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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biochem Biophys Res Commun. Author manuscript; available in PMC May 18, 2009.
Published in final edited form as:
PMCID: PMC2683343
NIHMSID: NIHMS31353

microRNAs and regeneration: let-7 members as potential regulators of dedifferentiation in lens and inner ear hair cell regeneration of the adult newt

Introduction

Among adult vertebrates, regeneration of body parts and organs is largely restricted to some salamanders [1,2]. These animals can regenerate many body parts including their limbs, hair cells of the inner ear and lateral line, tail (and spinal cord), eye retina and lens. They achieve this by the process of transdifferentiation of terminally differentiated cells at the site of the injury. During this process one cell type changes to another, by dedifferentiating [35]. It is conceivable that such an event may involve global changes in gene expression, implicating a large number of genes [6]. What could then be the regulators of changes of such magnitude?

To provide some answers to these questions we decided to look at the expression profiles of miRNAs during lens and inner ear hair cell regeneration. miRNAs are short RNAs (20–22 nt) which have complementary nucleotide sequences in target mRNAs. By binding to these target sequences miRNAs inhibit protein synthesis [7,8]. Since one miRNA can have target sequences in hundreds of different mRNAs they have been thought to act when rapid global regulation is needed. For example, miR430 can clear hundreds of maternal RNAs during development [9]. Likewise, differentiation of stem cells is linked to distinct miRNA expression [10,11]. Recently, miRNA presence in neoblasts (stem cells in planaria) has been associated with regenerative ability in that invertebrate animal [12]. Based on this work we believe that miRNAs may be key regulators during transdifferentiation, a process that requires regulation of many tissue-specific genes in a short time. To test this hypothesis we performed a microarray analysis of miRNAs during lens regeneration and inner ear hair cell regeneration.

After lentectomy, the dorsal iris pigment epithelial cells (PECs) dedifferentiate and then differentiate to lens-forming cells. Interestingly, the same cells from the ventral iris do not contribute to the process of regeneration [3,4]. After hair cell death due to aminoglycoside antibiotic treatment hair cells are regenerated by transdifferentiation of the supporting cells [13]. This event is mediated by direct transition without cell proliferation. We decided to investigate two different events of regeneration in order to compare possible common regulators. Our results provide strong evidence of association of miRNA expression and regeneration and imply a novel mechanism that might regulate transdifferentiation and regeneration.

Materials and Methods

Microarray analysis

We used mirVana miRNA Bioarrys V2 microarray slides from Ambion (Austin, TX), which contain most of the known mouse and human miRNAs along with others predicted by Ambion. The arrays were probed with RNA isolated from intact dorsal and ventral irises (day 0) and from dorsal and ventral irises taken 8 days after lentectomy. This time was selected because at day 8 the tip of the dorsal iris undergoes the crucial events of dedifferentiation, which will eventually lead to the regeneration of the new lens [3,4]. We then compared differences in the expression between intact dorsal and ventral irises as well as between 8-day dorsal and ventral irises. For hair cell regeneration, we isolated the labyrinths comprising the audutory and vestibular sensory epithelia. After treatment with 2mM gentamicin for 48 hours, the labyrinths were cultured in vitro. We used RNA samples from day 0 (end of antibiotic treatment), day 2, 7 and 12. Regenerated hair cells appear after 18 days in culture, so the period that we selected represents the time window of recovery, when the cells undergo ‘reprogramming’ for transdifferentiation [13].

Data normalization and analysis

The data were analyzed to identify differentially expressed miRNAs between dorsal and ventral tissues (intact or at day-8 post-lentectomy) as well as miRNAs at different times during hair cell regeneration Analyses were performed using R statistical software and the limma Bioconductor package [14]. Data normalization was performed in two steps for each microarray separately [1517]. First, background adjusted intensities were log-transformed and the differences (R) and averages (A) of log-transformed values were calculated as R = log2(X1) − log2(X2) and A = [log2(X1) + log2(X2)]/2, where X1 and X2 denote the Cy5 and Cy3 intensities after subtracting local backgrounds, respectively. Second, normalization was performed by fitting the array-specific local regression model of R as a function of A. Normalized log-intensities for the two channels were then calculated by adding half of the normalized ratio to A for the Cy5 channel and subtracting half of the normalized ratio from A for the Cy3 channel. The statistical analysis was performed for each time point comparison and for each gene separately by fitting the following Analysis of Variance (ANOVA) model [18]: Yijk = [Upsilon] + Ai + Sj + Ck+ ′Ωijk, where Yijk corresponds to the normalized log-intensity on the ith array, with the jth treatment condition, and labeled with the kth dye (k = 1 for Cy5, and 2 for Cy3). μis the overall mean log-intensity, Ai is the effect of the ith array, Sj is the effect of the jth treatment and Ck is the gene-specific effect of the kth dye. Resulting t-statistics for each comparison were modified using an empirical Bayesian moderated-T method [14]. This method uses variance estimates from all genes to improve the variance estimates of each individual gene. Estimates of fold-change and False Discovery Rates (FDR) were calculated [19].

Expression analysis via QPCR

In order to validate the microarray data, we examined the expression of selected miRNAs via QPCR. RNA from dorsal and ventral irises as well as from treated inner ear sensory epithelia was isolated using TRIreagent (Molecular Research Center, Cincinnati, OH) following the manufacturer’s instructions, with the exception that 2 volumes of ethanol were used instead of isopropanol at the precipitation step. QPCR was performed using Ambion’s mirVana qRT-PCR miRNA detection kit and mirVana qRT-PCR Primer Sets for miRNAs. We examined expression of miR148a and let-7. For normalization, Ambion’s mirVana qRT-PCR Primer Set for 5S rRNA was used. Real-time PCR was performed with iCycler (BioRad) and SYBR Green I fluorescent dye (Molecular Probes, Carlsbad, CA) [20,21].

Results and Discussion

Expression during lens regeneration

The microarray analysis revealed the regulation of several miRNAs between the intact dorsal and ventral irises and between the irises at day 8 of regeneration. For example, some miRNAs were more highly expressed in the intact dorsal iris while others were in the intact ventral irises. Similar regulation was observed in the 8-day irises as well (Table 1). For example a V/D fold change of 7.65 means that the levels in the ventral iris are 7.65 times more than in the dorsal. A negative sign would indicate the opposite, higher levels in the dorsal. It is interesting to note that miRNAs thought to be eye specific in mammals, such as miR184 are also found in our list [22]. Among others that have been found to be expressed during mouse eye development are: miR181, miR124, miR204, miR125 [22]. All these have been found in our microarray, indicating that our analysis is quite valid. Of interest are members of the let-7 family. Many of them (let-7a-g; highlighted with red color in Table 1) are found in the miRNA list, having the same overall expression pattern and deserve further attention. They were found in higher levels in the intact dorsal iris, but they were down-regulated in the dorsal iris during dedifferentiation. These miRNAs have been associated with control of cell cycle, which is a crucial event during dedifferentiation [23] and with oncogenic transformation [24]. Some other miRNA show an interesting up-regulation in the ventral iris. For example, miR148 is elevated in the intact ventral iris as well as in the 8-day ventral iris when compared with its dorsal counterparts. This might indicate that such miRNAs could be negative regulators (repress regeneration-related genes in the ventral iris). The expression patterns of miR148 were also verified by QPCR (Fig. 1a). We also examined expression of let7 and as it is shown in Fig.1b it was found to be expressed in the irises. We should draw attention here to the fact that the Ambion primers for let7 can pick several different members and that in principle QPCR and microarray are different techniques. Nevertheless, the QPCR data on let7 expression provide an independent verification of its expression in newt irises. In a separate study we have reported the cloning of most of these miRNAs, which as expected show identity in sequence with their mammalian counterparts [25].

Figure 1
Analysis of microRNAs expression by QPCR. a: miR148 during lens regeneration, b: let-7 during lens regeneration, c: let-7 during hair cell regeneration. The comparisons in a and b and between normal dorsal iris (ND), normal ventral irises (NV), dorsal ...
Table 1
Microarray analysis in the irises

Expression during hair cell regeneration

Several miRNAs were found to be regulated during the initial process of dedifferentiation (Tables 24). Interestingly, many of the regulated miRNAs have also been identified in another study during maturation in mice [26]. These miRNAs are very different than the ones identified during lens regeneration. The primary exception is the members of the let-7 family (highlighted red in Table 4). These miRNAs showed significant reduction in their expression levels at day 12 after antibiotic treatment, a period, which is characterized by the early events of regeneration. The expression was also verified via QPCR (Fig. 1c; see above for discussion). It is interesting to note here that this pattern is very similar in both lens and hair cell regeneration. Let-7 miRNAs are known to promote terminal differentiation, tumor suppression and re-entry to the cell cycle. Thus, their down-regulation might implicate them as regulators of dedifferentiation, a critical event for regeneration to occur. Their common expression in two different regeneration events in the newt may indicate that initiation of regeneration in different tissue involves common signals. This is of paramount importance in the field and for the first time alludes to a novel mechanism of vertebrate regeneration. These miRNAs may become indispensable tools to probe and understand the remarkable regenerative capabilities in salamanders and possibly allow extension in other animals. Since miRNAs are relatively short they can be easily transfected in vivo the same way as morpholinos [27]. As techniques to over-express or down-regulate miRNAs are becoming more advanced and available we will be able to delineate the function and role of these miRNAs in regeneration. Comparative studies with the axolotl (a lens regeneration-incompetent salamander) or even mouse might further allow us to understand why such regenerative capability is restricted.

Table 2
Microarray analysis during hair cell regeneration. Day 2 vs 0
Table 4
Microarray analysis during hair cell regeneration. Day 12 vs 0
Table 3
Microarray analysis during hair cell regeneration. Day 7 vs 0

Acknowledgments

This work was supported by NEI grant EY10540 and by a research contract from Wright State University to PAT and by RNID to RRT and AF.

Footnotes

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