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Let-7 repression leads to HMGA2 overexpression in uterine leiomyosarcoma

Overexpression of HMGA2 is common in uterine leiomyomas (ULM). The expression of HMGA2 in its malignant counterpart – uterine leiomyosarcomas (ULMS) remains undetermined. Recently it has been shown that repression of HMGA2 by microRNA let-7s is a critical molecular regulatory mechanism associated with tumour growth in many tumours and cell types, including leiomyomas. To test whether HMGA2 and let-7s play a role in ULMS, we examined the levels of endogenous HMGA2 and let-7 expression and found a significant correlation between these two molecules in a case-matched cohort of human ULMS. We found that overexpression of HMGA2 and let-7-mediated HMGA2 repression is a relevant molecular alteration in ULMS. Disrupting the control of HMGA2 and let-7 pairs promotes ULMS cell growth in vitro.


Introduction
Uterine leiomyosarcomas (ULMS) are rare neoplasms, representing 1 in 200 to 800 smooth muscle tumours or ~1% of all malignancies of the uterus [1]. Although the reported 5-year ULMS survival rates are variable, these tumours are clinically aggressive and have a high risk of recurrence and overall poor prognosis [2]. The cause or the molecular basis of human ULMS is largely unknown. While it was believed that most ULMS could arise de novo, some reports suggest that ULMS was progressive from pre-existing uterine leiomyomas (ULM) [3,4]. However, the conclusive evidence documenting the transformation of ULM to ULMS is lacking in human beings and it is thought that the incidence of transformation is Ͻ0.1% [5].
HMGA2, a high-mobility-group AT-hook (HMGA) protein, is considered to function as an oncogene strongly associated with many malignant epithelial and mesenchymal neoplasms [6]. 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 adult tissues [7,8]. HMGA2 is overexpressed in ULM due to chromosomal 12q15 changes [9][10][11][12]. Overexpression of HMGA2 in ULMS has been suggested, but not fully investigated [4,13]. HMGA2 is an important regulator of cell growth, differentiation, apoptosis and transformation [14]. New evidence suggest that dysregulation of microRNAs (miRNAs) may play a central role in tumour development [15]. Members of the let-7 miRNA family function as tumour suppressors through specific repression of its target gene, particularly of HMGA2 expression in some tumour cells both in vivo and in vitro [4,16,17]. The biological importance of molecular pairing of HMGA2::let-7 was further illustrated by the demonstration that repression of HMGA2 by let-7s impairs tumour cell proliferation in many different tumour types, including ULM [4,[16][17][18][19]. It would be of great interest to characterize whether overexpression of HMGA2 and disruption of the HMGA2::let-7 pairs contributes to the aggressive growth behaviour of ULMS. In this study, we examined the levels of endogenous HMGA2 and let-7 expression and analysed a potential correlation between these two molecules in a case-matched cohort of human ULMS. We demonstrate that overexpression of HMGA2 and let-7-mediated HMGA2 repression is important for molecular changes in ULMS. Disrupting the HMGA2::let-7 pairs promote ULMS cell growth in vitro.

Let-7 mimic and inhibitor
Mature double stranded miRNAs of let-7 and let-7 inhibitors were purchased from Dharmacon, Inc. (Lafayette, CO, USA). All experiments were controlled using a non-functional double-stranded random 22 nt RNA (Block-iT, Invitrogen, Carlsbad, CA, USA).

Primers and antibodies
Primers from HMGA2 and its alternative spliced transcripts were reported previously [19]. Primers for mature let-7 family (a-i) were purchased from Ambion, Inc.

Let-7c miRNAs in situ hybridization
The hybridization system and probes, miRCURY LNA, Let-7c, and U6, were purchased from Exiqon (Vedbaek, Denmark). The detailed procedure for in situ hybridization was followed as per manufacturer's protocol [20]. In brief, 4-m TMA slides were prepared. Following deparaffinization and deproteinization, the slides were pre-hybridized with 1ϫ hybridization buffer without probe. The hybridization was carried out overnight in a 1ϫ hybridization buffer (30-70 l) with pre-denatured miRCURY LNA, let-7c or U6 probes. After washing, the slides were blocked and incubated with AP conjugated anti-DIG Fab fragments (1:1500, Roche, Indianapolis, IN, USA) and visualized for colour detection.

qRT-PCR
For the detection of mature miRNAs, mirVana qRT-PCR primers and the mirVana qRT-PCR Detection Kits (Ambion, Inc.) were used and optimized according to the abundance of miRNAs in the samples. In brief, 50 to 100 ng of total RNA were reverse transcribed with specific miRNA primers. A total of 15-30 cycles were performed for quantitation. Primers for the common domain of HMGA2, the dominant transcript (HMGA2a), and the cryptic HMGA2 transcripts were described [19]. The abundances of cDNA products were detected by qRT-PCR and were normalized by the internal control products of U6 and ␣-Actin.

Immunohistochemistry
The TMA blocks from FFPE tissues were sectioned at 4 microns. After deparaffinization and antigen retrieval, all immunohistochemical staining was performed on a Ventana Nexus automated system.

Western blot analysis
Fresh frozen tissue or culture cell samples were homogenized at 4ЊC 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 to a PVDF membrane (Perkin Elmer Life Scientific Inc.). Development of the immunoblot with antisera against HMGA2 and negative control HMGA2 blocking peptide (provided by Dr. Masashi Norita and Santa Cruz Biotechnology, Inc., CA, USA) was tested and a single specific HMGA2 band at 25 kD was detected, as previously described.

Cell culture and miRNAs transfection
LMS cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% foetal bovine serum (Gemini, Calabasas, CA, USA) in 37ЊC incubators with 5% CO2 until the cells reached 30-40% confluence. Prior to transfection, cells were placed in standard media without antibiotics for 24 hrs. As per manufacturers' protocol, transfection was performed with the Lipofectamine system with miRNAs concentrations of 20-60 pmol/well in either 6-or 24-well plates. To estimate transfection efficiency, cotransfection with the block-iT fluorescent double-stranded random 22mer RNA from Invitrogen was performed. The FITC fluorescence was visualized by lex ϭ 494 nm and lem ϭ 519 nm to assess the percentage of cells that were successfully transfected. Cells receiving only the tagged random sequence double-strand 22mer were used as non-specific references at all data points. Following transfection, cells were harvested and analysed at the indicated times. 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 ϫ 10 3 cells/well for LNCaP and 1 ϫ 10 4

Statistical analysis
Mean and standard errors were calculated for the quantitative values. Statistical significance was analysed by a paired t-test and P-value Ͻ0.05 was considered significant.  Fig. 2A). Of same five tumours, two of them were positive for HMGA2 by Western blot analysis (Table 1, Fig. 2A). The karyotype analysis was carried out in the latter five cases (ULMS31-35). One (ULMS-34) of five ULMS had non-random chromosomal changes involving chromosome 12 (Fig. 2B). These non-random chromosome 12 changes were commonly seen in usual type ULM. Further analysis of this tumour revealed a high level of HMGA2 mRNA and protein ( Fig. 2A). This tumour had all the features of malignant uterine smooth muscle tumour, including tumour necrosis, cytological atypia, invasion and high mitotic counts (Fig. 2B). We speculated that overexpression of HMGA2 in ULMS-34 was contributed by chromosome 12 changes same as those seen in usual ULM.

HMGA2 and let-7 expression in ULMS
The levels of Let-7 expression in ULMS were low based on semi-quantitative RT-PCR analysis in 5 ULMS (Fig. 2A). Among five let-7 family members (let-7a, b, c, e, f-2) examined, let-7c transcript was detectable in all ULMS. We therefore selected let-7c to analyse its expression in 30 ULMS by TMA based in situ hybridization (see section 'Materials and methods'). We used U6 as tissue quality control (Fig.1B). The relative levels of let-7c were first normalized by U6 (let-7c/U6 ϫ 100%), and the net changes of let-7c in ULMS against matched myometrium (ULMS myometrium) were further calculated. Overall, there were slightly net losses of let-7c in ULMS in comparison to matched myometrium (Ϫ0.35 Ϯ 0.15). A case-matched analysis of HMGA2 and let-7c expression was performed. As illustrated in Fig. 1A, in those ULMS with positive immunoreactivity for HMGA2, net loss of let-7c expression was quite more common than in tumours negative for HMGA2. Correlation analysis showed an inverse association between HMGA2 and let-7c expression (r ϭ Ϫ0.39) (Table 1, Fig. 1A). In those five cases with RT-PCR data, higher levels of HMGA2 mRNA in ULMS-32 and -34 were accompanied by lower levels of let-7s ( Fig. 2A).

Translational regulation of HMGA2 by let-7s in ULMS cell lines
To test whether endogenous let-7s play a major role in repression of HMGA2 in ULMS, we selected three ULMS cell lines for the study. We first examined HMGA2 and its cryptic transcript (isoforms) [12,21] Table 1 Continued cultured leiomyoma cells [12], we used cultured leiomyoma cells as a positive control. We found all ULMS cell lines had a high abundance of HMGA2a mRNA (Fig. 3A). In addition, HMGA2 cryptic transcripts c, d and f showed moderate levels in these cell lines (Fig. 3A). In contrast, expression of let-7s was quite low in comparison to normal myometrium and ULM based on a semi-quantitative analysis (Fig. 3B). Let-7c and f were the most abundant transcripts of let-7 family in these cell lines.

Fig. 2 Semi-quantitative RT-PCR (upper panel) and Western blot (lower panel) analyses of HMGA2 and some let-7 family members in fresh frozen tissue samples of five ULMS. (A) HMGA2 mRNA was detectable in all 5 ULMS. One matched myometrium (MM) and one leiomyoma (ULM) were used as HMGA2 negative and positive control, respectively. Actin was used as loading control. Among let-7 members, let-7c was relatively higher than others in all ULMS. U6 was used as small RNA loading control. Western blot analysis of HMGA2 was examined in 4 ULMS (bottom). (B) Photographs illustrate gross appearance of a ULMS cross-section in case ULMS-34 and the corresponding karyotype (inserts with chromosome alterations) obtained in two different tumour regions.
and let-7 inhibitor in all cell lines. In untreated cell lines (controls), low levels of endogenous let-7s seemed not enough to destabilize HMGA2a and other cryptic mRNA (Fig. 3A and B), but were sufficient to repress HMGA2 translation (Fig. 3D, control lanes). When applying 40 pmol of exogenous let-7c, a significant reduction of HMGA2a, c, and f mRNAs (Fig. 3C) was observed, and a complete repression of HMGA2 protein was evident by a Western blot analysis (Fig. 3D, let-7c channel). When the cells treated with exogenous let-7 inhibitor (which can completely block endogenous let-7s), up to fivefold increases of HMGA2 protein were observed in comparison to the untreated cells (Fig. 3D). The findings further supported that HMGA2 repression by let-7s is specific regulatory mechanism for HMGA2 expression in uterine leiomyosarcoma cells.

Repression of HMGA2 inhibited ULMS cell growth
To test whether HMGA2 in ULMS cell lines plays a role in regulation of tumour growth rate, we examined the tumour cell proliferation by introducing exogenous let-7 and let-7 inhibitor, respec-tively. The cells were treated in triplicate with Block-iT (control), let-7c and let-7 inhibitor. Repression of HMGA2 by let-7c and induction of HMGA2 overexpression by let-7 inhibitor were validated as shown in Fig. 3C and D, we counted for the tumour growth rates at days 1, 2 and 3 in all three ULMS cell lines (Fig.  4). Starting from day 2 (48 hrs), cells treated with let-7c had significantly lower rates of tumour cell growth than control (P Ͻ 0.05). The differential growth rates were even wider at day 3 (72 hrs). Particularly, the growth rates in tumour cells treated with let-7 inhibitor were significantly higher than those in let-7c treated cells (P Ͻ 0.05), but at least two cell lines lower than those in control groups (Fig. 4). The later findings suggested that let-7 mediated tumour growth in ULMS is not solely through repression of HMGA2, and other let-7 target genes may be involved [22] (Fig. 4). changes involving chromosomal 12q13-15 region in leiomyosarcomas had been occasionally reported through traditional karyotype [23] and comparative genomic hybridization (CGH) analysis [4], in which a gain of chromosome 12q15 region including HMGA2 was identified. Examination of HMGA2 expression in anatomic sites other than the uterus revealed overexpression of HMGA2 mRNA in 57% of leiomyosarcomas by RT-PCR [13]. Examination of HMGA2 expression in ULMS has not been reported in the literature. This study is the first attempt to examine HMGA2 expression in ULMS. In this study, we observed that over one third of ULMS were immunoreactive for HMGA2. Furthermore, overexpression of HMGA2 mRNA was observed in all native ULMS and ULMS cell lines in a small scale analysis. Given that only one of five ULMS showed chromosomal 12 changes, overexpression of HMGA2 in ULMS seemed to be through an alternative mechanism.

ULMS usually have very complicated cytogenetic alterations involving many different chromosomal regions. Cytogenetic
The specific role of HMGA2 in tumourigenesis remains to be determined. Recent characterization of let-7 regulation of HMGA2 indicates that this molecular pair plays a critical role in the control of mitogenic activity in many different cell types and tumour cells [17,18,24,25]. Particularly, it has been found that dysregulation of let-7s is associated with HMGA2 expression in ULM and related to cell proliferation [17]. The later findings provided the evidence that this molecular pair is involved in benign uterine smooth muscle tumours. Further characterization of this molecular pair in its malignant counterpart ULMS may provide a new inside for our understanding of ULMS.
In this study, a seemly inverse association of HMGA2 protein and let-7c expression was observed. Although the global expression pattern of miRNA in ULMS remains to be determined, a loss of let-7 expression was identified in a proportion of ULMS. Particularly, we illustrate that exogenous let-7 can significantly reduce the tumour cell proliferation in leiomyosarcoma cell lines, largely through repression of HMGA2 expression (Figs 3 and 4). Therefore, it is speculated that disrupting of HMGA2 and let-7 pairs in ULMS could be an important molecular change responsible for ULMS growth. In our future study, it will be important to compare whether overexpression of HMGA2 and loss of let-7 are associated with unfavourable clinical outcome and specific histological grade in a large cohort of ULMS cases.
Another interesting finding is that the karyotype characterized by specific chromosome 12 changes, commonly seen in benign ULM, was also identified in one ULMS in our study. Further analysis revealed an overexpression of HMGA2 mRNA and protein in this tumour. The findings raise two questions: (1) [26]. The nature of LLA within ULMS has not yet been established. In the case with