Association between High miR-211 microRNA Expression and the Poor Prognosis of Oral Carcinoma

Abstract

MicroRNAs (miRNAs) are non-coding RNAs that play roles in gene silencing and may be involved in tumorigenesis. miR-211 was mapped to chromosome 15q13, a locus frequently altered in cancers. The role of miR-211in carcinogenesis has not been clearly defined, however. This study investigated the pathogenetic implications of miR-211 in oral carcinogenesis. An association was found between higher miR-211 expression and the most advanced nodal metastasis, vascular invasion, and poor prognosis of oral carcinoma. The function of enforced miR-211 expression in oral carcinoma cells was confirmed by the repression of LacZ in a reporter plasmid via miR-211 targeting. Enforced miR-211 expression significantly increased the proliferation, migration, and anchorage-independent colony formation of oral carcinoma cells, while it enhanced the tumorigenicity of only SAS high-grade oral carcinoma cells, but not OECM-1 non-tumorigenic cells. The findings suggest that high miR-211 expression may be associated with the progression of oral carcinoma and poor patient outcomes.

INTRODUCTION

Oral squamous cell carcinoma (OSCC) is a worldwide disease (Chen et al., 2004; Marcus et al., 2004; Liu et al., 2006; Shieh et al., 2007). Identification of genetic events involved in the pathogenesis of OSCC may be useful in the development of therapeutic targets. MicroRNA (miRNA) is a short, non-coding RNA that regulates gene expression by controlling mRNA translation (Dalmay and Edwards, 2006), either by translational repression of the targeted mRNA or by enhancing its degradation through an RNA interference mechanism (Dalmay, 2008). Various evidence suggests that miRNAs may be involved in both tumorigenesis and tumor suppression. For instance, an miRNA cluster, miR-17-92 (which includes miR-17 to miR-20 and miR-92), is overexpressed in certain cancers and is associated with the inhibition of apoptosis and an increased proliferation of cancer cells ( Hayashita et al., 2005; Dalmay, 2008). In contrast, the down-regulation of many miRNAs in tumors suggests that some may act as tumor suppressors, such as miR-15a and miR-16, which target Bcl-2 (Cimmino et al., 2005).

Alterations in miRNA expression have been found in cancers from different organs (Hayashita et al., 2005; Meng et al., 2006; Roldo et al., 2006; Volinia et al., 2006; Chen and Stallings, 2007; Dalmay, 2008). miR-98 was found to regulate HMGA2 expression in head and neck squamous cell carcinoma (HNSCC) cells (Hebert et al., 2007). miR-211 and miR-204 are in the same miRNA family, which might regulate cellular phenotypes by affecting targeted genes (Dalmay and Edwards, 2006; Volinia et al., 2006). Trpm is a family of cation channels acting as calcium and redox sensors (Bodding, 2007). miR-211 was localized in intron 6 of Trpm1 gene at 15q13–q14, which is a frequent locus of loss in neoplasms, including HNSCC (Feenstra et al., 1999; Natrajan et al., 2003; Lipton and Tomlinson, 2006; Poetsch and Kleist, 2006). This study investigated the clinical and functional implications of miR-211 in OSCC.

MATERIALS & METHODS

Participants

This study was approved by the institutional review board at Mackay Memorial Hospital, Taipei. Thirty-six persons with primary OSCC tumors (Table) and nine persons with oral pre-cancerous lesions (OPLs) (Appendix Table) gave informed consent for the use of tissue harvested at surgery.

miRNA Extraction and Stem-loop Quantitative (Q) RT-PCR

Total RNA was purified by acid-phenol/chloroform (Ambion, Austin, TX, USA) extraction. We subsequently used a mirVana™ PARIS™ Kit (Ambion) to isolate miRNA from the total RNA. RNA input was normalized with GAPDH. We used a TaqMan MicroRNA Assay kit (Applied Biosystems, Foster City, CA, USA) to quantitate miR-211 and miR-204 expression according to the manufacturer’s instructions. In brief, looped RT primers specific for each miRNA were used to produce a primer/mature miRNA-chimera that extended the 3′ end of the miRNA. The resulting amplicon presented a template amenable to subsequent TaqMan quantitative assays. Polymerase chain-reaction (PCR) was performed on the ABI Prism^® 7700 Sequence Detector system (Applied Biosystems), with let-7a miRNA as an internal control because of its stable expression across human tissues and cell lines, as suggested by the manufacturer and other investigators (Jiang et al., 2005). RNU19, a small RNA encoded in the intron of MATR3 in chromosome 5, was also used as an internal control (Chen and Stallings, 2007). We used the comparative threshold cycle (Ct) method to measure relative changes in miRNA expression. Ct is the number of the cycle at which the fluorescence signal of the amplification plot passes the fixed threshold. ΔCt = Ct(miR-211or miR-204) - Ct(Let7a or RNU19). ΔΔCt(OSCC or OPL) = ΔCt(OSCC or OPL) ΔCt (matched non-cancerous mucosa). Expression fold value = 2^−ΔΔCt. Relative quantity is the fold change of the test sample compared with the baseline sample (Cheng et al., 2005). All experiments were done in triplicate. A negative control without a template was run in parallel to assess the overall specificity of the reaction.

Construction of Plasmids

The sequences of mir-211 were amplified from genomic DNA with primers 5′-cgcctgggatccgagaatcagatctcgaagaatacattggtc-3′ and 5′-cttaaaggtaccttgatattttcaatctttaggaatatggtg-3′. Following digestion by BamH I and Kpn I restriction enzymes, the PCR products were cloned into a lentivirus vector, pLV-EF1a-GFP (a kind gift from Dr. W.-Y. Hu), to produce pLV-EF1a-GFP-miR-211 plasmid, in which the enforced GFP and miR-211 expression are driven by elongation factor 1α (EF1α) promoter. In addition, antisense sequences complementary to mature miR-211 sequences were cloned into a pCMV-LacZ reporter plasmid containing a multiple cloning site at the 3′-end of the lacZ coding sequence, to serve as a target for miR-211. The integrity of inserted sequences was confirmed by plasmid sequencing, and the plasmid was designated pCMV-LacZ-miR-211.

Cell Culture and Lentivirus Infection

SAS cells are high-grade tumorigenic OSCC cells, while OECM-1 and OC3 cells are non-tumorigenic OSCC cells (Lin et al., 2005; Shieh et al., 2007). Normal human oral keratinocytes (NHOKs) were cultured as previously described (Lu et al., 2006). pLV-EF1a-GFP vectors or pLV-EF1a-GFP-miR-211 plasmids were co-transfected with pCMVΔ8.2 (GAG-POL DNA), the vesicular stomatitis virus envelope plasmid pMD.G to 293T cells, to produce lentiviruses. Two days after transfection, the viral supernatants were harvested. SAS, OECM-1, and NHOKs were infected with the lentiviruses. The infectivity was determined by fluorescence detection. The expression of miRNAs was determined by stem-loop Q-RT-PCR assays.

Co-transfection of reporter plasmids with pCMV-Luc into lentivirus-transduced cells was performed with lipofectamine 2000 (Life Tech., Gaithersburg, MD, USA). Following normalization by the assessment of luciferase activity, lacZ gene expression, as represented by β-galactosidase activity, would be repressed if miR-211 is expressed and targeted to the complementary sequence.

Statistical Analysis

We used an unpaired t test to assess differences in miR-211 expression among the various clinicopathologic parameters. We used the Mann-Whitney test to assess the differences between experimental groups. We used a Kaplan-Meier survival analysis to evaluate the relationship between miR-211 expression and outcome. We performed a correlation analysis to determine the relationship between miR-211 expression and miR-204 expression in samples. A p value of < 0.05 was considered statistically significant. The methodologies for the other analyses are described in the APPENDIX.

RESULTS

Participants

Of the 36 persons with OSCC, 16 (44%) had cervical lymph node metastases and five (14%) had vascular invasion, according to histopathologic evaluation (Table). During the follow-up period, a median of 12.5 mos, ten (28%) members of the study group died. The expression of let-7a and RNU19 was very similar in tested samples. Thus, we used let-7a as an internal control.

miR-211 Expression in Microdissected Cells

The microdissected cells from OSCC, OPL (Appendix Table), and paired non-cancerous mucosa were processed for stem-loop Q-RT-PCR analysis. There were 29 (81%) OSCC samples exhibiting a decrease in miR-211 relative to their paired mucosa (Fig. 1a ). The remaining tumors exhibited higher miR-211 expression than corresponding non-cancerous mucosa. All OPL samples exhibited decreased miR-211 expression relative to their paired mucosa (Fig. 1a ). No significance difference in miR-211 expression level was found between OPL and OSCC, however. Tumors with N2 nodal metastasis or vascular invasion had significantly higher miR-211 expression than less aggressive tumors (Figs. 1a, 1b ). Survival analysis also revealed that persons having miR-211 expression higher than the median or higher in tumors than in paired non-cancerous mucosa had worse chances of survival than the other groups (Fig. 1c ). The expression of miR-211 was not associated with any other clinical parameter (not shown). Stem-loop Q-RT-PCR analysis for miR-204 expression was performed on tumor tissue pairs. It showed decreased miR-204 expression in 28 (78%) OSCC tumors compared with paired non-cancerous mucosa. A correlation between the expression of miR-211 and miR-204 was found in these samples (Fig. 1d ).

Enforced miR-211 Expression in Cells

SAS, OECM-1, and OC3 cells had lower endogenous miR-211 expression levels relative to NHOKs (Fig. 2b ), similar to those in tumors from our group. Lentiviral supernatants for enforced miR-211 expression were prepared. Five days after lentiviral infection, green fluorescence was detected in nearly all SAS cells, indicating efficient infection (Fig. 2a , upper). The stable cells were designated SAS-miR-211 and SAS-GFP (controls). Remarkable up-regulation of miR-211 in SAS-miR-211 relative to controls was demonstrated (Fig. 2b ). There was also a significant decrease in β-galactosidase activity for ~ 45% of stable SAS cells when transfected with miR-211 reporter plasmid. The results suggested the eligibility of miR-211 for targeting their antisense sequences and repressing LacZ expression (Fig. 2c ). A site-by-site experiment involving transfection of the miR-31 reporter plasmid available in our laboratory exhibited the same reporter activity in SAS-miR-211 and SAS-GFP cells (Fig. 2c ).

OECM-1-miR-211 and OECM-1-GFP cells were also established by lentiviral infection. The stable cells exhibited similar infectivity (Fig. 2a , middle). Marked up-regulation of miR-211 in OECM-1-miR-211 relative to controls was demonstrated (Fig. 2b ). There was also a significant decrease in β-galactosidase activity for ~ 45% of OECM-1-miR-211 cells after transfection with miR-211 reporter plasmid (Fig. 2d ), which validated the function of enforced miR-211expression.

Two distinctive NHOK lines (NHOK1 and NHOK2) were established by lentiviral infection. NHOK1-miR-211 and NHOK1-GFP exhibited similar infectivity (Fig. 2a , lower). Both NHOKs having enforced miR-211 expression exhibited only a slight up-regulation of miR-211 relative to controls (Fig. 2b ). Owing to the low transfection efficiency of these primary cells, the reporter assay for NHOK was not available.

Phenotypic Impact of Enforced miR-211 Expression

SAS or OECM-1 cells having enforced miR-211 expression exhibited significantly greater proliferation, migration, and anchorage-independent colony formation than did controls (Figs. 3a-3c ; Appendix Figs. 1a-1c). SAS-miR-211 cells also exhibited enhancement of xenograft tumor growth compared with control cells in nude mice (Fig. 3d ); however, OECM-1-miR-211 cells did not acquire such phenotypes in relation to controls (Appendix Fig. 1d).

No difference in growth rate and apoptosis rate was found between NHOK1-GFP and NHOK1-miR-211 (Appendix Figs. 2a, 2b). Late-RT analysis showed that NHOK2-miR-211 or NHOK2-GFP had ~ 10–30% higher viral integration than SAS-GFP or SAS-miR-211 cells (Appendix Fig. 2c). Although only a small amount of exogenous miR-211 was expressed, NHOK2-miR-211 exhibited a significant increase in proliferation and migration relative to NHOK2-GFP (Appendix Figs. 2d, 2f). NHOK2-GFP and NHOK2-miR-211 cells had similar apoptotic cellular fractions (Appendix Fig. 2e). The results for other analyses are described in the APPENDIX.

DISCUSSION

Alterations of miRNAs in various types of cancers have been reported (Hayashita et al., 2005; Meng et al., 2006; Volinia et al., 2006; Chen and Stallings, 2007), while little has been discovered about miRNA status in OSCC (Hebert et al., 2007). Therefore, this study explored the potential roles of miR-211 in the tumorigenesis of OSCC. We identified a decrease in miR-211 in the vast majority of primary OSCC tumor tissues relative to their non-cancerous counterparts, which may be consistent with the frequent loss of the 15q13–q14 allele in HNSCC (Feenstra et al., 1999; Poetsch and Kleist, 2006). miR-211 and miR-204 have been classified as part of the same miRNA family, owing to the high agreement in sequence (Dalmay and Edwards, 2006; Volinia et al., 2006). miR-204 expression was also found to be decreased in OSCC, and this decrease is correlated with the decrease of miR-211. Because this study also identified a conspicuous decrease of miR-211 expression in OPL, the results may substantiate the decrease of miR-211 expression in early carcinogenesis. A future study should elucidate whether genomic loss of the 15q locus could underlie the decrease of miR-211 expression in OPL and OSCC.

Due to the high endogenous miR-211 expression in NHOKs, we speculated that miR-211 expression could be important for the maintenance of non-neoplastic phenotypes in these primary cells. Since the viral integration in NHOKs was better than that in OSCC cells, our analysis suggested that the low level of enforced miR-211 expression in NHOK could be associated with the lesser activation in the EF1α promoter. Heterogeneity exists among different NHOKs, and more conspicuous miR-211 expression may be needed for some NHOKs to influence their phenotypes. Nevertheless, a slightly increased miR-211 expression appreciably affected growth and migration in certain NHOKs. Owing to low transfection efficiency, an initial attempt to deliver oligonucleotide blocker for the knockdown of miR-211 expression in NHOKs was not successful (not shown). Thus, the association between the miR-211 expression in NHOK and the resistance to tumorigenesis was unclear. Trpm family genes are involved in malignancies (Bodding, 2007). It would also be useful to determine the synchronized alterations of 15q13–q14, Trpm1, and miR-211 in oral carcinogenesis. Other factors that could decrease miR-211 expression during the neoplastic process of oral epithelial cells remain to be defined.

Overexpression of miR-211 has been found in insulinomas (Roldo et al., 2006). Inhibition of miR-211 and miR-204 decreased the growth of Hela cells (Cheng et al., 2005). SAS-miR-211 cells exhibited remarkable transformative advantages over control cells; however, a strong exogenous miR-211 expression induced OECM-1 tumorigenicity to only a limited extent. Since miR-211 was profoundly decreased in OSCC cells and tissues, the vigorous expression of miR-211 in SAS cells may surpass the biological regulations, because the OSCC cell lines being examined maintain relatively low miR-211, compelling evidence that the oncogenic role of miR-211 in oral carcinogenesis is still lacking. Identification of the concordance between miR-211 expression level and the malignant potential in multiple OSCC cell lines is required for further clarification of the oncogenic potential of miR-211. This study identified that more aggressive tumors had higher levels of miR-211 compared with levels in their less aggressive counterparts. If the allelic loss of 15q13–q14 is conducive to the establishment of OSCC in the early stage of oral carcinogenesis, it is plausible that expression of the remaining miR-211 locus could be activated later, playing a crucial role in the subsequent progression or maintenance of the established tumors. Moreover, whether miR-211 expression is more specifically involved in the advances of late-stage tumors, or other accessory factors are required for tumor establishment, deserves further elucidation.

miR-211 and miR-204 were predicted to target multiple genes, including Angiopoietin-1, that may regulate angiogenesis (Dalmay and Edwards, 2006). Bcl-2 is a target for several miRNAs (Cimmino et al., 2005). Owing to the inconsistent levels of Angiopoietin-1 and Bcl-2 in SAS-miR-211 and OECM-1-miR-211 cells relative to controls, these two proteins may not be targets of miR-211 in OSCC cells. Tcf-12 is a member of the Tcf/β-catenin transcriptional complex, with unknown activity in oral tumorigenesis (Yang et al., 2006). Our preliminary analysis also excluded Tcf-12 as a miR-211 target in OSCC. The increase of cyclin D1 in OSCC cells with enforced miR-211 expression seemed to be correlated with their proliferative enhancement.

This study demonstrated that miR-211 expression could be a valuable prognostic indicator of OSCC. Due to the discrepancies in miR-211 expression during various stages of oral carcinogenesis in tissues and cell lines, and the limited tumor induction in OECM-1 cells by miR-211 transduction, it is still difficult to conclude that miR-211 is oncogenic. Further resolution of the mechanisms involved in miR-211 regulation, identification of its targets and characterization of their functions, and clarification of the behavior of miR-211 in various types of cancer may eventually elucidate its pathogenetic roles.

Table.

Clinicopathological Features of OSCC

Parameters	OSCC
Age (yrs)	40–77
Mean ± SD	52.0 ± 8.1
Follow-up (mos)	2.2 – 41.1
Mean ± SD	14.3 ± 8.0
Site
Gingiva	12
Buccal mucosa	8
Tongue	8
Others	8
Differentiation
Well	11
Moderate and Poor	25
Stage
I–III	7
IV	29
Primary tumor
T1–T3	6
T4	30
Lymph node metastasis
N0	20
N1	6
N2	10
Vascular invasion
Absence	31
Presence	5
Survival status
Alive	26
Dead	10

Figure 1.

miR-211 expression in microdissected cells. (a) We performed stem-loop Q-RT-PCR analysis to detect expression of miR-211 in microdissected cells. The scattered dot plots of miR-211 expression in OPL and OSCC cells appear in the left columns. Each dot represents the mean value of triplicate analyses. The miR-211 expression of N0 plus N1 tumors and N2 tumors are shown as box- and whisker-plots. miR-211 expression is higher in N2 tumors than in the other tumors (p = 0.011). (b) miR-211 expression is significantly higher in tumors with vascular invasion than in the other tumors (p = 0.002). Box- and whisker-plot. In (a,b), horizontal lines, median; +, mean. (c) Kaplan-Meier analysis. The tumors were divided into two groups with the median or 0 of ΔΔCt as cut-offs. Tumors with higher miR-211 expression (Δ ΔCt > median) and tumors with miR-211 higher than matched non-cancerous mucosa (Δ ΔCt > 0) were significantly associated with worse survival. (d) Scattered dot plot of miR-211 expression and miR-204 expression in all OSCC samples, showing a significant correlation between the expression of miR-211 and miR-204. Dot lines indicate 95% confidence interval. **p < 0.01; unpaired t test.

Figure 2.

Enforced miR-211 expression in cells. (a) Green fluorescence in SAS-GFP and SAS-miR-211 cells (upper), OECM-1-GFP and OECM-1-miR-211 cells (middle), and NHOK1-GFP and NHOK1-miR-211 cells (lower). Bars, 100 μm. (b) Stem-loop Q-RT-PCR analysis detected up-regulation of miR-211 in SAS-miR-211, OECM-1-miR-211, NHOK1-miR-211, and NHOK2-miR-211 cells compared with controls. SAS-GFP is the baseline. (c) Reporter assay in SAS cells. Each miR-31 or miR-211 reporter plasmid was co-transfected with pCMV-Luc to SAS-GFP and SAS-miR-211 cells. The luciferase activity was used to normalize transfection efficiency. The β-galactosidase activity in cells transfected with empty pCMV-LacZ vector was used to normalize β-galactosidase activity. Compared with controls, SAS-miR-211 cells having specific miR-211 targeting on the antisense sequences of miR-211 significantly repressed β-galactosidase activity (p = 0.025). The antisense sequences of miR-31 did not affect β-galactosidase activity in SAS-miR-211 cells. (d) Reporter assay in OECM-1 cells showing significant repression of β-galactosidase activity in OECM-1-miR-211 cells in relation to controls (p = 0.005). Data shown are mean ± SE from triplicate analyses (in b) and two distinctive triplicate analyses (in c,d). *p < 0.05, **p < 0.01; Mann-Whitney test.

Figure 3.

Phenotypic changes of SAS cells with enforced miR-211 expression. (a-c) Cell proliferation, migration, and anchorage-independent colony formation, respectively. Significant increase in proliferation (at day 6 to day 8), migration, and anchorage-independent colony formation was noted in SAS-miR-211 cells relative to SAS-GFP cells. SAS-miR-211 cells grew to a plateau phase after day 8. Data shown are mean ± SE from two distinctive triplicate analyses. (d) Tumorigenesis assay showed greater tumorigenicity in SAS-miR-211 xenografts than in SAS-GFP xenografts following the injection of 1 x 10⁵ cells (solid lines) or 2.5 x 10⁵ cells (dotted lines). Data shown are mean ± SE from 5 or 6 nude mice. *p < 0.05; **p < 0.01; ***p < 0.001; Mann-Whitney test.

Footnotes

Notes

A supplemental appendix to this article is published electronically only at .

#

authors contributing equally to this paper

Acknowledgements

This study was supported by grants NS-96-2628-B-010-033-MY3 and NSC-96-2314-B-195-018-MY3 from the National Science Council, by the Aim for the Top University Plan from the Department of Education, and by Grant 9117 from Taipei Mackay Hospital, Taiwan.

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