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Molecular Cancer Research 6, 663-673, April 1, 2008. doi: 10.1158/1541-7786.MCR-07-0370
© 2008 American Association for Cancer Research
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Signaling and Regulation

Antiproliferative Effects by Let-7 Repression of High-Mobility Group A2 in Uterine Leiomyoma

Yi Peng, Jordan Laser, Guizhi Shi, Khush Mittal, Jonathan Melamed, Peng Lee and Jian-Jun Wei

Department of Pathology, New York University School of Medicine, New York, New York

Requests for reprints: Jian-Jun Wei, Department of Pathology, New York University School of Medicine, 462 First Avenue, NB4W1, New York, NY 10016. Phone: 212-562-7893; Fax: 212-263-7573. E-mail: weij03{at}med.nyu.edu, or Peng Lee, Department of Pathology, New York University School of Medicine. E-mail: peng.lee{at}med.nyu.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
High-mobility group A2 (HMGA2) is commonly overexpressed in large leiomyomas. HMGA2 is an important regulator of cell growth, differentiation, apoptosis, and transformation. As a predicted target of Let-7 microRNAs (Let-7s), HMGA2 can be repressed by Let-7s in vitro. MicroRNA profiling analysis revealed that Let-7s were significantly dysregulated in uterine leiomyomas: high in small leiomyomas and lower in large leiomyomas. To evaluate whether Let-7 repression of HMGA2 plays a major role in leiomyomas, we analyzed the molecular relationship of HMGA2 and Let-7s, both in vitro and in vivo. We first characterized that exogenous Let-7 microRNAs could directly repress the dominant transcript of HMGA2, HMGA2a. This repression was also identified for two cryptic HMGA2 transcripts in primary leiomyoma cultures. Second, we found that the endogenous Let-7s were biologically active and played a major role in the regulation of HMGA2. Then, we illustrated that Let-7 repression of HMGA2 inhibited cellular proliferation. Finally, we examined the expression levels of Let-7c and HMGA2 in a large cohort of leiomyomas (n = 120), and we found high levels of Let-7 and low levels of HMGA2 in small leiomyomas, and low levels of Let-7 and high levels of HMGA2 in large leiomyomas. Our findings suggest that the Let-7–mediated repression of HMGA2 mechanism can be an important molecular event in leiomyoma growth. (Mol Cancer Res 2008;6(4):663–73)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Uterine leiomyomas are a major public health problem, represented by a high incidence (1), a high rate of hysterectomies for symptomatic disease (2), and a high medical cost (3, 4). About 20% of patients with leiomyomas experience clinical symptoms and seek medical treatment. The severity of symptoms is often associated with large leiomyomas.

Overexpression of high-mobility group A2 (HMGA2) is very common in uterine leiomyomas and it is induced by chromosomal changes involving 12q13-15 (5, 6). Most of the 12q15 breakpoints in leiomyomas are located 5' to the HMGA2 locus (7, 8), giving rise to a nontruncated HMGA2 overexpression, and are strongly associated with large leiomyomas (9, 10). Leiomyomas with and without HMGA2 expression have distinctly different gene expression profiles (11, 12), indicative of the unique molecular role of HMGA2 in the tumorigenesis of uterine leiomyomas.

HMGA2, a high-mobility-group AT-hook (HMGA) protein, is a non-histone DNA binding factor. It has three AT-hook DNA binding domains through which HMGA2 binds to AT-rich sequences in the minor groove of the DNA helix. HMGA2 is expressed in embryonic tissue but not in most normal adult tissues (13, 14). As an important regulator of cell growth, differentiation, apoptosis, and transformation (15), HMGA2 interacts with many different transcription factors and influences numerous gene expression patterns (15).

Overexpression of HMGA2 in human uterine leiomyomas is mainly contributed by its dominant transcript HMGA2a by Northern blot analysis (14). In addition to the major transcript HMGA2a, several "cryptic" HMGA2 (HMGA2b-HMGA2g) isoforms can be transcribed by alternative splicing from the large intron 3 (16, 17). These cryptic transcripts can only be detected in very low levels in fetal cells and some cultured cells (16).

Let-7 microRNAs (Let-7s) are one of the first microRNAs identified in Caenorhabditis elegans and is highly conserved in C. elegans, Drosophila, zebrafish, and humans (18-20). Let-7s regulate target genes through either posttranscriptional repression (mRNA; refs. 21-23) or translational repression (protein; refs. 22, 23). In normal embryonic development, temporal up-regulation of Let-7s is required for terminal differentiation. In humans, various Let-7 genes have been reported to map to regions deleted in many human cancers (24), such as lung cancer (25), in which the loss of Let-7 expression results in the up-regulation of RAS oncogenes (26). There are several predicted Let-7 complementary sites (LCS) at the 3' untranslation region (3'-UTR) of human HMGA2. Recently, several studies have illustrated that Let-7s can regulate HMGA2 expression in squamous cell carcinoma (27) and in vitro systems (28, 29).

In our microRNA profiling study, we found that several Let-7 members were significantly dysregulated in uterine leiomyomas: high levels in small leiomyomas and low in large ones (30). To evaluate the biological role of Let-7s in the regulation of leiomyoma growth, we analyzed the relationship between Let-7s and HMGA2 and their roles in cellular proliferation, both in vitro and in vivo. Our data showed that Let-7s could be a key negative regulator for HMGA2 expression and cellular proliferation. Our findings provide additional support that repression of HMGA2a by Let-7s is a specific molecular mechanism responsible for leiomyoma growth.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Let-7 microRNAs Regulate the Dominant Transcript HMGA2a and Two Additional Cryptic Transcripts in Leiomyomas
Several recent studies (27, 29), including ours (30), illustrate that Let-7 microRNAs can directly repress HMGA2 expression at the translational level. Lee and Dutta (28) further characterized that the repression of HMGA2 was mostly through destabilization of HMGA2 mRNA in HeLa cells. We previously found that transfection of exogenous Let-7c in primary leiomyoma culture cells can repress HMGA2 translation (30) with minimal effect on HMGA2 mRNA levels (Fig. 1A ). Provided that the presence of Let-7 LCS at the 3'-UTR of HMGA2 is required for Let-7 activity, several potential mechanisms can explain the overexpression of HMGA2 in uterine leiomyomas: (a) loss or reduction of Let-7 expression; (b) the truncated transcripts of HMGA2 with a partial or complete loss of Let-7 LCS, secondary to chromosomal 12q13-15 changes (7, 31); and (c) expression of cryptic HMGA2 isoforms (HMGA2b-HMGA2g; refs. 17, 32) that may lack Let-7 LCS.


Figure 1
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  		FIGURE 1. Transcriptional regulation of HMGA2 and its alternatively spliced transcripts by Let-7 in culture and nonculture leiomyoma cells. A. Photonegative images illustrate minimal change in HMGA2 mRNA levels (common domain from exon 1 and exon 3; see Table 1) in primary culture leiomyoma cells in the absence (lanes 1-3) or presence (lanes 4 and 5) of exogenous Let-7c treatment. Mature Let-7c and HMGA2 in each cell sample were examined by RT-PCR and the relative abundance of each cDNA product was scored under each band. B. Differential expression of HMGA2a and cryptic transcripts (alternatively spliced transcripts of HMGA2; see Table 1) in native and cultured leiomyoma cells (ULM). C. Photonegative images illustrated posttranscriptional regulation of the endogenous HMGA2a and cryptic transcripts by transient transfection of mature let-7c mimic in primary culture leiomyoma cells. D. Posttranscriptional regulation of the endogenous HMGA2a mRNA by transient transfection of control RNA, mature let-7c mimic, and let-7 inhibitor in two additional cultured tumor cells. Actin and U6 were examined as RNA loading controls for mRNA and microRNA.

 

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  		Table 1. RT-PCR Primers for HMGA2

 
To exclude leiomyomas with truncated HMGA2 transcripts, which may have partial or complete loss of the HMGA2 3'-UTR, we carried out reverse transcription-PCR (RT-PCR) with primers from exon 3 and exon 5 (see Table 1 ). These primer pairs were chosen because they cover almost the entire HMGA2a 3'-UTR. We found no loss of the Let-7 LCS in our study.

Little is known about the expression of HMGA2 isoforms in uterine leiomyomas. Studies showed that HMGA2 isoforms might be induced in tissue culture conditions (16, 17). To test whether HMGA2 isoforms were differentially expressed in native leiomyomas versus culture conditions, we examined six HMGA2 mRNA transcripts (primers for each isoforms were listed in Table 1). We found that noncultured leiomyomas contained a high abundance of HMGA2a mRNA and a low abundance of HMGA2b and HMGA2c isoforms. Minimal or no expression of transcripts HMGA2d, HMGA2e, and HMGA2f was identified (Fig. 1B). In examination of an additional 10 HMGA2-positive leiomyomas, similar HMGA2 expression patterns were observed (data not shown).

We then examined the expression of HMGA2 isoforms in three leiomyomas under culture conditions. We found that all HMGA2 mRNA isoforms except for transcript HMGA2e were detectable by RT-PCR with 32 amplification cycles (Fig. 1B). The full-length HMGA2 mRNAs have only been published for isoforms HMGA2a and HMGA2b (NM_003483 and NM_003484). Sequence analysis shows that HMGA2a, but not HMGA2b, contains the Let-7 LCS. To determine which of the HMGA2 transcripts can be destabilized by Let-7s, we examined the levels of the HMGA2 isoforms by RT-PCR in cultured leiomyoma cells following transient transfection of Let-7c. Under these culture conditions, a reduction of HMGA2 transcripts HMGA2a, HMGA2c, and HMGA2f was observed, and the reduction of HMGA2c and HMGA2f seemed to be dose dependent. No change of transcripts HMGA2b and HMGA2d was seen (Fig. 1C). These findings showed that not all of the HMGA2 isoform mRNAs can be destabilized by exogenous Let-7s.

In another two independent experiments, let-7c repression of HMGA2a mRNA was reproducible (Fig. 1D). However, the levels of HMGA2a mRNA reduction by exogenous Let-7c were not as efficient as we saw in HMGA2a stable cell lines (see below).

Let-7 microRNAs Are Major Players in the Regulation of HMGA2 Expression in Leiomyomas
By computer searches (TargetScan and miRanda; refs. 33, 34), it has been shown that, in addition to Let-7s, HMGA2 can be potentially regulated by more than 30 other microRNAs. Among these 30 microRNAs, only three of them were found to be significantly overexpressed in leiomyomas (miR-30a, miR-34, and miR-24; ref. 30). These microRNAs (miR-30a, miR-34, and miR-24) were shown to have a much lower binding score to HMGA2 than Let-7s.

To evaluate the role of endogenous Let-7 microRNAs in the regulation of HMGA2a expression, we prepared the pGL3 constructs with the luciferase (LCF) reporter gene under control of the HMGA2a 3'-UTR (Fig. 2A ). For this assay, we used HeLa cells because (a) HeLa cells have a moderate level of endogenous Let-7 expression (35), and (b) HeLa cells have minimal HMGA2 expression by RT-PCR and Western blot analysis (data not shown). We found that in cells transfected with LCF + HMGA2a 3'-UTR, there was 5.2-fold reduction of luciferase expression in comparison with baseline control (LCF only; Fig. 2B). To test whether repression of luciferase expression with HMGA2a 3'-UTR was mainly contributed by Let-7, cotransfection of Let-7 inhibitor at various concentrations was done and the luciferase expression was measured. As illustrated in Fig. 2B, the luciferase expression was rescued by anti–Let-7 treatment, and the level of luciferase expression was dose dependent. Of note is the fact that the level of luciferase activity did not return to baseline (Fig. 2B). This suggests that additional minor regulatory mechanisms for HMGA2 may exist in HeLa cells, such as long 3'-UTR reducing translation efficiency and other microRNA or non-microRNA regulators.


Figure 2
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  		FIGURE 2. Repression of luciferase expression under HMGA2a 3'-UTR control by endogenous Let-7 microRNAs in HeLa cells and five leiomyomas. A. Sketch diagram illustrates the luciferase (LCF) reporter gene constructs with and without HMGA2a 3'-UTR in pGL3. B. Luciferase expression (y axis) was measured in LCF vehicle alone (empty box) and LCF + HMGA2a 3'-UTR (shadowed boxes) by administration of five different doses of Let-7 inhibitor (0-60 pmol). Fold changes were indicated on top of the bars. ***, P < 0.001. C. Expression analysis of endogenous mature Let-7c (RT-PCR) and HMGA2 protein (Western blot) in five HMGA2 mRNA positive leiomyomas corresponding to tumor sizes of 1 cm (T1), 2 cm (T2), 5 cm (T5), 7 cm (T7), and 10 cm (T10). U6 and extracellular signal–regulated kinase (ERK) were loading controls for microRNA and total protein in each sample. D. Luciferase expression (y axis) analysis in these five tumors (T1.0-T10.0) and normal myometrium (MM) in triplicate following transfection with LCF vehicle only (luc+), LCF + HMGA2a 3'-UTR (luc+/3-UTR), and LCF + HMGA2a 3'-UTR with Let-7 inhibitor (anti–Let-7). Bars, SE. Control LCF expression (vehicle alone) was set at 100 in y axis.

 
To determine the relationship of Let-7c and HMGA2 in vivo, we selected five HMGA2 (mRNA) positive leiomyomas of varying tumor sizes (1-10 cm; T1.0, T2.0, T5.0, T7.0, and T10.0 cm). The endogenous Let-7c and HMGA2 proteins were semiquantitatively evaluated in each tumor and the expected inverse relationship was identified (Fig. 2C). Namely, large leiomyomas contained low levels of endogenous Let-7c and high levels of HMGA2, and small leiomyomas contained high levels of Let-7c and low levels of HMGA2 (Fig. 2C).

After establishing primary cultures from these five leiomyomas, transient transfections of control (LCF only), test 1 (LCF + HMGA2a 3'-UTR), and test 2 (LCF + HMGA2a 3'-UTR and Let-7c inhibitor) were done in each tumor in triplicate. The luciferase expression was measured after 48 hours of transfection. The reduction in luciferase expression was significantly greater in small leiomyomas (high Let-7c levels) than in larger tumors (low Let-7c levels; 3.3- and 2.6-fold reduction in 1.0- and 2.0-cm tumors versus 1.2- to 1.5-fold in the 5-, 7-, and 10-cm tumors; r = 0.86; Fig. 2D). Similar to previous cell line experiments, cotransfection with Let-7 inhibitor (40 pmol) rescued the luciferase activity up to 80% of the original levels (Fig. 2D).

The findings indicate that (a) the level of HMGA2a repression is correlated with the level of endogenous Let-7 microRNA; (b) repression of HMGA2a in leiomyomas was largely contributed by Let-7 microRNAs; and (c) there was an inverse association of endogenous Let-7c and HMGA2a protein in native leiomyomas.

Let-7 Inhibits Tumor Growth through Repression of HMGA2a
Studies showed that overexpression of HMGA2 is strongly associated with large leiomyomas (9, 10). It is important to determine whether the regulatory mechanism of Let-7 repression of HMGA2 affects tumor growth. There were a couple of limitations in the analysis of the Let-7::HMGA2 regulation in primary leiomyoma cell cultures: First, primary cultures showed a slow growth rate, and second, the culture induced cryptic HMGA2 transcripts that may not respond to Let-7 due to lack of Let-7 LCS in some transcripts (Fig. 1C). We therefore chose PC3 and LNCaP cell lines for our proliferation assays. The rationale for using these two cell lines is that, first, these prostate cell lines have minimal or no endogenous HMGA2 and let-7 expressions (36); particularly, there are no culture-induced HMGA2a and other cryptic transcripts commonly seen in leiomyoma primary cultures. Second, the constructions of HMGA2 with and without 3'-UTR in these cell lines using retroviral pBabe vectors (Fig. 3A ) will allow us to further characterize the specificity for let-7 repression of HMGA2a in the presence and absence of let-7 LCS. Stable overexpression of HMGA2a in these cell lines was shown by both RT-PCR and Western blot (Fig. 3B).


Figure 3
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  		FIGURE 3. Antiproliferation analysis of Let-7c in PC3 and LNCaP cell lines with stable overexpression of HMGA2a. A. Sketch diagram illustrates HMGA2a constructs with and without Let-7 LCS in retrovirus pBabe. B. Established PC3 (left) and LNCaP (right) cell lines with stable overexpression of HMGA2a. C and D. Repression of HMGA2a by exogenous Let-7c in HMGA2 + PC3 (C) and HMGA2 + LNCaP (D) cells with Let-7 LCS. Samples were loaded from left to right as nontreated, Block-iT, Let-7c (40 pmol), Let-7c (40 pmol) + Let-7c inhibitor (40 pmol), and Let-7c inhibitor (40 pmol) only. The levels of Let-7c and HMGA2 protein were determined by RT-PCR and Western blot analysis. U6 and actin were used as RNA loading controls. E and F. Cell growth curves in PC3 (E) and LNCaP (F) cells with pBabe (PC3+pBabe; left), stable overexpression of HMGA2a without 3'-UTR (PC3+HMGA2-3'-UTR; middle), and stable overexpression of HMGA2a with 3'-UTR (PC3+HMGA2+3'-UTR; right). Cells were treated by control RNA (dotted lines), Let-7c (solid lines), and Let-7 inhibitor (dashed lines) in triplicate. Cell counts (y axis) were quantitatively measured by colorimetric WST-1 stain (see Materials and Methods) and time points (x axis) were at 24, 48, 72, and 96 h. Cell types were listed below the diagrams.

 
Confirmation that the Let-7c::HMGA2 regulatory mechanism is preserved in these transduced cells was provided by transfection of exogenous Let-7c and Let-7c inhibitor. Of note was that HMGA2a expression could be completely repressed in both cell lines (Fig. 3C and D).

To evaluate the role of the Let-7c::HMGA2 regulatory mechanism in cellular proliferation, we determined the proliferation rate of PC3 and LNCaP cell lines by controlling HMGA2a expression. In PC3 and LNCaP cells with pBabe only (control cell lines), transfection of exogenous Let-7c resulted in up to 12% reduction in the proliferation rate after 24 to 48 hours (Fig. 3E and F, left). In PC3 and LNCaP cell lines with stable overexpression of HMGA2a without 3'-UTR (lack of Let-7 LCS), transfection of exogenous Let-7c had similar reductions of proliferation rate as pBabe control (Fig. 3E and F, middle), whereas in the cell lines with stable overexpression of HMGA2a with 3'-UTR (presence of Let-7 LCS), transfection of exogenous Let-7c (Fig. 3C and D) resulted in up to a 35% reduction in the proliferation rate (Fig. 3E and F, right). There were minimal differences of tumor cell growth rates between control and anti–Let-7 groups. This could be explained by very low levels or no endogenous Let-7 expression in these cell lines.

To test whether Let-7 microRNAs affect the proliferation in leiomyomas, we compared the correlation between endogenous Let-7 microRNAs and the cell proliferation index in leiomyomas. We selected 36 leiomyomas, in which the relative abundance of Let-7 microRNAs was scored based on a microRNA gene microarray analysis (30). By determining the proliferation rate using immunostains for Ki-67 (a proliferation marker), we found that there was a weakly negative correlation between Let-7 microRNAs (Let-7a-1, Let-7b, Let-7c, Let-7d, Let-7e, Let-7f-1, and Let-7f-2) and Ki-67 (r = –0.15 to –0.33; data not shown). By comparing the Ki-67 index (by immunohistochemistry) and Let-7c expression (by in situ hybridization) in a large cohort of leiomyomas, an inverse association was observed (Table 2 ). These findings suggest that the Let-7c::HMGA2 regulatory mechanism may play a role in governing proliferation in leiomyomas.


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  		Table 2. Expression of HMGA2 and Ki-67 (Immunohistochemistry) and Let-7c (Tissue In situ Hybridization) in 180 Leiomyomas

 
Correlation of HMGA2 and Let-7 Expression in a Large Leiomyoma Cohort
Protein levels of HMGA2 were determined via immunohistochemistry. Overall, 45% of leiomyomas had recognizable nuclear immunoreactivities for HMGA2 (Fig. 4A ). Among them, 37.8% (68 of 180) of leiomyomas had an immunointensity score of 1+ and above. These findings are consistent with previously published data (37).


Figure 4
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  		FIGURE 4. Differential expression of HMGA2 with respect to leiomyoma size. A. Photomicrographs illustrated examples of immunoreactivity for HMGA2 in leiomyomas (positive) and matched myometrium (negative). B. Columns, mean immunoreactivities for HMGA2 gene products (y axis) in four groups of different leiomyoma sizes; bars, SE. Significance of immunoscores (P < 0.05) between tumor sizes of 1-3 and 7-9 cm was shown above the columns. C. Dot plot analysis of HMGA2a mRNA expression in small leiomyomas (≤3 cm; middle), large leiomyomas (≥10 cm; right), and matched myometrium (left). Each dot represents the relative amount of cDNA scaled by semiquantitative RT-PCR products (y axis) by density photometry in each tissue sample. Short horizontal bars, mean values in each group.

 
The 180 leiomyomas were arbitrarily divided into four groups, depending on the size of the tumor: 1-3 cm (n = 47), 4-6 cm (n = 53), 7-9 cm (n = 50), and ≥10 cm (n = 30). The mean immunointensity and positive rate of HMGA2 in each group were calculated (Fig. 4B), with the highest immunoreactivities for HMGA2 present in the 7-9 cm group (2.45 ± 0.47) and the lowest in the ≤3 cm group (1.16 ± 0.39; P < 0.05). The percentage of HMGA2-positive cases was 25.9% (7 of 27) in tumors of ≤3 cm and 61.1% (11 of 18) in tumors of ≥10 cm.

HMGA2 mRNA levels, with respect to tumor size, were determined by RT-PCR with primers from exons 4 and 5 (Table 1). A total of 60 leiomyomas [30 large (≥10 cm) and 30 small (≤3 cm)] with matched myometrium were analyzed. HMGA2a mRNA was detected in 38% (23 of 60) of tumors with a similar proportion in both the large and small groups. Minimal expression of HMGA2a mRNA in myometrial controls was identified (Fig. 4C). The expression of HMGA2a mRNA varied greatly and the difference between the large and small groups was insignificant (P = 0.89; Fig. 4C). These results suggest that Let-7c regulated HMGA2 expression mostly via inhibition of translation.

Our previous studies identified that 7 of the 11 Let-7 microRNA family members were significantly overexpressed in leiomyomas via microRNA microarray analysis (30). Of these seven, five (Let-7c, Let-7d, Let-7e, Let-7f-1, and Let-7f-2; Ambion) were statistically significant with respect to tumor size, where smaller tumors had higher levels (Fig. 5A ). To further evaluate the differential expression of Let-7 microRNAs in different tumor sizes, we examined the mature Let-7a, Let-7c, and Let-7f-2 by semiquantitative RT-PCR (mirVana microRNA PCR, Ambion). With an appropriate U6 control, small (<3 cm) tumors had higher Let-7 expression than the large (>10 cm) tumors (Fig. 5B).


Figure 5
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  		FIGURE 5. Differential expressions of Let-7 microRNAs with respect to leiomyoma size. A. Unsupervised dendrogram cluster analysis of seven Let-7 family members (row) in a total of 48 leiomyomas (column) of small (1-3 cm), intermediate (4-9 cm), and large (≥10 cm) leiomyomas (the tumor sizes were indicated in colored bars underneath). The net changes of Let-7 microRNAs are marked in red (overexpression), black (no change), and green (underexpression). B. Photonegative images of Let-7a, Let-7c, and Let-7f-2 by RT-PCR in randomly selected eight small (S1-S8) and eight large (L1-L8) leiomyomas. U6 was used as an RNA loading control. C. Photomicrographs illustrating examples of Let-7c expression in myometrium (left) and small (middle) and large (right) leiomyomas (ULM) by microRNA in situ hybridization (details in Materials and Methods). Intensity of the purple color represents the relative amount of Let-7c expression. D. Semiquantitative analysis of Let-7c expression based on the intensity of microRNA in situ hybridization signals (see C). The net changes of Let-7 expression in leiomyomas were normalized against the matched myometrium (y axis). The means of Let-7c in each of four tumor size groups are given (x axis). The fold changes between the smallest leiomyomas and other sizes of leiomyomas were listed above. *, P < 0.05.

 
Let-7c was the most abundant microRNA in Let-7 family in leiomyomas (Fig. 5B). To further characterize differential expression of Let-7c in leiomyomas, we examined Let-7c by microRNA in situ hybridization (Exiqon) in a total of 120 tumors (Fig. 5C and D). The intensity of Let-7c microRNA was scored in a semiquantitative method in triplicate tissue cores from each case. The matched myometrium was used as an internal control and the net gain or loss of Let-7c was calculated. Overexpression of Let-7c was observed in this large cohort. When the levels of Let-7c microRNA were analyzed with respect to leiomyoma size (≤3, 4-6, 7-9, and ≥10 cm), a reduction of Let-7c was substantial in larger leiomyomas in comparison with small tumors (Fig. 5D). There was a 2.6-fold difference of Let-7c net loss between the smallest and the largest leiomyomas (P < 0.05). These findings are consistent with our previous global microRNA profiling analysis.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
HMGA2 is normally expressed in embryonic tissue but not in normal adult tissue. Proper regulation of HMGA2 is imperative for its biological function in the development of specific cell types. Previous studies have shown that HMGA2 is abnormally overexpressed in many benign and malignant neoplasms, particularly in uterine leiomyomas. Although its role in tumorigenesis is not fully understood, overexpression of HMGA2 is found to be associated with large tumor size in leiomyomas (16, 37), suggesting its role in promoting tumor growth.

MicroRNAs are important small molecules known to participate in the fine-tuning of functional gene activity. Because HMGA2 is one of the major targets of Let-7 microRNAs (27-30), and large leiomyomas have high levels of HMGA2 (16, 37) and a high Ki-67 index (38), we speculate that dysregulation of the Let-7::HMGA2 regulatory mechanism may be one of key genetic events promoting leiomyoma growth. Characterization of Let-7 repression of HMGA2 in leiomyomas can provide a better understanding of HMGA2 function.

The HMGA2 gene consists of five exons and spans >200 kb with a large intronic sequence (>100 kb) between exons 3 and 4. The major transcript is HMGA2a, a 4.1-kb mRNA from five exons that translates to a 108-amino-acid HMGA2 protein. The first exon encodes the first 37 amino acid residues for the first AT-hook domain. The second and third AT-hook domains are encoded by the second and third exons, respectively. Exon 4 encodes 12 amino acids to separate the AT-hooks from the COOH-terminal amino acid domain, encoded by exon 5 (39). Many cryptic transcripts seem to have unique 3' exons along the large intron 3. Although the full-length mRNA sequences from most cryptic transcripts remain undetermined, we illustrate that, in addition to the dominant transcript, HMGA2a, two other minor transcripts, HMGA2c and HMGA2f, can be destabilized by Let-7 microRNAs in leiomyomas. Apparently, regulation of HMGA2 transcripts can be complicated by its genetic and transcriptional mechanisms, depending on whether Let-7 LCS exists.

Several attempts to characterize the molecular regulation of Let-7::HMGA2 pairing in leiomyomas have been conducted in this study. We characterized that HMGA2, in natural leiomyomas and stable HMGA2a expression cell lines, can be repressed by exogenous Let-7 microRNAs. Endogenous Let-7 microRNAs in leiomyoma tumor cells can repress luciferase activity when HMGA2 3'-UTR is present. In HMGA2a-positive leiomyomas, the levels of endogenous Let-7 microRNAs determine the levels of HMGA2a gene products. Because HMGA2 overexpression and a loss of Let-7 microRNA expression are more common in large leiomyomas (Fig. 5), we proposed that disrupting the pairing between Let-7 and HMGA2 is an important molecular mechanism in promoting leiomyoma growth. A supporting factor arises from the inverse association between Let-7 expression and the Ki-67 index in leiomyomas (Table 2). Although we do not have a good in vitro model to show that HMGA2 promotes leiomyoma growth (due to a slow growth rate of leiomyoma primary culture), the mitogenic role of HMGA2 was examined in the HMGA2-positive PC3 and LNCaP cell lines (Fig. 3) and in the NIH 3T3 cell line in xenografts of nude mice (29). The findings from this study and other recent publications (27-29) strongly suggest that Let-7 microRNAs are important molecules that regulate HMGA2 expression and tumor cell growth.

Let-7 microRNAs may regulate 500 to 600 genes (PicTar and TargetScan). Of the 1,780 significantly dysregulated genes in leiomyomas (40, 41), 33 are predicted targets of Let-7 microRNAs. Of these, only a few are significantly dysregulated in leiomyomas and are therefore likely biological targets. In addition to HMGA2, Let-7 regulation of leiomyoma growth may also be mediated through other predicted targets. Although RAS genes have been characterized as Let-7 targets (42), no evidence of abnormal expression of RAS genes in leiomyomas has been documented.

Regulation of Let-7 microRNAs is not a well established phenomenon. In humans, various Let-7 genes have been reported to map to regions deleted in many human cancers (24, 25). Therefore, direct genomic alterations could result in a loss of Let-7 expression. Recently, Kulshreshtha et al. (43) conducted a global analysis of microRNA expression and identified a group of microRNAs that can be regulated by hypoxia (hypoxia-regulated microRNAs) in colon and breast cancer cells. Of interest, Let-7 microRNAs can be regulated by hypoxia, and Let-7 hypoxia-regulated microRNAs seem to be tissue specific. For example, in squamous cell carcinoma, hypoxia induces Let-7s overexpression (27); in comparison, in nasopharyngeal carcinoma, down-regulation of Let-7s by hypoxia is evident (44). The loss of Let-7 in large leiomyomas requires further study.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Patient and Tissue Samples
Leiomyoma tissue samples were collected from hysterectomy specimens removed for symptomatic disease. Of the 30 cases selected, fresh frozen tissues from one large (≥10 cm) and one small (≤3 cm) leiomyoma and matched myometrium were collected. All tissue samples were collected within 1 to 4 h of surgery. Tissues were stored at –80°C before being used to examine mRNA, microRNA, and protein by RT-PCR, Northern blot, and Western blot analyses, respectively. Eight HMGA2-positive leiomyomas were selected for additional transfection and luciferase assays.

Two large tissue microarrays (TMA1 and TMA2), prepared from paraffin-embedded tumors and matched myometrium, were used to examine HMGA2 and Let-7c microRNA expression. TMA1 contained 60 cases (45) and TMA2 contained 120 cases (46). Details about mean age and tumor sizes are summarized in Table 3 . The study was approved by the NYU Medical Center Institutional Review Board.


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  		Table 3. Summary of Patients and Tissue Samples

 
Let-7 MicroRNA and let-7 Inhibitors
Mature double-stranded microRNAs of Let-7 and Let-7 inhibitors were purchased from Dharmacon, Inc. All experiments were controlled using a nonfunctional double-stranded random 22-nucleotide RNA (Block-it, Invitrogen).

HMGA2 cDNA Constructs
The PCR products of full-length HMGA2a (forward primer, 5'-ATGAGCGCACGCGGTGAGGGC-3'; reverse primer, 5'-GTTAGAAGACACTCAAAGGAACAG-3') and HMGA2a 3'-UTR (forward, 5'-GAGGAAACTGAAGAGACATCCTC-3'; reverse, 5'-GTTAGAAGACACTCAAAGGAACAG-3', containing 5 Let-7 LCS; Table 1) were cloned into the pDNA3.1 construct (Invitrogen). The HMGA2a 3'-UTR cDNAs were prepared with the XbaI linker and further subcloned into the Luciferase Reporter Vector pGL3 (Promega). Retroviral pBabe-puro vectors were treated with NdeI and BamHI, releasing the full-length HMGA2a cDNA insert from pcDNA3.1 and subsequently subcloning it into the pBabe-puro vector. The orientation and fidelity of the cDNA sequence were verified by sequence analysis.

PC3 and LNCaP Cell Lines with Stable Overexpression of HMGA2a
To produce viral vectors, pBabe retroviral constructs containing HMGA2a, with and without the 3'-UTR, were transfected onto the Phoenix amphotropic packaging cell line (American Type Culture Collection) per manufacturer's protocol for Lipofectamine-mediated transfection. Subsequently, LNCaP and PC3 cells were infected with a mixture of viral supernatant and fresh medium at a ratio of 1:4 in the presence of 4 µg/mL polybrene for 24 to 48 h. Single colonies with proven expression of HMGA2 by Western blot were isolated and further cultured for use in proliferation assays.

Let-7c MicroRNA In situ Hybridization
The hybridization system and probes, miRCURY LNA, Let-7c, and U6, were purchased from Exiqon. The detailed procedure for in situ hybridization was done per manufacturer's protocol (47). In brief, 4-µm TMA2 slides were prepared. Following deparaffinization and deproteinization, the slides were prehybridized with 1x hybridization buffer without probe. The hybridization was carried out overnight in a 1x hybridization buffer (30-70 µL) with predenatured miRCURY LNA, Let-7c, or U6 probes. After washing, the slides were blocked and incubated with alkaline phosphatase–conjugated anti-DIG Fab fragments (1:1500; Roche) and visualized for color detection.

Quantitative RT-PCR
For the detection of mature microRNAs, mirVana quantitative RT-PCR primers and the mirVana Quantitative RT-PCR Detection Kits (Ambion) were used and optimized according to the abundance of microRNAs in the samples. In brief, 50 to 100 ng of total RNA were reverse transcribed with specific microRNA primers. A total of 15 to 30 cycles were done for quantitation. Primers for the common domain of HMGA2, the dominant transcript (HMGA2a), and the cryptic HMGA2 transcripts were designed (Table 1). The abundances of cDNA products were detected by quantitative RT-PCR and were normalized by the internal control products of U6 and {alpha}-actin.

Immunohistochemistry
The TMA blocks from formalin-fixed and paraffin-embedded tissues were sectioned at 4 µm. After deparaffinization and antigen retrieval, all immunohistochemical staining was done on a Ventana Nexus automated system.

Western Blot Analysis
Fresh frozen tissue or culture cell samples were homogenized at 4°C in a protein lysis buffer (0.5 g tissue in 1-2 mL). Identical amounts of total proteins from each sample were separated through a 12% SDS-PAGE gel and then transferred onto a polyvinylidene difluoride membrane (Perkin-Elmer Life Scientific, Inc.). Development of the immunoblot with antisera against HMGA2 and negative control HMGA2 blocking peptide (from Dr. Alfredo, Universitá degli Studi di Napoli, Napoli, Italy, and Santa Cruz Biotechnology, Inc.) was tested and a single specific HMGA2 band at 25 kDa was detected, as previously described.

Primary Cell Culture
Selected leiomyomas were chopped into small fragments (1 mm) and placed in DMEM (Invitrogen) containing 10% fetal bovine serum (Gemini) and 250 units/mL collagenase V (Sigma-Aldrich) for 2 to 4 h in a 37°C incubator. Cells were washed and maintained in 37°C incubators with 5% CO2 until the cells reached 30% to 40% confluence. For long-term storage, cells were trypsinized, resuspended in freezing medium, and stored in liquid nitrogen.

MicroRNA Transfection
Before transfection, cells were placed in standard medium without antibiotics for 24 h. Per manufacturers' protocol, transfection was done using the Lipofectamine system with microRNA concentrations of 20 to 60 pmol/well in either 6-well or 24-well plates. To estimate transfection efficiency, cotransfection with the Block-iT Fluorescent double-stranded random 22-mer RNA (Invitrogen) was done. The FITC fluorescence was visualized by lex = 494 nm and lem = 519 nm to assess the percentage of cells that were successfully transfected. Cells receiving only the tagged random sequence double-strand 22-mer were used as nonspecific references at all data points. Following transfection, cells were harvested and analyzed at the indicated times.

Luciferase Transfection Assays
HeLa cells and primary leiomyoma cell cultures were split at a cellular density of 1 x 104 per well into 24-well plates ~24 h before transfection. After washing with PBS, cells in each well were transfected with 200 ng of either the luciferase reporter pGL3 control (Promega) or the pGL3 HMGA2 3'-UTR construct, as well as with 1 ng of the pRLuc internal control plasmid (Biosignal). To block the endogenous effect of Let-7, cotransfection of Let-7 inhibitors at various doses (0, 20, 30, 40, and 60 pmol; Dharmacon) was also done. Cells were maintained in transfection conditions for 48 h and harvested for the dual luciferase assay (Promega). Briefly, cells were washed and lysed in 100 µL of 1x lysis buffer. The lysis mixture was transferred into an Eppendorf tube containing 100 µL of solution A. The luciferase expression was determined as recommended by Promega or by a Western blot analysis of HMGA2.

Cellular Proliferation Assay
PC3 and LNCaP cell lines of control (with pBabe) and of tests (stable overexpression of HMGA2a with and without Let-7 LCS) were passed in 24-well plates in triplicate at densities of 5 x 103 per well for LNCaP and 1 x 104 per well for PC3 cells. Cells were subsequently transfected with control RNA (nonfunction; Invitrogen), Let-7c, and/or Let-7 inhibitors (Dharmacon) at a dose of 40 pmol/well. Cellular proliferation was counted at 24, 48, 72, and 96 h using the colorimetric WST-1 assay (Cell Proliferation Reagent, Roche). Briefly, the cells were rinsed with PBS and then incubated with 10% WST-1 reagent in a serum-free medium for 2 h. Aliquots were then transferred to 96-well plates and the samples were read in a spectrophotometric plate reader at 450 nm (FLUOstar OPTIMA, BMG Lab Technologies).

Statistical Analysis
Mean and SEs were calculated for the quantitative values. Statistical significance was analyzed by a paired t test and P < 0.05 was considered significant.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. Eva Hernando for the scientific discussions, and Drs. Patricia Soteropoulos, Virginie Aris, Tongsheng Wang, Luis Chiriboga, Peng Lan, and Huihui Ye for providing technical assistance in our early microRNA profiling analysis, TMA preparation, microRNA in situ hybridization, and immunostains.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: This work was presented in part at the 96th Annual Meeting of United States and Canadian Academy of Pathology, March 24–30, 2007, San Diego, CA.

Y. Peng and J. Laser contributed equally to this work.

Received 8/ 7/07; revised 11/27/07; accepted 11/30/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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