA interference mechanism [
4]. Evidence suggests that miRNAs can be functionally classified as proto-oncogenes or tumor suppressor genes and aberrantly expressed in different cancer types Dysregulation of these cancerous miRNAs is involved in tumor initiation and progression by facilitating an inappropriate cellular program that promotes uncontrolled cell proliferation, favors survival, inhibits differentiation, or induces invasive behavior [
5‐
7].
Previous studies characterized microRNA-99a (miR-99a) as a tumor suppressor in several human cancers, including childhood adrenocortical tumors [
8], prostate [
9], liver [
10], head and neck [
11], and oral [
12] cancers. These studies showed that expression of miR-99a could reduce the expression of a number of proto-oncogenes by targeting their 3′ untranslated region (3′UTR) [
8‐
13]. Several groups further identified the downregulation of miR-99a in clinical samples of oral or head and neck squamous cell carcinoma (HNSCC) of different stages using miRNA microarray analysis [
11,
12,
14‐
16]. However, most of the biological functions of miR-99a in OSCC remain unknown.
Considering the diverse and overlapping biological functions of the identified targets of miR-99a, it is possible that an additional miR-99a target might contribute to OSCC tumorigenesis. In this study, we identified IGF1R as a specific target of miR-99a. The IGF1R is overexpressed in several cancers, including in OSCC tissues and cell lines [
17,
18], and essential for malignant transformation and progression [
19]. In the study by Lara et al., IGF1R expression was a predictor of clinical outcome in patients with locally advanced OSCC [
20].
Insulin-like growth factor 1 receptor is a transmembrane tyrosine kinase receptor with a heterodimer of α- and β-chains, and activated by its ligands insulin–like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2) [
21]. Insulin-like growth factor 1 receptor signaling pathways influence cancer cell proliferation, adhesion, migration, and cell death, and are critical in tumor cell survival and metastasis [
22]. Activation of IGF1R leads to activation of the Ras, Raf, and mitogen-activated protein kinase (MAPK) pathway, resulting in increased proliferation and stimulation of the phosphatidylinositol 3-kinase (PI3K) pathway, which subsequently leads to the inhibition of apoptosis [
23]. The overexpression of a constitutively active form of IGF1R in the mouse mammary gland can initiate tumorigenesis [
24], whereas expression of a dominant negative IGF1R inhibits Ras-induced cell transformation [
25]. In this study, we demonstrated that miR-99a is downregulated in OSCC tissues and cell lines and can function as a tumor metastasis suppressor for migration, invasion and lung colonization in OSCC cells. Using bioinformatic prediction and luciferase reporter assays, we further confirmed that miR-99a negatively regulates IGF1R protein levels by specifically binding to the 3′UTR of IGF1R mRNA. We identified the negative regulation of miR-99a expression by IGF1-induced signaling and proposed a reciprocal regulation responsible for the mutual regulation of miR-99a and IGF1R.
Discussion
Previous studies used miRNA microarray and qRT-PCR analyses to characterize the miRNA expression profiles in oral cancer cell lines and clinical samples of different stages [
27,
28]. Their findings suggested the dysregulation of miRNAs in the initiation and progression of oral cancers. Similar to the results from a study by Wong et al. [
14], our data showed that miR-99a is frequently downregulated in OSCC cell lines and tissues, especially in OSCC patients with lymphovascular invasion (Figure
1 and Additional file
1: Table S1). This suggested that the downregulation of miR-99a might play a critical role in invasion and metastasis of OSCC and supported evidence that ectopic miR-99a expression inhibits cell migration, invasion and lung colonization in OSCC cells (Figures
2D-G). Li et al. reported that low miR-99a expression in hepatocellular carcinoma (HCC) tissues was associated with worse prognosis in HCC patients [
10]. However, in this study, we observed insignificant correlation between miR-99a expression and most of clinicopathological parameters (Additional file
1: Table S1).
The biological roles of miR-99a in oral tumorigenesis remain unclear. Previous studies described that ectopic miR-99a expression slowed keratinocyte growth and liver cancer cell growth by blocking the cell cycle at the G1/S transition [
10,
29]. In OEC-M1 and CGHNC9 cells, ectopic miR-99a expression had no significant effects on cell growth (Figures
2B and C), cell cycle (Additional file
7: Figure S6A) and cell morphology (Figure
3A and Additional file
2: Figure S1) even though ectopic expression of miR-99a did subtly affect the expression of cell cycle-related molecules (Additional file
7: Figure S6B) and EMT-related proteins (Figure
3). However, ectopic miR-99a expression decreases cell migration, invasion and lung colonization (Figures
2D-G). These observations suggested that the diverse biological functions of miR-99a may be cell type- and context- dependent. Because of low miR-99a expression in all tested OSCC cells, we were unable to observe any significant knockdown of miR-99a in OSCC cells in response to anti-miR-99a expression (data not shown).
With bioinformatic prediction and experimental validation using microarray and polyribosomal loading analysis, Sun et al. identified that IGF1R and mTOR are the direct targets of miR-99a [
9]. In our study, the expression of miR-99a correlated inversely with IGF1R, but not mTOR protein expression (Figures
4B and
4C). Ectopic expression of miR-99a decreased the levels of IGF1R protein (Figure
4D and Additional file
4: Figure S3), suggesting that IGF1R, but not mTOR, is a specific target of miR-99a in OSCC cells. However, it remains possible that miR-99a might functionally target mTOR in other cell types or in different biological systems [
12]. It is, therefore, possible that other molecules or signaling pathways influenced by miR-99a could be involved in OSCC pathogenesis, and that some of them have yet to be identified.
Insulin-like growth factor 1 receptor is overexpressed in several malignancies and plays a crucial role in promoting cell proliferation and metastasis [
30]. Our results demonstrated that miR-99a expression decreases IGF1R protein levels by directly targeting the 3′UTR of IGF1R mRNA (Figure
4E). Previous studies have identified that IGF1R is also a specific target of miR-145 in colon cancer cells [
31], miR-122 in liver cancers [
32], miR-7 in tongue squamous cell carcinoma cells [
28], miR-375 in esophageal squamous cell carcinoma [
33], and let-7c in regulation of glucose metabolism [
34]. A recent study showed that the administration of tumor-suppressive miRNA to mice could effectively suppress tumorigenesis in liver cancer without causing toxicity [
35]. This indicated the possibility of using miRNA to target IGF1R in cancer therapy.
Although previous studies identified reduced miR-99a expression in several types of tumors [
8,
9,
11,
12,
14,
36‐
38], the detailed mechanisms underlying its repression remain unclear. As previously reported, epigenetic mechanisms, including DNA methylation and histone modification [
39], chromosome deficiency [
40], and transcriptional regulation [
41] can influence the downregulation of miRNA. Yamada et al. showed that the gene encoding miR-99a, located in chromosome 21q21, is frequently deleted in lung cancers [
36]. In this study, we examined 40 paired tissue samples from OSCC patients for chromosome deletions using array comparative genomic hybridization and failed to identify any obvious deletion in chromosome 21q21 (data not shown). We then considered the hypermethylation of the CpG islands surrounding miRNA genes as markers for the investigation of epigenetically silenced miRNAs. However, reactivated expression of miR-99a was undetectable in oral cancer cells after treatment with 5-Aza-dC (Figure
5A). Similarly, Li et al. suggested that there was little evidence of the contribution of methylation to downregulation of miR-99a [
10]. These findings indicated that promoter methylation did not cause the downregulation of miR-99a.
Reduced endogenous miR-99a in OSCC cells retreated with IGF1 after serum starvation (Figure
6A), increased miR-99a expression in cells treated with PI3K or MAPK kinase inhibitors (Figures
6C and
6D), and a reduced level of miR-99a in OSCC cells provided further evidence of the regulation of levels of miR-99a by IGF1R signaling. We proposed a model to describe the combining of the functions of miR-99a and IGF1R to achieve a maximal effect in OSCC cells in response to IGF1R activation (Figure
6E). Activation of the IGF1R signaling pathway initiated the inhibitory signal for miR-99a expression. Decreased miR-99a expression then increased the levels of IGF1R protein. Inhibition of the IGF1R signaling pathway by depletion of growth factors or specific chemical inhibitors increased miR-99a expression because of the loss of the inhibitory signal. This then reduced the levels of IGF1R protein. This reciprocal regulation, therefore, maximizes the activation of the IGF1R signaling pathway in OSCC cells in response to IGF1 stimulation. Due to tumor heterogeneity, however, our results indicated a reciprocal regulation that mutually regulates the levels of miR-99a and IGF1R in a part of OSCC cells. Different from our hypothesis, Lerman et al. described that the activation of IGF1 signaling increased the expression of miR-99a, which then repressed the expression of IGF1R in psoriatic skin [
29]. Huang et al. showed that IGF1 significantly accelerated the upregulation of miR-133, which targeted IGF1R during skeletal myogenesis [
42]. Both studies described the reciprocal regulation of miRNA and IGF1R during proliferation and differentiation in normal tissues. Collectively, this reciprocal regulation is potentially involved in a number of genetic pathways involving miRNA, and might enhance the functionality and robustness of gene networks [
43]. Other previous studies described similar findings involved in the regulation of let-7 g and lectin-like oxidized LDL receptor-1 (LOX-1) [
44]; let-7 and Fas [
45]; ZEB1-SIP and the miR-200 family [
46].
The mechanisms for the negative regulation of miR-99a by the IGF1R signaling pathway remain unclear. Several studies described the positive correlation between the expression of mature miR-99a and primary miR-99a and that of their host gene C21orf34/LINC00478 in liver and prostate cancer tissues, suggesting the possible cotranscription of miR-99a with C21orf34 [
10,
47]. Willimott et al. reported that stromal cell contact induced the expression of the miRNA cluster miR-99a/let-7c/miR-125b, indicating the involvement of transcriptional regulation in the induction of this miRNA cluster [
48]. Future investigation to evaluate the possible promoter regions and to identify the transcription factors binding to the region upstream of the miR-99a gene is, therefore, warranted.
Collectively, we demonstrated that miR-99a is frequently down-regulated and functions as a tumor metastasis suppressor in OSCC cells. Also, miR-99a mutually regulates its own target, IGF1R expression within a reciprocal regulation, suggesting that the possibility of miR-99a for targeting IGF1R in cancer therapy.
Materials and methods
Clinical samples and patient characteristics
Paired tumor specimens and their adjacent nontumorous epithelia were derived from 40 primary OSCC patients who received curative surgery from 2002 to 2009 at National Cheng Kung University Hospital. Fresh frozen tissues were preserved in liquid nitrogen until use. Clinical parameters, including age, sex, social history, pathological features, and TMN stage, were retrospectively collected by reviewing patients’ charts. The study protocol was approved by the Institutional Human Experiment and Ethics Committee. Informed consent was obtained from each patient.
Oral squamous cell carcinoma cell lines
Human oral keratinocytes (HOK) were purchased from ScienCell Research Laboratories and cultured in an oral keratinocyte medium (OKM; ScienCell Research Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. OSCC cell lines, including CGHNC9 [
28], OC3 [
49], OEC-M1 [
50], TW2.6 [
51], FaDu [
52], KB [
53], SCC-4, SCC15 [
54], SCC9, SCC25 [
55], UT-MUC-1 [
56], YD-15 [
57], DOK [
58], Tu183 [
59], UMSCC1 [
60] and HSC3 [
61], were cultured at 37°C in a 5% CO
2 atmosphere within 3 months of resuscitation from the frozen aliquots, with lower than 20 passages in each experiment.
Quantification of miRNA
Total RNA molecules were polyadenylated and reverse transcribed using poly (A) polymerase and MMLV reverse transcriptase in a Mir-X™ miRNA First-Strand Synthesis kit (Clontech, Madison, WI, USA) according to the manufacturer’s manual. Real-time quantitative polymerase chain reactions were performed using a Fast SYBR® Green Master Mix (Applied Biosystems, Foster City, CA, USA) for amplification. Specific miRNA sequences in the cDNA were quantified using miRNA-specific sequences as 5′ primers. The forward primer used for miR-99a was 5′ AACCCGTAGATCCGATCTTGTG. The U6 was used as a reference for miRNA quantification and was supplied in the kit. All amplifications were performed in triplicate and values were normalized to an endogenous control U6. The relative expression of miRNA was normalized to that of the control in each experiment.
Immunoblot analysis
Immunoblot assays were performed as described previously [
62]. Primary antibodies were used as followed: anti-E-cadherin (610182, BD, San Jose, CA, USA), anti-α-catenin (610193, BD), anti-N-cadherin (610920, BD), anti-Vimentin (MS-129-P0, Thermo Scientific, Cheshire, UK), anti-Twist (sc-15393, Santa Cruz, Santa Cruz, CA, USA), anti-Snail (3895, Cell Signaling, Danvers, MA, USA), anti-Slug (AP2053a, Abgent, San Diego, CA, USA), anti-MMP2 (#4022, Cell Signaling), anti-MMP9 (#2551-1, Epitomics, Burlingame, CA, USA), anti-phospho-mTOR (Ser2448) (#5536, Cell Signaling), anti-mTOR (#2972, Cell Signaling), anti-phospho-IGF1 Receptor β (Tyr980) (#4568, Cell Signaling), anti-IGF1 Receptor β (#3018, Cell Signaling), anti-p21 (#2946, Cell Signaling), anti-p27 (#2552, Cell Signaling), anti-cyclin D1 (#2926, Cell Signaling), anti-cyclin E (sc-247, Santa Cruz), anti-β-actin (sc-1615, Santa Cruz) and anti-α-tubulin (MS-581-P0, Thermo Scientific). Protein levels were determined by measuring the intensity of bands on the blots using Image J (National Institutes of Health, Bethesda, Maryland, USA). Protein levels were normalized against an internal control β-actin or α-tubulin. The ratio was determined by dividing the normalized protein levels in expressing cells with that in control cells. The mean of ratio was obtained by averaging the ratios from several independent blots.
Cell proliferation
Cell proliferation was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay as described previously [
63]. 10
3 cells were seeded onto 96-well plate and measured their growth by CellTiter 96 Aqueous Non-Radioactive Cell Proliferation assay (Promega, Madison, WI, USA) for 4–5 days according to the manufacturer’s instructions. The proliferation curves were determined by calculating the mean value of absorbance measurement at 490 nm using a 96-well plate reader.
Migration and invasion assay
Migration and invasion assays were performed using transwells as described previously [
62]. Briefly, cells in 0.5 mL of serum-free medium were plated into the inserts (Matrigel-coated inserts for invasion assay). Inserts were placed in wells with 0.75 mL of complete medium containing 10% FBS as a chemoattractant. After culture for 20 to 24 hours at 37°C, cells were fixed with methanol for 8 min and stained with Giemsa regent (Sigma, St Louis, MO, USA). Cells on the upper sides of the inserts were removed with a cotton swab, and the insert membranes were cut and mounted on glass slides. The numbers of migrated or invaded cells on the membranes were determined by counting the cell numbers in the field at 100X magnification under light-field microscope. The relative migration/invasion activity was measured by normalizing the mean of total migrated/invaded cells per insert in overexpressing cells to that in the corresponding controls. The data shown represent the average of at least 3 repeated experiments.
Lung colonization assay
Lung colonization assay was performed as described previously [
63]. All animal studies were followed by the guidelines for the Care and Use of Laboratory Animals of National Health Research Institutes, Taiwan. The protocol was approved by the Institutional Animal care and Use Committee of National Health Research Institutes (Protocol No: NHRI-IACUC-100047-A). All efforts were made to minimize suffering. Briefly, 10
5 cells suspended in phosphate buffered saline (PBS) were injected into nude mice (National Laboratory Animal Center, Taiwan) via tail vein injection. The whole lungs were harvested for paraffin embedding, sectioning, and histological examination after H&E staining (Pathology Core, National Health Research Institutes, Taiwan). 6 animals were included in each group. The number of tumor nodules in every lung section was counted under the light-field microscope. The colony number/per lung section was determined by averaging the numbers of tumor nodules from independent lung sections.
Immunofluorescence
Cultured cells on slips were fixed and followed the protocols as previous described [
62]. After incubation with Alexa Fluro 488 phalloidin (1:200, Molecular Probes, Ungene, OR, USA), anti-β-tubulin (MS-581-P0, Thermo Scientific), anti-E-cadherin (610182, BD) and anti-FAK (sc-557, Santa Cruz), the slips were mounted with antifade onto the slides and viewed under a fluorescence confocal microscope.
Lentiviral infection of miR-99a
The expression construct of miR-99a and non-silencing microRNA were kindly donated by Dr Michael Hsiao, Academic Sinica, Taiwan and transfected into the packaging cell line 293FT, along with pMD.G and pCMVΔR8.91 plasmids, using the Polyjet transfection reagent (SignaGen Lab, Ijamesville, MA, USA). After 48-hour incubation, viral supernatants were transferred to target cells and infected cells were cultured in the presence of different concentrations of puromycin (Calbiochem, La Jolla, CA, USA) depending on the cell lines.
Luciferase reporter assay
The IGF1R (nucleotides 9543 to 9833) 3′UTR sequence (IGF1R 3′UTR) and deletions in the 3′UTR-predicted miR-99a binding sites (ΔIGF1R 3′UTR) were cloned in psiCHECK-2 vector (Promega) and donated by Dr Enzo Lalli [
8]. The OEC-M1 cells with ectopic miR-99a (OEC-M1 miR-99a) or non-silencing microRNA expression (OEC-M1 NS) were transfected in 6-well plates according to the manufacturer’s protocol. Rellina and firefly luciferase assays were performed using the Dual-Luciferase Reporter® Assay System (Promega), using a luminometer for measurement of luminescence. In each sample, the luciferase activity was determined by normalizing the Renilla luciferase activity to the firefly luciferase activity. The relative luciferase activity was determined by normalizing the activity in cells with wild type 3′UTR expression (IGF1R 3′UTR) to that in cells with the mutant 3′UTR (ΔIGF1R 3′UTR). Experiments for each construct were repeated 2 to 4 times.
Transient expression of Insulin-like growth factor I receptor
Expression construct of IGF1R was kindly obtained from Dr. Lu-Hai Wang, National Health Research Institutes, Taiwan. The plasmids were transiently transfected into cells using the Polyjet transfection reagent (SignaGen Lab). After 48-hour incubation, cells were collected to perform the subsequent experiments.
Prediction of miR-99a targets
Using miRWalk (
http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/), a comprehensive database with eight established programs, including DIANA-microT (version 3.0), miRanda (August 2010), miRDB (April 2009), PicTar (March 2007), PITA (August 2008), RNA22 (May 2008), RNAhybrid (version 2.1) and Targetscan (version 5.1), miR-99a targets were predicted in 10 datasets (DIANA-mT, miRanda, miRDB, miRwalk, RNAhybrid, PICTAR4, PICTAR5, PITA, RNA22, TargetScan) for binding sites on 3′UTR of mRNA. For correlations, the expression of miR-99a and interested mRNA was analyzed in 40 pairs of OSCC patients (GSE37991) using Pearson correlation.
Drug treatment
Cells were seeded and treated with 5-Aza-dC (5 μM; Sigma). After incubating for 96 hours with a change of culture medium every 24 hours, cells were collected for further analysis.
Cells were seeded overnight and refreshed with serum-free medium, then subjected to serum starvation for 12 hours. After starvation, cells were treated with vehicle, 10% fetal bovine serum (FBS) or 10 nM IGF1 (Sigma) for different durations. For experiments involving inhibition of signaling pathways, cells were cultured in the presence of PD98059 (Sigma) or LY294002 (Sigma), specific inhibitors of MAPK kinase and PI3K, respectively, for 1 hour prior to IGF1 stimulation.
Statistical analysis
Data are presented as the mean ± standard error of the mean (SE) from at least 2 independent experiments. For comparisons between 2 groups, the differences between the groups were analyzed using 2-tailed student’s t test. Linear regression, Pearson correlation, and Spearman correlation were used to evaluate the correlation between 2 variants. Analysis was performed using GraphPad Prism version 5.01 (GraphPad Software, La Jolla, CA, USA). For all comparisons, p < 0.05 was considered statistically significant.
Acknowledgments
The authors would like to thank Wei-Chi Shiah and Dr Hung-Che Chiang for providing reagents and technical support, Dr Michael Hsiao for donating the miR-99a expressing construct, Dr Enzo Lalli for donating the plasmids for the analysis of luciferase reporter assays, Dr Lu-Hai Wang for proving the IGF1R construct, Dr Shih-Sheng Jiang for assistance with statistical analysis, and Dr Shankuang Lin for critical review of the manuscript. We are also grateful to the Tissue Bank, Research Center of Clinical Medicine, National Cheng Kung University Hospital for providing clinical samples.
Grant support
This study was supported by grants NSC 99-2314-B-006-018-MY3, NSC 100-2320-B-400-008, NHRI CA-102-PP-03, NHRI CA-101-PP-03, and DOH102-TD-C-111-004 from National Science Council, National Health Research Institutes and Department of Health, Taiwan.
Competing interests
The authors declare that they have no conflict of interest.
Authors’ contributions
YCY carried out the molecular and cellular biology studies and helped to draft the manuscript. SGS performed the bioinformatic analysis and qRT-PCR. HCC performed Western blots. YMH performed the immunofluorescence. JRH collected the information of OSCC patients and helped to draft the manuscript. JYC participated in design of the study and collection of OSCC patients. WCH helped to draft the manuscript. CTL participated in establishment of cell lines and helped to draft the manuscript. AJC participated in establishment of cell lines. YCL participated in establishment of cell lines. YWC participated in the design of the study and coordination and drafted the manuscript. All authors read and approved the final manuscript.