High Mobility Group AT-hook 2 mRNA Research Articles

METTL3-mediated m6A modification of HMGA2 mRNA promotes subretinal fibrosis and epithelial-mesenchymal transition.

Subretinal fibrosis is a major cause of the poor visual prognosis for patients with neovascular age-related macular degeneration (nAMD). Myofibroblasts originated from retinal pigment epithelial (RPE) cells through epithelial-mesenchymal transition (EMT) contribute to the fibrosis formation. N6-methyladenosine (m6A) modification has been implicated in the EMT process and multiple fibrotic diseases. The role of m6A modification in EMT-related subretinal fibrosis has not yet been elucidated. In this study, we found that during subretinal fibrosis in the mouse model of laser-induced choroidal neovascularization, METTL3 was upregulated in RPE cells. Through m6A epitranscriptomic microarray and further verification, high mobility group AT-hook 2 (HMGA2) was identified as the key downstream target of METTL3, subsequently activating potent EMT-inducing transcription factor SNAIL. Finally, by subretinal injections of adeno-associated virus vectors, we confirmed that METTL3 deficiency in RPE cells could efficiently attenuate subretinal fibrosis in vivo. In conclusion, our present research identified an epigenetic mechanism of METTL3-m6A-HMGA2 in subretinal fibrosis and EMT of RPE cells, providing a novel therapeutic target for subretinal fibrosis secondary to nAMD.

Green Tea Epigallocatechin Gallate Inhibits Preadipocyte Growth via the microRNA-let-7a/HMGA2 Signaling Pathway.

This study investigates the effect of epigallocatechin gallate (EGCG) on white and beige preadipocyte growth and explores the involvement of the miR-let-7a/HMGA2 pathway. 3T3-L1 and D12 cells are treated with EGCG. The effect of EGCG on cell proliferation and viability is evaluated, as well as microRNA (miRNA)-related signaling pathways. EGCG inhibits 3T3-L1 and D12 preadipocyte growth, upregulates miR-let-7a expression, and downregulates high-mobility group AT-hook 2 (HMGA2) mRNA and protein levels in a time- and dose-dependent manner. In addition, overexpression of miR-let-7a significantly inhibits the growth of 3T3-L1 and D12 cells and decreases HMGA2 mRNA and protein levels. MiR-let-7a inhibitor antagonizes the inhibitory effects of EGCG on the number and viability of 3T3-L1 and D12 cells. Furthermore, miR-let-7a inhibitor reverses the EGCG-induced increase in miR-let-7a expression levels and decrease in HMGA2 mRNA and protein levels. HMGA2 overexpression induces an increase in cell number and viability and antagonizes EGCG-suppressed cell growth and HMGA2 expression in 3T3-L1 and D12 preadipocytes. EGCG inhibits the growth of 3T3-L1 and D12 preadipocytes by modulating the miR-let-7a and HMGA2 pathways.

Therapeutic role of adipose-derived mesenchymal stem cells-derived extracellular vesicles in rats with obstructive sleep apnea hypopnea syndrome.

Obstructive sleep apnea hypopnea syndrome (OSAHS) is an underestimated sleep disorder that leads to multiple organ damages, including lung injury (LI). This paper sought to analyze the molecular mechanism of extracellular vesicles (EVs) from adipose-derived mesenchymal stem cells (ADSCs) in OSAHS-induced lung injury (LI) via the miR-22-3p/histone lysine demethylase 6B (KDM6B)/high mobility group AT-hook 2 (HMGA2) axis. ADSCs and ADSCs-EVs were separated and characterized. Chronic intermittent hypoxia (CIH) was used to mimic OSAHS-LI, followed by ADSCs-EVs treatment and hematoxylin and eosin staining, TUNEL, ELISA, and assays of inflammation and oxidative stress (MPO/ROS/MDA/SOD). The CIH cell model was established and treated with ADSCs-EVs. Cell injury was assessed by the assays of MTT, TUNEL, ELISA, and others. Levels of miR-22-3p, KDM6B, histone H3 trimethylation at lysine 27 (H3K27me3), and HMGA2 were determine by RT-qPCR or Western blot analysis. The transfer of miR-22-3p by ADSCs-EVs was observed by fluorescence microscopy. Gene interactions were analyzed by dual-luciferase assay or chromatin immunoprecipitation. ADSCs-EVs effectively alleviated OSAHS-LI by reducing lung tissue injury, apoptosis, oxidative stress, and inflammation. Invitro, ADSCs-EVs increased cell viability and reduced apoptosis, inflammation and oxidative stress. ADSCs-EVs delivered enveloped miR-22-3p into pneumonocytes to upregulate miR-22-3p expression, inhibit KDM6B expression, increase H3K27me3 levels on the HMGA2 promoter, and decrease HMGA2 mRNA levels. Overexpression of KDM6B or HMGA2 attenuated the protective role of ADSCs-EVs in OSAHS-LI. ADSCs-EVs transferred miR-22-3p to pneumonocytes and reduced apoptosis, inflammation, and oxidative stress through KDM6B/HMGA2, mitigating OSAHS-LI progression.

HMGA2-Snai2 axis regulates tumorigenicity and stemness of head and neck squamous cell carcinoma

Cancer stem cells (CSCs) are a tumorigenic cell subpopulation, which contributes to treatment resistance, tumor recurrence, and metastasis. This study aimed to investigate the role and underlying molecular targets of high mobility group AT-hook 2 (HMGA2) in the progression and CSCs regulation of head and neck squamous cell carcinoma (HNSCC). HMGA2 mRNA and protein expression levels were examined in HNSCC specimens and cells by qRT-PCR, Western blot, and immunohistochemistry. The roles of HMGA2 were validated via loss-of-function and exogenous overexpression experiments in vitro and in vivo, and CSCs properties were assessed by tumorsphere formation assay. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays provided further insight into the molecular mechanisms by which HMGA2 regulates stemness. HMGA2 was abnormally overexpressed in HNSCC, and it promoted the expression of the CSCs markers including SOX2, CD133, CD44, ALDH1A1, and Bmi1. HMGA2 was correlated with stemness, malignant progression, and reduced survival in HNSCC. Luciferase reporter assay indicated that Snai2 was a direct downstream target gene of HMGA2. Mechanistically, ChIP-qPCR assay showed that HMGA2 was recruited to three binding sites on the Snai2 promoter, directly facilitating the transcription of Snai2 in HNSCC. Snai2 overexpression reversed the inhibitory effect of HMGA2 interference on the proliferation, invasion, and metastasis of HNSCC and CSC marker expression in vitro and in vivo. HMGA2 promoted the malignant progression of HNSCC and acquired CSCs properties through direct regulation of Snai2, thereby suggesting that targeting the HMGA2-Snai2 axis might be a promising therapeutic strategy for HNSCC.

155 A biomarker function of HMGA2 in cutaneous squamous cell carcinoma development

The high mobility group AT-hook 2 (HMGA2) gene encodes a transcription factor that is expressed during embryonic development but down-regulated in adult tissues. Re-expression of HMGA2 in adult tissues is often associated with both benign and malignant tumor formation. HMGA2 has been identified as a biomarker of melanoma progression and prognosis, but its role in non-melanoma skin cancer development remains controversial. In this study, we measured HMGA2 expression in normal human keratinocytes and found a significant decrease in HMGA2 mRNA expression in adult keratinocytes compared to neonatal keratinocytes. HMGA2 is highly expressed in human cutaneous squamous cell carcinoma (SCC) cell lines and primary human SCC tumors, but not detected in adjacent normal skin. In mouse skin, Hmga2 has been found to be translocated from the cell membrane into the nucleus during DMBA/TPA-induced skin tumorigenesis. While Hmga2 expression is absent in the hair follicle in non-UV-irradiated mouse, UV irradiation increased Hmga2 expression in both the epidermis and hair follicles. In agreement, we found enhanced Hmga2 expression in UV-induced mouse skin SCCs and also Hmga2 reactivation in the dermis from UV-irradiated mice. Furthermore, oncogene depletion such as FOXM1 and TRIP13 in human SCC cell lines using CRISPR/Cas9 decreased the expression of HMGA2 coupled with decreased cell proliferation and survival. Taken together, these results demonstrate that the upregulation of HMGA2 is an important biomarker of skin SCC tumorigenesis. Moreover, understanding of the mechanism for HMGA2 overexpression in skin hair follicle cells can develop the new drug for skin cancer treatment.

MicroRNA-34a-5p suppresses tumorigenesis and progression of glioma and potentiates Temozolomide-induced cytotoxicity for glioma cells by targeting HMGA2

Glioma is a frequently diagnosed brain tumors and Temozolomide (TMZ) is a common chemotherapeutic drug for glioma. High mobility group AT-hook 2 (HMGA2) was reported to be linked with glioma pathogenesis and Temozolomide (TMZ)-induced cytotoxicity. Our present study aimed to further search for the upstream regulatory microRNAs (miRNAs) of HMGA2 in glioma. RT-qPCR assay was conducted to measure the expression of HMGA2 mRNA and microRNA-34a-5p (miR-34a-5p). HMGA2 protein expression was examined by western blot assay. Cell proliferative ability and cell viability was assessed by CCK-8 assay. Cell migratory and invasive capacities were estimated by Transwell migration and invasion assay. Bioinformatics analysis and luciferase reporter assay was conducted to investigate the potential interaction between miR-34a-5p and HMGA2. Mouse xenograft experiments were performed to further test the roles of TMZ, miR-34a-5p and HMGA2, alone or in combination, in glioma tumorigenesis in vivo. We found HMGA2 expression was notably upregulated in glioma tissues and cells, and associated with glioma grade and poor prognosis. HMGA2 knockdown or miR-34a-5p overexpression inhibited migration, invasion, proliferation and enhanced TMZ-induced cytotoxicity in glioma cells. Moreover, HMGA2 was a target of miR-34a-5p. And, miR-34a-5p expression was remarkably reduced in glioma tissues and cells. MiR-34a-5p exerted its function through targeting HMGA2 in glioma cells. HMGA2 knockdown or miR-34a-5p overexpression inhibited tumor growth and enhanced TMZ-mediated anti-tumor effect in glioma xenograft models. We concluded MiR-34a-5p suppressed tumorigenesis and progression of glioma and potentiated TMZ-induced cytotoxicity for glioma cells by targeting HMGA2, deepening our understanding on molecular basis of HMGA2 in glioma.

MiR-302a-5p/367-3p-HMGA2 axis regulates malignant processes during endometrial cancer development

BackgroundMetastasis is one of the main reasons for treatment failure in endometrial cancer. Notably, high mobility group AT-hook 2 (HMGA2) has been recognized as a driving factor of tumour metastasis. microRNAs (miRNAs) are powerful posttranscriptional regulators of HMGA2.MethodsThe binding sites of miR-302a-5p and miR-367-3p on HMGA2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. The expression levels of miR-302a-5p and miR-367-3p were detected using quantitative real-time PCR and in situ hybridization. Western blotting and immunohistochemistry were used to detect the levels of HMGA2 and epithelial-mesenchymal transition pathway-related proteins. Co-immunoprecipitation was used to detect protein interactions. The roles of miR-302a-5p and miR-367-3p in the regulation of HMGA2 during the progression of endometrial cancer were investigated using both in vitro and in vivo assays.ResultsIn the present study, high HMGA2 expression was correlated with poor clinical outcomes in endometrial cancer. The binding sites of miRNAs on HMGA2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. In the endometrial cancer cell lines Ishikawa and HEC-1A, the overexpression of miR-302a-5p/367-3p significantly inhibited the expression of HMGA2 mRNA. In endometrial cancer tissues, we showed that miR-302a-5p and miR-367-3p were significantly downregulated and thus inversely correlated with HMGA2. The miR-302a-5p and miR-367-3p expression levels were closely correlated with FIGO stage and lymph node metastasis. High expression of miR-302a-5p/367-3p was correlated with high survival rates in endometrial cancer. In addition, miR-302a-5p/367-3p suppressed the malignant behaviour of endometrial carcinoma cells via the inhibition of HMGA2 expression.ConclusionOur findings indicate that miR-302a-5p/367-3p-mediated expression of HMGA2 regulates the malignant behaviour of endometrial carcinoma cells, which suggests that the miR-302a-5p/367-3p-HMGA2 axis may be a predictive biomarker of endometrial cancer metastasis and patient survival and a potential therapeutic target in metastatic endometrial cancer.

Circulating cell-free high mobility group AT-hook 2 mRNA as a detection marker in the serum of colorectal cancer patients.

Detection of circulating cell-free mRNA serves as noninvasive tools for cancer diagnosis. As an oncofetal protein, HMGA2 (high mobility group AT-hook 2) is upregulated in colorectal cancer (CRC) tissues. However, it is not clear whether the increased levels of circulating cell-free HMGA2 mRNA functions as potential biomarkers for improved diagnosis of CRC. To assess its clinical significance in diagnosis and prediction, we evaluated serum levels of circulating HMGA2 mRNA in CRC patients and in healthy controls. In this study, 83 CRC patients and 11 normal controls were enrolled in this study. We used real-time quantitative reverse transcription-PCR to evaluate the plasma mRNA levels of HMGA2 and analyze the correlation between their expression and clinicopathologic characteristics. We found that the levels of HMGA2 mRNA were significantly higher in CRC patients compared with healthy volunteers. The patients with right-sided CRC, colon cancer, positive nerve infiltration, positive vascular invasion, negative microsatellite instability (MSI), and increasing in serum carbohydrate antigen (CA) 199 had higher levels of plasma HMGA2 mRNA. A strong positive correlation between circulating cell-free HMGA2 mRNA and CA199 level in serum was found in our study. Furthermore, statistical analysis revealed that levels of HMGA2 mRNA in plasma and in tumors were strictly correlated. Collectively, our data suggested that cell-free HMGA2 mRNA in plasma might function as a novel diagnostic marker for CRC.

MiR-106a-5p Suppresses the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting HMGA2.

We aim to investigate the effect of miR-106a-5p on the proliferation, migration, and invasion of osteosarcoma (OS) cells by targeting high-mobility group AT-hook 2 (HMGA2). Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used for detecting the expressions of miR-106-5p and HMGA2 in 137 OS and adjacent normal bone tissues. Immunohistochemistry was applied for the HMGA2 protein expression detection. Luciferase reporter gene assay was conducted for verifying whether miR-106-5p targeted HMGA2. MG63 and U2SO cells were respectively divided into five groups: Blank, miR-106a-5p, scramble, HMGA2-siRNA, and miR-106a-5p+HMGA2 groups. RT-qPCR and western blot were applied for detecting the expressions of miR-106a-5p and HMGA2 in five groups. Proliferation rate, cell cycle, invasion, and migration ability of OS cells were detected using methyl thiazolyl-tetrazolium, 5-ethynyl-2'-deoxyuridine (Edu) assay, flow cytometry, and Transwell. Compared with adjacent normal tissues, OS tissues presented with decreased miR-106a-5p expressions, elevated HMGA2 mRNA, and positive expressions (all p < 0.05). The sensitivity and specificity of miR-106a-5p were 97.8%, 93.43%, and HMGA2 mRNA were 97.8%, 99.27%, separately. miR-106a-5p and HMGA2 expressions were associated with tumor size, Enneking stage, distant metastasis, and lung metastasis. Expressions of HMGA2 in OS cells in miR-106a-5p and HMGA2 siRNA groups were both significantly decreased with the same downregulation level, and the proliferation rates in both groups were obviously slowed down after 48 h (both p < 0.001). Edu positive cells, S phase cells (majority of cells blocked at G0/G1 phase), migratory and invasive cells were obviously decreased (all p < 0.05). Downregulation of miR-106a-5p was found in OS tissues, and upregulation of miR-106a-5p can inhibit the proliferation, migration, and invasion by targeting HMGA2 in OS cells.

Downregulating HMGA2 attenuates epithelial-mesenchymal transition-induced invasion and migration in nasopharyngeal cancer cells

BackgroundEpithelial-mesenchymal transition (EMT) is associated with invasion and metastasis of cancer cells. High-mobility group AT-hook 2 (HMGA2) has been found to play a critical role in EMT in a number of malignant tumors. However, whether HMGA2 regulates the EMT in human nasopharyngeal carcinoma (NPC) is unclear. ObjectiveThe aim of this study was to investigate the effect and mechanism of HMGA2 in inducing invasion and migration in NPC. MethodsIn NPC tissues samples, the association of HMGA2 mRNA expression with clinicopathological characteristics were estimated by real-time quantitative RT-PCR(qRT-PCR). In vitro, following the silencing of HMGA2 in CNE-1 and CNE-2 cell lines, the viability and metastatic ability were analyzed using Cell Counting Kit-8 (CCK8), colony formation assay, and transwell assay. EMT and transforming growth factor-beta (TGFβ)/Smad3 signaling pathway-related protein expression changes were evaluated using western blot. ResultsHMGA2 was upregulated in NPC cell lines and clinical specimens (P < 0.01), and HMGA2 expression correlated significantly with metastasis (P = 0.02) and disease-free survival of NPC (hazard ratio: 3.52; 95% confidence interval: 1.34–7.79; P = 0.01). In addition, following in vitro knockdown of HMGA2, the aggressiveness of cells was markedly inhibited, Vimentin and Snail level was downregulated and E-cadherin expression was upregulated. Moreover, the expression of key proteins TGFβRII and p-Smad3 of the TGFβ/Smad3 signaling pathway was inhibited by the downregulation of HMGA2. ConclusionHMGA2 might maintain EMT-induced invasion and migration through the TGFβ/Smad3 signaling pathway in NPC cell lines.

IMP-3 Promotes Migration and Invasion of Melanoma Cells by Modulating the Expression of HMGA2 and Predicts Poor Prognosis in Melanoma

IGF II mRNA-binding protein 3 (IMP-3) has been reported to be a marker of melanoma progression. However, the mechanisms by which it impacts melanoma are incompletely understood. In this study, we investigate the clinical significance of IMP-3 in melanoma progression and also its underlying mechanisms. We found that IMP-3 expression was much higher in advanced-stage/metastatic melanomas and that it was associated with a poor prognosis (P=0.001). Univariate analysis showed that IMP-3 expression was associated with stage III/IV melanomas (odds ratio=5.40, P=0.031) and the acral lentiginous subtype (odds ratio=3.93, P=0.0034). MeWo cells with overexpression of IMP-3 showed enhanced proliferation and migration and significantly increased tumorigenesis and metastatic ability in nude mice. We further demonstrated that IMP-3 could bind and enhance the stability of the mRNA of high mobility group AT-hook 2 (HMGA2). It was also confirmed that IMP-3 had an important role in melanoma invasion and metastasis through regulating HMGA2 mRNA expression. IMP-3 expression was positively correlated with HMGA2 expression in melanoma cells and also in melanoma tissues. Our results show that IMP-3 expression is a strong prognostic factor for melanoma, especially acral lentiginous melanoma.

Dysregulation of the Let-7/HMGA2 Axis with Methylation of p16 Promoter As a Possible Target of Histone Deacetylase Inhibitor in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal hematological disorders characterized by proliferation of mature blood cells. Recently, several agents that influence epigenetic modifications, such as histone deacetylase inhibitors (HDACi), as well as JAK2 inhibitors, have been investigated for high-risk MPNs. For example, an HDACi, panobinostat has shown significant efficacy including nearly complete response in PMF (Mascarenhas et al, BJH, 2013), but molecular targets of HDACi remain largely unknown. The High Mobility Group AT-hook 2 (HMGA2) is a non-histone chromatin protein that modulates transcriptions of various genes and contributes to chromatin modification and epigenetic regulation including DNA methylation (Fusco et al, Nat Rev Cancer, 2007; Sun et al, PNAS 2013). Let-7 micro RNAs (miRNAs) negatively regulate expression of HMGA2 through 3’UTR of HMGA2 mRNA, although HMGA2 mRNA consists of both the major variant containing 3’UTR with let-7-specific sites (variant 1) and some minor variants without 3’UTR. We previously reported that overexpression of HMGA2 due to transgenic expression of Hmga2 cDNA without 3’UTR caused proliferative hematopoiesis with providing a clonal advantage to hematopoietic stem cells in mice (Ikeda et al, Blood, 2011). We also showed a deregulation of HMGA2 mRNA expression due to reduced let-7 miRNA level in granulocytes from patients with almost all of PMF and over 20% of PV and ET (Harada-Shirado et al, Blood [Abst], 2013), being associated with splenomegaly, elevated serum LDH values, and methylation of p16 promoter. Thus, we hypothesized that HMGA2 may be a candidate gene as a therapeutic target in MPNs. Since association of HDAC with HMGA2 has been reported in cord blood-derived cells (Lee et al, Cell Mol Life Sci, 2011), we here studied effects of the panobinostat on expressions of HMGA2 and let-7 in HMGA2-expressing myeloid cells including PMF-derived CD34+ cells. First, we found significantly higher HMGA2 mRNA levels in CD34+ cells from 2 PMF patients compared with CD34+ cells from 2 healthy individuals (P<0.001), as well as U937 cells compared with HL60 cells (P<0.001). Thus, we used CD34+ cells from one of these 2 PMF patients and U937 cells for further experiments. Interestingly, treatment with panobinostat at the concentration of 40 nM for 8 hours significantly increased expressions of let-7a (P<0.001 and P=0.003, respectively), -7b (P<0.001 and P<0.001), and -7c (P<0.001 and P=0.06) in U937 cells and PMF CD34+ cells, compared with samples without the treatment. In contrast, Western blotting showed clearly reduced expression of HMGA2 protein in U937 cells after the treatment with panobinostat. Moreover, we found that variant 1 of HMGA2 mRNA with 3’UTR was significantly reduced by the treatment with panobinostat, compared with samples without the treatment in both U937 cells and PMF CD34+ cells (P<0.001 and P<0.001, respectively), while expression levels of variant 2 lacking let-7-specific sites were not changed by the treatment. These findings strongly suggested that panobinostat decreased expression of HMGA2 through 3’UTR of HMGA2 mRNA by increasing expressions of let-7 miRNAs. Of note, we next found much higher expression of variant 1 of HMGA2 mRNA than variant 2 in granulocytes from 15 of 17 (88%) MPN patients whose HMGA2 mRNA levels were higher than controls in our previous study (Blood [Abst], 2013). We also assessed if treatment by panobinostat for the dysregulated let-7/HMGA2 axis may be a therapeutic option for MPNs with respect to DNA methylation. Panobinostat treatment substantially reduced the expressions of DNMT1 and DNMT3a as well as HMGA2 proteins, significantly demethylated the p16 promoter (P<0.001), and decreased survival (P<0.001) in U937 cells. Moreover, knocking-down of HMGA2 with small interfering RNA in U937 cells significantly increased expression of TET3 mRNA and demethylated the p16 promoter, suggesting that HMGA2 expression may contribute to methylation of the p16 promoter. In conclusion, deregulated expression of HMGA2 due to downregulation of let-7 miRNAs, which may lead to some epigenetic modifications such as methylation of the p16 promoter, is a possible therapeutic target of HDACi in MPNs. DisclosuresIkeda:Novartis Pharmaceuticals: Other.

MicroRNA-26a acts as a tumor suppressor inhibiting gallbladder cancer cell proliferation by directly targeting HMGA2

MicroRNAs (miRNAs) are a class of small, single-stranded, non-coding RNA molecules which can act as oncogenes or tumor suppressor genes in human cancer. However, the possible functions and mechanisms of miRNA action in gallbladder cancer (GBC) have not been elucidated. In the present study, it was found that miR-26a was often downregulated in GBC and the expression of miR-26a was associated with neoplasm histological grade. miR-26a significantly inhibited the proliferation of GBC cells based on the gain-of-function assays. Furthermore, we demonstrated that high mobility group AT-hook 2 (HMGA2) was a direct target of miR-26a. The results showed that HMGA2 mRNA levels and miR-26a levels were negatively correlated. In addition, we confirmed that reintroduction of HMGA2 antagonized the inhibition of miR-26a to GBC cell proliferation and all these effects were achieved through the cell cycle. Together, all these results suggest that miR-26a expression contributes to GBC proliferation by targeting HMGA2. miR-26a shows promise as a prognosis factor and therapeutic target of GBC patients.

Frequent Overexpression of HMGA2 in Human Atypical Teratoid/Rhabdoid Tumor and Its Correlation with let-7a3/let-7b miRNA

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive pediatric malignancies characterized by biallelic inactivation of the SMARCB1 tumor suppressor gene. We searched for novel genomic aberrations by investigating the copy number and expression alterations of let-7a3/let-7b microRNA (miRNA) and correlated these with expression of high-mobility group AT-hook 2 (HMGA2) oncoprotein, a target of let-7 miRNA family, in 18 AT/RT samples to elucidate potential roles of HMGA2 in the pathogenesis of AT/RT. Genomic aberrations, let-7a3/let-7b miRNA and HMGA2 expression in AT/RT tissues were identified using quantitative PCR, reverse transcription PCR (RT-PCR), and immunohistochemistry. The impact of let-7b miRNA on HMGA2 expression and the malignant potential of human rhabdoid tumor cell G401 (SMARCB1(-/-)) were investigated by antisense inhibition and ectopic overexpression studies. The copy number of let-7a3/let-7b miRNA was substantially decreased in 4 of 11 AT/RT samples. A significantly inverse correlation between let-7a3/let-7b miRNA expression and HMGA2 mRNA expression was observed in AT/RT tissues (R = -0.34; P < 0.05). Immunohistochemistry analysis demonstrated that HMGA2 was highly overexpressed in 83.3% (15 of 18) of AT/RT tissues. Restoration of let-7 miRNA or knockdown of HMGA2 expression significantly suppressed proliferation and colony formation, and almost abolished the invasive potential of G401 cells. Reduction of let-7a3/let-7b miRNA may be one of mechanisms leading to overexpression of HMGA2 in AT/RT tissues. HMGA2 oncoprotein plays critical roles in the pathogenesis of AT/RT development; and reconstitution of let-7 miRNA or knockdown of HMGA2 oncoprotein may provide a novel therapeutic strategy for the treatment of patients with AT/RT.

The Role Of Deregulated HMGA2 Expression With Promoter Methylation Of p16 In Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPNs) are characterized by clonal proliferative hematopoiesis with increased mature blood cells. The signal-activating mutations such as JAK2V617F increase blood cells, but it remains uncertain how an abnormal hematopoietic cell clone expands in MPNs. We have recently showed that overexpression of the high mobility group AT-hook 2 (HMGA2) causes proliferative hematopoiesis with providing a clonal growth advantage to hematopoietic cells in mice (Ikeda et al, Blood, 2011), suggesting the possibility that HMGA2 contributes to the pathogenesis of MPNs. However, since only a few studies have evaluated expression of HMGA2 mRNA in patients with MPNs, the role of HMGA2 in the pathogenesis of MPNs is yet unclear. MPNs also show mutations in epigenetic modifiers involving DNA methylation such as polycomb group genes (PcG) and aberrant expressions of micro RNAs (miRNA) that negatively regulate expressions of targeted genes. Interestingly, deficiency in either PcG-related BMI1 (Oguro et al, J Exp Med, 2012) or let-7-family miRNA (Mayr et al, Science, 2007) causes deregulation of HMGA2 expression, leading to its oncogenic activity in part by negatively regulating tumor suppressor p16. Thus, in this study, to clarify the role of HMGA2 in MPNs, we investigated expression of HMGA2 mRNA in peripheral granulocytes of 56 patients with MPNs including 23 polycythemia vera (PV), 26 essential thrombocythemia (ET) and 7 primary myelofibrosis (PMF) along with clinical findings, JAK2V617F allele burden, expressions of BMI1 mRNA and let-7-family miRNAs, and promoter methylation of p16. Quantitative RT-PCR (qPCR) showed significantly higher expression of HMGA2 mRNA relative to internal control HPRT1 mRNA in PMF (mean ± SD; 31.7 ± 42.8, p<0.01), but not PV (15.7 ± 53.2) or ET (2.14 ± 7.70), compared with 12 healthy volunteers (HV; 0.431 ± 0.366). In addition, deregulated HMGA2 expression (>1.2), which was determined as relative expression level above mean + 2SD of HMGA2 mRNA in 12 HV, was most frequently detected in patients with PMF [7/7 (100%)] (p<0.01), compared with PV [5/23 (21.7%)] and ET [6/26 (23.1%)]. We also found a significant positive correlation in expression levels of HMGA2 mRNA with serum LDH values (r=0.531, p<0.01) rather than JAK2V617F allele burden (r=0.25, p=0.08). These data suggested that expression of HMGA2 mRNA independently correlated with disease phenotype and status in MPNs. We next explored the cause of deregulated expression of HMGA2 mRNA and found lower expression of let-7a (0.19 ± 0.13 vs. 0.42 ± 0.39, p=0.04) and -7c (0.57 ± 0.60 vs. 1.14 ± 0.94, p=0.06) rather than -7b (p=0.2) by qPCR, in patients with deregulated expression of HMGA2 mRNA compared with other patients. However, HMGA2-involved chromosomal abnormality in 12q13-15 was not detected in any patient, and there was no difference in expression of BMI1 mRNA between patients with deregulated expression of HMGA2 mRNA and other patients. Thus, decreased expression of let-7 miRNAs might contribute to deregulated expression of HMGA2 mRNA in MPNs. Finally, we investigated correlation of deregulated expression of HMGA2 mRNA with promoter methylation of p16. Methylation-specific PCR assay detected promoter methylation of p16 in 17/56 (30.4%) patients with MPNs. Strikingly, patients with deregulated expression of HMGA2 mRNA significantly more often showed promoter methylation of p16 compared with other patients [10/18 (55.6%) vs. 7/38 (18.4%), p<0.01]. Furthermore, patients with promoter methylation of p16 showed higher expression levels of HMGA2 mRNA than patients without the methylation, especially in patients with PMF (2.33 ± 0.90 vs. 70.9 ± 38.3, p=0.01). In conclusion, deregulated expression of HMGA2 in association with decreased expression of let-7 miRNAs may play a crucial role in the pathogenesis of MPNs possibly through p16. Disclosures:No relevant conflicts of interest to declare.

Features of Myeloproliferative Neoplasms and Clonal Advantage of Hematopoietic Stem Cells In Mice Expressing An Hmga2 cDNA Lacking the 3′UTR.

AbstractAbstract 1161Overexpression of high mobility group AT-hook 2 (HMGA2) has been identified in patients with paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndromes, and myeloproliferative neoplasms (MPN). Overexpression is often associated with truncation or deletions of the 3′ untranslated region (UTR) of HMGA2 and is regulated by the binding of let-7-family micro RNAs to the 3′UTR of HMGA2 mRNA. PNH is an acquired hemolytic anemia caused by clonal expansion of a hematopoietic cell (PNH cell) with a somatic mutation in the X-linked PIGA gene occurring in the background of bone marrow (BM) failure. The PIGA gene is essential in the synthesis of GPI-floats, and therefore progeny cells of this clone lack all GPI-linked proteins. However, the nature of the clonal growth advantage of a PNH cell is unclear. Here, to clarify the role of expression of 3′UTR-truncated HMGA2 in hematopoiesis and clonal expansion of hematopoietic cells, we generated transgenic mice (ΔHmga2 mice) expressing an Hmga2 cDNA with a truncation which removes six of the seven let-7-complementary sequences in the 3′UTR, which is similar to the truncations of HMGA2 found in two patients with PNH (Inoue et al., Blood, 2006). Expression of Hmga2 mRNA and protein were both significantly higher in BM, spleen, and thymus of ΔHmga2 mice compared to wild-type (WT) mice, with overexpression being more prominent in protein rather than mRNA. Unexpectedly, ΔHmga2 mice showed proliferative hematopoiesis that mimicked an MPN-like phenotype with increased numbers of peripheral white blood cells, red blood cells, and platelets, and low serum erythropoietin (EPO) values, hypercellular BM, and splenomegaly with extramedullary erythropoiesis compared to WT mice. Furthermore, colony assays of BM cells from ΔHmga2 mice showed significantly higher numbers of burst forming-unit erythroid, colony forming-unit erythroid (CFU-E), and CFU granulocyte-macrophage compared to those from WT mice. Formation of EPO-independent CFU-E was also observed. In competitive repopulation assays after BM transplant (BMT), BM cells from ΔHmga2 mice had a growth advantage over those from WT mice in all lineages. Surprisingly, cells derived from ΔHmga2 BM took over most of hematopoiesis during serial BMT with a ratio of 1:9 donor:recipient cells at the initial BMT, suggesting that overexpression of Hmga2 confers a clonal advantage to hematopoietic cells at the level of the hematopoietic stem cell (HSC). We found significantly higher expression of phosphorylated Stat3 in BM HSC-enriched KLS (c-kit+lineage-Sca-1+) cells and phosphorylated Akt in total BM cells from ΔHmga2 mice compared to WT mice without stimulation by cytokines, suggesting that intrinsic activation of Stat3 and Akt may contribute to proliferative hematopoiesis in ΔHmga2 mice. Microarray analysis of KLS cells and megakaryocyte-erythrocyte progenitor (MEP) cells showed a wide range of differences, in part mimicking MPN. When we bred our mice with circulating PNH cells (Piga- mice) with ΔHmga2 mice, the ΔHmga2+Piga+ cells and ΔHmga2+Piga- cells showed similar growth kinetics, indicating that the lack of GPI-linked proteins did not add an additional clonal advantage to the ΔHmga2+ cells. In summary, our results showed that the overexpression of a 3′UTR-truncated Hmga2 leads to proliferative hematopoiesis with clonal advantage at the level of HSC, which may also explain the effect of HMGA2 expression on clonal expansion in PNH or MPN. Disclosures:Bessler:Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.

HMGA2 gene expression analysis performed on cytologic smears to distinguish benign from malignant thyroid nodules

Up to 80% of thyroid nodules with an indeterminate diagnosis on fine-needle aspiration (FNA) (eg, "suspicious for follicular neoplasm") prove to be benign at the time of surgical resection. Ancillary tests in current use are limited in their ability to improve the preoperative detection of malignant follicular thyroid nodules. Studies using paraffin-embedded tissue have indicated that high mobility group AT-hook 2 (HMGA2) overexpression is present in a high percentage of malignant thyroid neoplasms but not in benign thyroid neoplasms. In the current study, the ability of HMGA2 overexpression analysis to preoperatively distinguish benign from malignant thyroid nodules by reverse transcriptase-polymerase chain reaction (RT-PCR) on suspicious cytologic smears was evaluated. Patients who underwent thyroid FNA and subsequent thyroid resection from 2001 through 2007 were identified. A subset of these patients who had a cytologic diagnosis of "suspicious" underwent HMGA2 expression analysis. HMGA2 expression was detected on cells scraped from cytologic smears with 1-step, real-time quantitative RT-PCR. Of the 125 cases identified, RNA extraction and HMGA2 analysis were successful in 115 cases. With an HMGA2 overexpression change of 5.9-fold or greater compared with a thyroid tumor cell line as a positive cutoff, the test was found to have the following overall performance for detecting malignant nodules: sensitivity of 71%, specificity of 97%, positive predictive value of 94%, and negative predictive value of 84%. HMGA2 overexpression was found to have low sensitivity for detecting Hurthle cell carcinoma (33%). HMGA2 mRNA expression analysis can be performed on cytologic smears and demonstrates a high specificity and positive predictive value and relatively high sensitivity and negative predictive value for detecting malignancy in "suspicious" thyroid aspirate specimens.

Dysregulation of BMPR2, Arginase II and HMGA2 Expression in Idiopathic Myelofibrosis and Secondary Myelofibrosis.

Idiopathic myelofibrosis (IMF) is a fatal clonal myeloproliferative disorder, characterized by variable pancytopenia, splenomegaly, leukoerythroblastic blood smear, marrow fibrosis (MF), and myeloid metaplasia. Like all myeloproliferative disorders, it is due to an acquired somatic mutation of a single hematopoietic progenitor. MF has also been reported in systemic lupus and metastatic malignancies involving bone marrow. We have recently demonstrated the occurrence of MF in 85% patients with pulmonary hypertension (PH), which (unlike IMF) is associated with polyclonal hematopoiesis and normal number of circulating CD34+ cells. The pathophysiological mechanisms resulting in IMF and secondary MF (SMF) in PH remain still unclear. To date, 52 heterozygous germline mutations in the gene (BMPR2) encoding the bone morphogenetic protein type II receptor (BMPR-II) have been found to characterize many cases of familial (Lane, 2000) and sporadic primary PH (PPH) (Thomson, 2000). Recently, increased activity of arginase in pulmonary tissue of patients with PH was found. This may contribute to the pathogenesis of pulmonary hypertension by decreasing the substrate for NO synthesis. We examined BMPR2 and Arginase II mRNA levels by quantitative real-time RT-PCR in mononuclear cells (MNC), platelets (PLT) and granulocytes (GNC) from IMF patients and PH patients with secondary MF. We show for the first time that the mRNA level of BMPR2 is increased in PLT and GNC of IMF compared with normal controls. Arginase II mRNA was significantly increased in GNC of IMF as well as PH patients with SMF. Rearrangement and over expression of high-mobility group AT-hook2 (HMGA2) gene in myeloid progenitors in two patients with IMF was recently reported. This gene is known to play a major role in fetal growth and development, and is expressed in trace amount in adult lung, kidney, and synovia. We found significantly increased HMGA2 mRNA expression in MNC of IMF patients. In summary, the biomarkers studied here can be abnormal in both IMF and PH with secondary MF and may not be useful to discriminate between the two types of MF. Dysregulation of BMPR2, Arginase II and HMGA2 expression may play a pathophysiological role in MF of both IMF and PH.Expression of BMPR2, Arginase II and HMGA2 in Blood Cells of IMF and SMFGeneCellsControl (ΔCT)Idiopathic MF (ΔCT)Secondary MF (ΔCT)BMPR2PLT18.5±1.216.7±1.4 (p=0.038) *18.4±2.2 (p=0.881)GNC19.2±1.018.3±1.3 (p=0.009) *19.1±1.2 (p=0.893)MNC18.3±1.219.2±1.8 (p=0.059)19.0±2.7 (p=0.554)Arginase IIGNC24.5±0.621.9±1.7 (p=0.000) *22.6±1.2 (p=0.004) *MNC24.4±3.524.4±3.9 (p=0.965)22.5±2.4 (p=0.222)HMGA2MNC32.4±1.629.3±1.7 (p=0.000) *30.8±1.8 (p=0.085)*The P value represents a significant difference of gene expression in IMF or SMF compared with normal controls. A lower ΔCT indicates a higher gene expression.

Fenofibrate increases the expression of high mobility group AT-hook 2 (HMGA2) gene and induces adipocyte differentiation of orbital fibroblasts from Graves' ophthalmopathy.

Expansion of adipose tissue in the orbits is a key feature of Graves' ophthalmopathy. Recent evidence shows that orbital fibroblasts are committed to differentiate into adipocytes under appropriate stimuli. Rosiglitazone, an agonist of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma) is able to induce both differentiation of orbital fibroblasts into mature adipocytes and expression of the TSH receptor (TSHr) gene. Several studies have suggested an important role of the high mobility group AT-hook 2 (HMGA2) gene in adipocytic cell growth and development. To investigate further the association between adipogenesis-related genes and orbital fibroblasts, we treated fibroblasts from Graves' ophthalmopathy (FGOs) and from normal orbital tissues with fenofibrate, a specific agonist for PPARalpha. We then evaluated the expression of the PPARalpha, PPARgamma2, HMGA2, leptin and TSHr genes before and after 24 h of fenofibrate treatment, using semiquantitative and real-time PCR. For up to 96 h after exposure to fenofibrate, FGOs differentiated into adipocytes. PPARalpha and PPARgamma2 were expressed more in FGOs than in normal cultures, whereas TSHr mRNA was detected only in FGOs. Expression of HMGA2 mRNA and protein was significantly increased in FGOs from 6 to 24 h after fenofibrate, confirming its role in the early phase of adipocyte differentiation. Treatment with fenofibrate for 24 h significantly increased the expression of leptin and TSHr genes. Moreover, TSH treatment significantly increased the accumulation of cAMP, demonstrating that FGOs express functional TSHr. The high level of expression of PPARalpha other than PPARgamma2 transcripts and the stimulating effect of fenofibrate on adipogenesis and on HMGA2, leptin and TSHr genes also indicate that the PPARalpha pathway plays an important part in the adipocyte differentiation of FGOs. These findings suggest that novel drugs to antagonize PPARalpha, other than the PPARgamma signalling system, may also need to be considered in the treatment or prevention of Graves' ophthalmopathy.