MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers

Abstract

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation −0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation −0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3′-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

Introduction

Annexin A1 (ANXA1), also known as lipocortin or p35, is a well-characterized member of the calcium- and phospholipid-binding protein family of annexins and is involved in modulating arachidonic acid metabolism and the epidermal growth factor receptor tyrosine kinase pathway. Cloned as a phospholipase A2 inhibitor (Wallner et al., 1986), ANXA1 was identified as a critical mediator of apoptosis (Solito et al., 2001) and subsequently implicated in the actions of glucocorticoids, including the inhibition of cell proliferation and regulation of cell migration (Parente and Solito, 2004). In addition, ANXA1 is also shown to be involved in varied biological functions including membrane trafficking, exocytosis, signal transduction, cell differentiation and apoptosis (Gerke and Moss, 2002).

Suppression or loss of ANXA1 expression reported in different cancers such as prostate (Kang et al., 2002), breast (Shen et al., 2006), esophageal cancers (Paweletz et al., 2000; Xia et al., 2002; Hu et al., 2004) and B-cell non-Hodgkin's lymphoma (Vishwanatha et al., 2004) attributes tumor suppressive function to ANXA1 in these cancers. Furthermore, ANXA1 overexpression promoted caspase-mediated apoptosis in bronchoalveolar cells (Debret et al., 2003) and macrophages (Solito et al., 2001) and facilitated H2O2-induced apoptosis in thymocytes (Sakamoto et al., 1996). In prostate cancer cell lines, ANXA1 overexpression resulted in reduced cell viability and colony formation, enhanced apoptosis and suppressed response to epidermal growth factor signaling (Hsiang et al., 2006). The above-mentioned reports suggest a proapoptotic and tumor suppressive role for ANXA1.

In contrast to the suppressed expression of ANXA1 observed in the above-mentioned cancers, increased ANXA1 expression has been reported in pancreatic (Bai et al., 2004), hepatic (Masaki et al., 1996), glial (Johnson et al., 1989) and stomach cancers (Sinha et al., 1998), suggesting that the functional role of ANXA1 may be tissue- and cell type-specific. These findings further highlight the complexity and highly specific nature of ANXA1 physiological functions.

In esophageal cancers, several published reports confirm that the loss of ANXA1 is an early event in tumorigenesis (Paweletz et al., 2000; Xia et al., 2002; Hu et al., 2004). Western blotting and immunohistochemical analysis showed a drastic decrease or complete loss of ANXA1 levels in esophageal tumors and high-grade dysplasia when compared to the paired normal epithelium, implying that ANXA1 plays a functional role in maintaining the normal status of esophageal epithelium (Paweletz et al., 2000). A comparative protein profiling study also identified lower ANXA1 protein levels in esophageal carcinomas than in the paired normal epithelia (Xia et al., 2002). In their study, ANXA1 levels were found to be suppressed in 75% of well-differentiated, 94% of moderately differentiated and 100% of the poorly differentiated tumors analysed, thus correlating with the extent of tumor differentiation. A significant correlation was also observed between ANXA1 expression and the status of tumor differentiation, with the ANXA1 levels increasing with the extent of tumor differentiation. In addition, studies from our laboratory have shown that ANXA1 was downregulated in esophageal cancers resistant to preoperative chemoradiotherapy (Luthra et al., 2006).

Little is known about the underlying mechanisms responsible for the loss or decrease of ANXA1 expression in cancers. In esophageal carcinomas, allelic loss at the ANXA1 locus has been observed, but this loss does not correlate with ANXA1 protein loss, suggesting that other mechanisms such as epigenetic silencing or microRNA (miRNA)-mediated regulation are at play (Hu et al., 2004). However, CpG islands, the potential sites of gene methylation, were not present in either the promoter or the coding region of ANXA1, thus eliminating methylation as a possible silencing mechanism (Paweletz et al., 2000). Therefore, we explored miRNA-mediated repression as a potential mechanism of reduced ANXA1 expression.

MicroRNAs are a class of small (≈19–25 nt), non-coding regulatory RNAs that regulate gene expression by complementary base pairing with the 3′-untranslated region (UTR) of target mRNAs and causing their degradation or suppressing mRNA translation, which consequently leads to a decrease in target protein levels (Ambros and Chen, 2007; Rana, 2007). In cancer and metastasis, several miRNAs have been implicated in the deregulation of gene expression, thus establishing them as a relatively new and important class of oncogenes and tumor suppressors (Esquela-Kerscher and Slack, 2006). We investigated whether decreased ANXA1 expression was mediated by miRNAs and identified miR-196a, miR-196b and miR-584 as potential miRNAs to target ANXA1 mRNA using the Sanger miRNA registry at http://microrna.sanger.ac.uk/, which predicts the potential miRNAs for a given target based on the extent of their base complementarity to 3′-UTR of target mRNA. Correlating the levels of miRNA with ANXA1 levels in cancer cell lines and in esophageal cancers, we identified miR-196a as the most likely candidate targeting ANXA1. We then performed experiments to demonstrate direct targeting of ANXA1 mRNA by mir-196a. Further, we discovered that increased miR-196a expression is a characteristic molecular change in esophageal cancers and investigated the effect of enhanced levels of miR-196a on growth characteristics of cancer cell lines.

Results

Computational analysis

The Sanger database predicts the potential miRNAs that target mRNA of any given gene by comparing the complementarity of the miRNA sequence to the 3′-UTR of the mRNA. The potential is scored depending on the extent of complementarity between the sequences at the 5′-end of the miRNA and the binding region in the 3′-UTR of the mRNA. The database predicted miR-584, miR-196a and miR-196b as potential miRNAs to target ANXA1 mRNA, with miR-584 showing the highest complementarity score. The base complementarities of these three miRNAs to the target 3′-UTR region of ANXA1 mRNA are shown in Figure 1.

Figure 1
figure1

Sequence complementarity of miRNAs 196a (a), 196b (b) and 584 (c) to the 3′-UTR of ANXA1 mRNA. Nucleotide sequences of miR-196a, -196b and -584, computationally predicted by the Sanger miRNA registry as potential miRNAs targeting ANXA1 mRNA. The base complementarity between the three miRNAs and 3′-UTR of ANXA1 is schematically shown. ANXA1, annexin A1; miRNAs, microRNAs.

Correlation of miR-196a, miR-196b and miR-584 levels with ANXA1 expression in cancer cell lines

Western blot analysis of the 12 cell lines representing esophageal, breast and endometrial cancers revealed that ANXA1 levels varied considerably among the cell lines, with a few cell lines showing negligible ANXA1 expression (Figure 2a). Levels of ANXA1 mRNA measured by real-time quantitative PCR (qPCR) followed the pattern seen at protein level indicating that ANXA1 mRNA levels are a good measure of the protein levels (Figure 2b).

Figure 2
figure2

Correlation of miR-196a, -196b and -584 to ANXA1 in esophageal, breast and endometrial cancer cell lines. (a) Western blotting analysis of the comparative levels of ANXA1 protein levels in esophageal, breast and endometrial cancer cell lines. Real-time qPCR analysis of ANXA1 mRNA levels (b) shows inverse correlation with the levels of miR-196a (c) (correlation coefficient=−0.66, P=0.019) and miR-196b (d) (correlation coefficient=−0.25, P=0.428) in the 12 cell lines comprising of esophageal (left panels), breast (middle panels) and endometrial cancers (right panels). (e) No negative correlation was observed between ANXA1 mRNA levels and the miR-584 levels (correlation coefficient=0.228, P=0.475). Note that relative expression levels of microRNA were multiplied by a factor of 1000 to simplify data representation. The levels of ANXA1 protein corresponded with the observed pattern of ANXA1 mRNA levels in the cell lines. ANXA1, annexin A1; qPCR, quantitative PCR.

miR-196a levels correlated inversely with the ANXA1 mRNA levels across all the 12 cell lines tested (Figure 2c). In the esophageal cancer cell lines OE33 and SEG-1, the ANXA1 mRNA levels were high (15.47 and 17.49, respectively), which corresponded to low miR-196a (0.046 and 0.041, respectively). In contrast, BIC-1 and SKGT-5 esophageal cancer cells had low ANXA1 mRNA (3.23 and 0.0026, respectively) and correspondingly high levels of miR-196a (0.13 and 0.11, respectively) (Figures 2b and c, left panels). The breast cancer cell lines, T47D and MDA-453, had highly suppressed ANXA1 levels (0.06) with comparatively high levels of miR-196a (relative expression 0.1), whereas MDA-231 and MDA-435 cells, which exhibited elevated ANXA1 mRNA levels (>20.0), showed lower levels of miR-196a (relative expression 0.03) (Figures 2b and c, middle panels). Similarly, in endometrial cell lines HEC-1A and HEC-1B, high levels of ANXA1 mRNA (>20.0) corresponded with very low levels of miR-196a (0.001), whereas lower levels of ANXA1 mRNA (<12.0) in Ishikawa and AM3CA cells correlated with relatively higher levels of miR-196a (0.003) (Figures 2b and c, right panels), confirming mutually inverse correlation between miR-196a and ANXA1 mRNA. Pearson's correlation analysis, used to examine the correlation between miR-196a and ANXA1 mRNA in all 12 cell lines, yielded a correlation of −0.66, with a P-value of 0.019, indicating a statistically significant negative linear correlation between levels of ANXA1 mRNA and miR-196a. This was also confirmed by a simple analysis of variance linear regression model (P=0.019).

An inverse correlation was also observed between ANXA1 mRNA levels (Figure 2b) and miR-196b (Figure 2d). However, the correlation was not statistically significant (Pearson's correlation of −0.25, P=0.428). Even though miR-584 had the highest predictive score of the miRNA tested to potentially target ANXA1, it showed no inverse correlation with ANXA1 mRNA (Figures 2b and e), indicating that this miRNA is unlikely to target ANXA1.

Inverse correlation between miR-196a and ANXA1 mRNA levels in esophageal cancers

To further confirm the negative correlation observed between miR-196a and ANXA1 mRNA, their levels were measured in 10 esophageal tumor tissues with varied levels of ANXA1 mRNA (Figures 3a and b). As in the cell lines, an inverse correlation was observed between ANXA1 mRNA and miR-196a level in the tumor specimens. Tumors T1 through T5 with low levels of ANXA1 mRNA showed relatively high miR-196a levels. In contrast, tumor specimens T6 through T10 with high levels of ANXA1 mRNA showed very low levels of miR-196a. The inverse correlation was statistically significant (Pearson's correlation −0.64, P=0.047).

Figure 3
figure3

Inverse correlation of miR-196a and ANXA1 in esophageal cancers. Real-time qPCR analysis of the levels of ANXA1 mRNA (a) and miR-196a (b) in a set of 10 esophageal tumor samples. Tumors with relatively low levels of ANXA1 mRNA (T1 to T5) showed comparatively high levels of ANXA1, whereas those with relatively higher levels of ANXA1 mRNA (T6 to T10) consistently showed lower levels of miR-196a, confirming the inverse correlation (correlation coefficient=−0.64, P=0.047). (c) ANXA1 mRNA levels were consistently suppressed in esophageal adenocarcinoma tissue samples in comparison to their paired normal mucosa as seen in a set of 10 paired normal and tumor tissue samples. (d) A distinct increase of miR-196a levels (10- to 100-fold) was consistently observed in the esophageal tumors in comparison to their respective paired normal mucosa. The inverse correlation observed in each esophageal tissue sample tested reinforced the possibility of miR-196a as a candidate miRNA targeting ANXA1 mRNA. ANXA1, annexin A1; qPCR, quantitative PCR; miRNAs, microRNAs.

Loss of ANXA1 expression is a frequent molecular change observed in esophageal cancer (Paweletz et al., 2000; Xia et al., 2002; Hu et al., 2004). To further confirm this observation and to investigate the possible role of miR-196a in this molecular change, the levels of ANXA1 mRNA and miR-196a were analysed in paired normal (esophageal or gastric) and tumor tissues from 10 patients. ANXA1 mRNA levels were indeed suppressed in the esophageal tumors compared with the normal tissue (Figure 3c, P=0.004). Interestingly, a concomitant elevation of the miR-196a levels was observed in the tumor tissue, with miR-196a levels increasing by 10- to 100-fold compared to those in corresponding normal tissue (Figure 3d, P=0.003). This drastic increase in the tumor levels of miR-196a indicated not only a signature change of this miRNA in esophageal cancers but also the potential role of this miRNA in decreased ANXA1. In light of this inverse correlation between miR-196a and ANXA1 mRNA, further experimental efforts were focused in establishing ANXA1 mRNA as a direct target of miR-196a.

RNA mimics of miR-196a suppress ANXA1 mRNA levels in cancer cell lines

To confirm that miR-196a specifically regulates ANXA1, the gain-of-function effects of miR-196a on ANXA1 mRNA were studied. To achieve elevated levels of miR-196a in cells, two mimics of miR-196a were transfected into esophageal, endometrial and breast cancer cell lines and the consequent effect on the levels of ANXA1 mRNA was monitored by real-time qPCR and western blotting analysis. The miRNA mimics were synthetic double-stranded RNA oligonucleotides, which on delivery generated higher levels of miR-196a in the cells, thus amplifying its effects. It is of interest to note that mature miR-196a can be generated by two distinct precursor RNAs that originate from two different loci, one located on chromosome 17 and the other on chromosome 12. However, the final mature miR-196a sequence generated from both precursors is identical. To determine if the source of mature miRNA affected the target levels, we used both mimic 196a1 and mimic 196a2, which corresponded to the two different precursor sequences.

As shown in Figure 4a, transfection of cancer cells with miR-196a mimics resulted in a reproducible decrease of ANXA1 mRNA by 60–65% in BIC-1, 30–35% in SEG-1, 30–35% in MDA-231 and 40–45% in HEC1B cells when compared to the respective controls that were transfected with a nonspecific negative control RNA. The decrease in the ANXA1 mRNA levels was accompanied by a decrease in ANXA1 protein levels in these four cell lines (Figure 4b). On the whole, these experiments demonstrated that increasing the level of miR-196a resulted in concomitant suppression of the mRNA as well as protein levels of ANXA1 in cell lines from three different cancer types. This finding provides further evidence that ANXA1 is indeed a target of miR-196a.

Figure 4
figure4

Transfection with two RNA mimics of miR-196a decreases ANXA1 mRNA and protein levels. (a) Increasing levels of miR-196a in esophageal (BIC-1, SEG-1), breast (MDA-231) and endometrial (HEC-1B) cancer cell lines by the transfection of two different mimics of miR-196a resulted in a reproducible decrease of ANXA1 mRNA levels as studied by real-time PCR analysis. (b) Consistent decrease of ANXA1 protein levels was also observed in these cell lines on transfection of the two RNA mimics of miR-196a. The western blotting data for ANXA1 and control β-actin are shown and the quantification of relative levels of ANXA1 protein levels (ANXA1 versus β-actin) under each condition is provided above. ANXA1, annexin A1; qPCR, quantitative PCR.

RNA mimics of miR-196a directly target the 3′-UTR of ANXA1 mRNA

To evaluate if the negative regulation of ANXA1 by miR-196a was a direct effect, we subcloned a 284-bp fragment of the 3′-UTR region of ANXA1 mRNA that included the predicted miR-196a recognition site (Figure 1a) into a luciferase reporter plasmid designated as PGL3-ANXA1-LUC (Figure 5, upper panel). This luciferase reporter construct was cotransfected with miR-196a1 and miR-196a2 mimics into two esophageal cell lines, BIC-1 and OE33 with high and low levels of endogenous miR-196a levels, respectively. Both mimics caused greater than 90% decrease in the luciferase activity compared to the negative mimic control (Figure 5, lower panel). A similar suppression was also observed in the breast cancer cell line MDA-453. This experiment demonstrated clearly that miR-196a affects ANXA1 expression by directly binding to and targeting the complementary 3′-UTR region of ANXA1 and validated that ANXA1 was a bonafide target of miR-196a.

Figure 5
figure5

RNA mimics of miR-196a directly target 3′-UTR of ANXA1 mRNA. Luciferase assay was conducted in MDA-MB-453 breast cancer cells using the PGL3 luciferase vector, in which a 284-bp section of the 3′-UTR region of the ANXA1 mRNA was cloned downstream of the luciferase gene. A schematic representation of the principle behind the assay employed has been provided (upper panel). Cotransfection of either of the mimics for miR-196a resulted in a drastic suppression of the luciferase activity, indicating the direct targeting of the 3′-UTR of ANXA1 by miR-196a in esophageal (BIC-1 and OE33) and breast cancer cell lines (MDA-453) (lower panels). ANXA1, annexin A1.

RNA mimics of miR-196a enhance cell proliferation in cancer cell lines

To test whether increased levels of miR-196a might be involved in increasing the proliferative potential of cancer cells, we assessed the effect of miR-196a RNA mimics on proliferation in six cell lines representing esophageal, breast and endometrial cancers. A consistent increase in the cell proliferation was observed in all the cell lines transfected with miR-196a RNA mimics (Figure 6a). The two RNA mimics increased proliferation within 96 h of transfection in comparison to mimic negative control in three breast cancer cell lines MDA-453, MDA-435 and MDA-231 by 20–45% (Figure 6a, upper panel), in two esophageal cancer cell lines OE33 and SEG-1 by 25–30% (Figure 6a, lower panel) and in an endometrial cancer cell line HEC-1B by 20% (Figure 6a, lower panel). This consistent trend of enhanced proliferation within 96 h after transfection of the mimics showed that miR-196a could be physiologically involved in increasing the proliferative potential of cancer cells. Consequently, the characteristic increase of miR-196a levels observed in esophageal carcinomas (Figure 3) could be one of the important physiological changes occurring during the neoplastic transformation of normal esophageal tissue.

Figure 6
figure6

Increased levels of miR-196a increased cell proliferation and suppressed apoptosis. (a) Transfection of both the mimics of miR-196a resulted in a consistently elevated proliferative potential of esophageal, breast and endometrial cancer cell lines within 96 h of transfection, indicating that elevated levels of miR-196a might be involved in neoplastic transformation in cells. The cell proliferation was tested by MTS assay. Results showed are an average of two separate experiments and observed increase in proliferation was statistically significant (P<0.05). (b) In SEG-1 cells, overexpression of miR-196a suppressed apoptosis induced by staurosporine (100 nM). Reduced extent of cell death was evident after staurosporine treatment in 196a mimic-transfected cells in comparison to the cells transfected with mimic negative control. Cell death was measured by trypan blue dye exclusion assay.

RNA mimics of miR-196a enhance colony-forming ability in esophageal cancer cell lines

To further characterize the effects of miR-196a on the growth characteristics of esophageal cancer cells, we determined the effect of miR-196a overexpression on anchorage-independent growth of BIC-1 and SEG-1 by examining their colony-forming ability after transfection with the mimics. At 2 weeks of growth in methylcellulose, a clear trend of increase in both the number and the size of the colonies was observed. In BIC-1 cells, the mimics boosted the colony-forming ability as evident by the increase in the colony size and colony numbers (>2-fold) (Figure 7, upper panels). Similar increase in the colony number and more notably in colony size was observed in SEG-1 cells (Figure 7, lower panels).

Figure 7
figure7

miR-196a stimulated anchorage-independent colony formation in esophageal cancer cells. BIC-1 and SEG-1 cells were counted and seeded into methylcellulose 24 h after transfection with miR-196a mimics 1 and 2 or mimic negative control. The numbers of colonies were counted after 2 weeks of growth. A distinct increase in the colony-forming ability was observed in both the cell lines transfected with RNA mimics of miR-196a, as evident by the enhanced colony number and size. Colonies were counted under microscope. Colony numbers and representative pictures under each condition are shown.

Both the mimics in general exhibited similar trend of effects in our assays, with small differences (<10%) in the extent of the effects proving that an effective increase in the level of miR-196a could be achieved by employing any one of the two mimics.

RNA mimic of miR-196a shows suppression of apoptosis

To test if increased miR-196a levels result in reduced apoptosis, the effect of miR-196a overexpression on apoptosis was studied in esophageal cancer cell line SEG-1. Twenty-four hours following transfection with miR-196a mimic 2, apoptosis was induced by treating cells with 100 nM staurosporine, a potent inhibitor of protein kinase C. The extent of cell death was assayed by trypan blue staining. After 5 h of staurosporine treatment, cells transfected with the mimic showed 5% cell death compared to 8% in control. After 20 h, 25% cell death was observed in mimic-transfected cells as compared to 41% in control (Figure 6b). This suggests that miR-196a has an antiapoptotic effect.

Discussion

In the present study, we have investigated the potential involvement of an miRNA-mediated mechanism in the reduced expression of ANXA1 in cancers. We identified by computational analysis three candidate miRNAs which could potentially target ANXA1 mRNA. Though computational algorithms have played a central role in predicting miRNA targets, virtually all available programs generate some level of false predictions (Lim et al., 2005). Hence, we first performed correlative studies to assess negative correlation between levels of these three miRNAs and ANXA1 in cancer cell lines and tumors from patients with esophageal cancer. Our results demonstrated a significant inverse correlation between ANXA1 mRNA levels and miR-196a in 12 different esophageal, breast and endometrial cancer cell lines and in esophageal tumors from patients, which supports the putative role of miR-196a in regulating ANXA1 expression. The 10- to 100-fold increase in miR-196a seen in esophageal tumors compared to normal tissue and the fact that ANXA1, a potential tumor suppressor, is a direct target of miR-196a suggest that miR-196a may possess growth-promoting physiological functions in the cell. The ability of miR-196a to enhance cell proliferation and colony formation and suppress apoptosis further supports the role of this miRNA in cancer growth.

Functional analysis of miR-196a using specific mimics validated its role in targeting the ANXA1 mRNA. By employing the PGL3 vector with the cloned target 3′-UTR region of ANXA1 mRNA, we demonstrated that the negative effect of miR-196a on ANXA1 levels in the cell was the result of direct targeting of ANXA1 mRNA by miR-196a and not due to indirect targeting of proteins that may be involved in the positive transcriptional regulation of ANXA1.

The identification of proteins with aberrant expression during malignant transformation is of great importance in the ongoing effort to detect cancer in its early stages. Our analysis of normal and esophageal tumor tissues demonstrates decreased ANXA1 expression with a concomitant increase in miR-196a levels in tumors compared to the matched normal tissue. In light of the report that ANXA1 mRNA levels progressively decrease from normal esophageal epithelium to Barrett's esophagus (BE) and from BE to adenocarcinoma (Kimchi et al., 2005), it would be interesting to determine if miR-196a level can be used as early marker of esophageal cancer. Though our study predominantly focused on esophageal adenocarcinomas, previous studies have reported an association between ANXA1 loss and tumorigenesis of squamous cell carcinoma of the esophagus (Paweletz et al., 2000; Xia et al., 2002; Hu et al., 2004). It is conceivable that miR-196a plays a similar role in this other major subtype of esophageal cancer as seen in adenocarcinoma subtype. However, further studies are required to confirm this possibility.

It is also of interest to note here that transfection of RNA mimics of miR-196a in all the cell lines tested consistently resulted in decreased ANXA1 mRNA levels. Until recently, it was generally believed that when there is partial complementarity between miRNA and its target, the miRNA functions by binding to the target mRNA and blocking translation of the target protein rather than by inducing degradation of the target mRNA. However, several recently published reports contradict this notion and demonstrate that despite partial base pairing, the binding and degradation of the target mRNA levels is one of the important modes by which miRNAs decrease the levels of their targets in the cell (Bagga et al., 2005; Lim et al., 2005; Giraldez et al., 2006). The decrease in ANXA1 mRNA levels caused by elevated miR-196a levels in our study reinforces this concept.

Our study has added ANXA1 mRNA as one more bonafide target of miR-196a, which has also been implicated in targeting HOXB8, HOXC8, HOXD8 and HOXA7 mRNAs (Yekta et al., 2004). A high degree of negative correlation found between miR-196a and HOX genes in acute myeloid leukemia provides further confirmation of its role in the regulation of HOX gene expression (Debernardi et al., 2007). The elevated expression of HOX genes in leukemogenesis is very well established (Thorsteinsdottir et al., 1997, 2002), and in that respect, as a negative regulator of HOX gene expression, miR-196a might be considered a potential tumor suppressor in leukemia. Our study, on the other hand, shows that an increase in miR-196a levels is characteristic of esophageal cancers, where it may be functioning as an oncogenic miRNA. As a single miRNA can potentially target many genes, the classification of any miRNA as oncogenic or tumor suppressive might be strictly cancer type-specific and dependent on the target gene whose expressions it may be modulating. Recent studies have also shown that miRNA-196a levels are inversely correlated with survival in pancreatic adenocarcinoma patients (Bloomston et al., 2007).

ANXA1 is known to be a mediator of apoptosis and suppressor of cell proliferation (Solito et al., 2001). By targeting and suppressing ANXA1 levels, miR-196a may promote deregulated growth characteristics in cells. Stimulation of cell proliferation, anchorage-independent growth and suppression of apoptosis by miR-196a observed in this study provide persuasive proof for a potential oncogenic role of miR-196a. Thus, continued efforts to identify targets of miR-196a and elucidate its physiological role will help in establishing miR-196a as an important ‘oncomir’ in cancer.

To summarize, our study is the first to identify an miRNA-mediated mechanism behind the frequently observed suppression of ANXA1 levels in esophageal cancers. The consistent increase in miR-196a levels in esophageal cancers, accompanied by a decrease in ANXA1 levels, suggests that miR-196a may serve as a molecular marker of neoplastic transformation in esophageal cancers. We also have provided evidence for the potential oncogenic role of miR-196a in cancer.

Materials and methods

Cell lines

The 12 cell lines included in the study were esophageal adenocarcinoma cell lines SEG-1 and BIC-1 (Dr U Raju, MD Anderson Cancer Center, Houston, TX, USA) and OE33 and SKGT-5 (Dr J Izzo, MD Anderson Cancer Center), breast cancer cell lines MDA-231, MDA-453, MDA-435, MCF7 and T47D (Dr J Liu, MD Anderson Cancer Center) and endometrial cancer cell lines HEC-1A, HEC-1B, Ishikawa and AM3CA (Dr R Broaddus, MD Anderson Cancer Center).

Patient samples

Institutional database at Department of Pathology, The University of Texas MD Anderson Cancer Center was searched to identify patients with esophageal adenocarcinoma who underwent esophagectomy without prior chemoradiation. All patients had clinically localized disease as per the endoscopic ultrasonography and imaging findings, which included positron emission tomography scan. Esophageal adenocarcinomas from 20 patients were included in the study. Ten of the specimens had matched gastric (six cases) or esophageal (four cases) tissue available to use as normal control tissue. Five-micron thick slides were prepared from the paraffin block and RNA was isolated following manual microdissection. All tissue specimens were collected through a protocol approved by MD Anderson Cancer Center's Institutional Review Board.

Real-time quantitative PCR for ANXA1 mRNA and miRNA expression analysis

Total RNA was extracted from the cell lines using Trizol (Gibco BRL, Life Technologies, Gaithersburg, MD, USA) as per the manufacturer's protocol. For cDNA synthesis, 100 ng total RNA from each cell line was reverse transcribed in a final volume of 20 μl using random primers and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA). The Taqman minor groove binder probe and the ABI Prism 7900 HT Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA) were used for performing real-time PCR. The primers and probe for ANXA1 mRNA and an internal control glucuronidase beta (GUSB) mRNA, were obtained from PE Applied Biosystems through their Assays-on-Demand gene expression products services. PCR assays included 10 μl of Taqman Universal Master Mix No Amperase UNG (2 × ), 1 μl of 2 × Assays-on-Demand Gene Expression Assay Mix and 2 μl of cDNA diluted in RNase-free water, in a final volume of 20 μl. The PCR thermalcycling conditions were as follows: 10 min at 95 °C for AmpliTaq Gold activation and 40 cycles for the melting (95 °C, 15 s) and annealing/extension (60 °C, 1 min) steps. Each target was amplified individually and in duplicate. The relative levels of ANXA1 mRNA were calculated based on the difference between amplification of ANXA1 and GUSB mRNA using the delta CTCT) method.

RNA from formalin-fixed paraffin-embedded tissues was isolated after manual microdissection using the RecoverALL Total Nucleic Acid isolation kit (Ambion/Applied Biosystems, Austin, TX, USA). Reverse transcription was performed as described above, except with 300 ng of total RNA. Real-time qPCRs for ANXA1 and GUSB mRNAs were performed using 5 μl of cDNA for each target.

The relative levels of miR-584, miR-196b and miR-196a were determined by stem loop real-time qPCR using gene-specific primers according to the TaqMan MicroRNA Assay protocol (PE Applied Biosystems). For reverse transcription, 7 ng of total RNA was used for cell lines, whereas 50 ng of total RNA was used for formalin-fixed paraffin-embedded samples. miR-16 was selected as the normalizer, as this miRNA showed minimal variation in expression among different cell lines and cancer specimens (data not shown). Each miRNA was amplified individually and in duplicate. The relative levels of individual miRNAs with reference to miR-16 were calculated using the ΔCT method.

Western blot analysis

Cell lysates from the cancer cell lines were prepared using Beadlyte Universal Lysis buffer (Upstate, Lake Placid, NY, USA) with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Total proteins were resolved by SDS–polyacrylamide gel electrophoresis using a 12% acrylamide gel and transferred to a nitrocellulose membrane. The membranes were probed with antibodies for ANXA1 (BD Biosciences, San Jose, CA, USA) and β-actin (Sigma-Aldrich, St Louis, MO, USA).

Transfection with miR-196a mimics

Two RNA mimics for miR-196a (designated as mimics 196a1 and 196a2) were purchased from Dharmacon Inc. (Chicago, IL, USA). The mimics were transfected into cultured cells using DharmaFECT Duo transfection reagent (Dharmacon Inc.). The final concentration of the mimics was 40 nM. After 48 h, the cells were harvested to measure ANXA1 protein and mRNA levels as described above. A nonspecific miRNA mimic was used as an appropriate negative control.

Cell proliferation assay

The proliferation assay was done in a 96-well format using CellTitre 96 One solution Cell proliferation assay kit (Promega Corporation, Madison, WI, USA). Mimics of miR-196a were transfected as described above and after 96 h, the proliferation of the cells was assayed. In an individual experiment, proliferation under each condition was studied in triplicate and the overall experiment was repeated at least twice.

Cell death assay

Twenty-four hours post-transfection with RNA mimics of miR-196a and the mimic negative control, the cells were treated with 100 nM staurosporine (Calbiochem, San Diego, CA, USA) and the extent of cell death was estimated by trypan blue staining after 5 and 20 h using Vi-Cell cell counter (Beckman-Coulter, Fullerton, CA, USA).

Colony-formation assay

Twenty-four hours post-transfection with RNA mimics, cells were counted and seeded in methylcellulose. After 14 days, colonies were counted and photographed under microscope.

Cloning of the 3′-UTR of ANXA1 into PGL3 vector and luciferase assay

The 284-bp 3′-UTR region of ANXA1 containing the putative miR-196a recognition site was amplified from the genomic DNA of MCF-7 breast cancer cells. The amplified fragment was then cloned into the pGL3 control vector (Promega Corporation) at the XbaI site and confirmed by sequencing. The primers used were as follows: forward sense primer, 5′-IndexTermGCATCTAGAACATTCCCTTGATGGTC-3′ and reverse antisense primer, 5′-IndexTermCCGCATCTAGAGTGACGTCATTTTATTTTC-3′. Cotransfection of the plasmid and the RNA mimics into the cells was accomplished using DharmaFECT Duo transfection reagent (Dharmacon Inc.). Luciferase assay was performed 48 h after transfection using the Dual Luciferase Reporter Assay System kit (Promega Corporation).

Statistical analysis

Pearson's product-moment correlation coefficient was used to measure the degree of the linear relationship between mRNA levels of miR-196a, miR-196b and miR-584 as compared to ANXA1 across the 12 cell lines. These correlations were tested for significance under the assumption that under the null hypothesis of no linear relationship, the test statistic

follows a t-distribution with (n−2) degrees of freedom when the sample correlation r is based on a sample from a bivariate normal distribution. Pearson correlation was also used to measure correlation between ANXA1 mRNA and miR-196a levels in esophageal cancers. A paired t-test was used to investigate expression level differences between normal and cancerous tissue. P-values<0.05 were considered statistically significant.

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Acknowledgements

We thank Dawn Chalaire for critical editing of the manuscript and James M Gilbert and Kim-Anh Vu for assistance with preparation of the manuscript and illustrations. We also thank Drs Raju, Izzo and Liu of MD Anderson Cancer Center for providing the cell lines used in the study.This study was supported by Goodwin Fund for Target Molecular Diagnosis and Institutional Research Grant (CT Albarracin), NIH 1P50CA098258-01, SPORE in Uterine Cancer (RR Broaddus).

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Correspondence to R Luthra.

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Luthra, R., Singh, R., Luthra, M. et al. MicroRNA-196a targets annexin A1: a microRNA-mediated mechanism of annexin A1 downregulation in cancers. Oncogene 27, 6667–6678 (2008) doi:10.1038/onc.2008.256

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Keywords

  • annexin A1
  • miR-196a
  • esophageal cancer
  • breast cancer
  • endometrial cancer

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