HMGA2 mRNA Expression Research Articles

Silencing LINC00987 ameliorates adriamycin resistance of acute myeloid leukemia via miR-4458/HMGA2 axis

BackgroundMost patients with acute myeloid leukemia (AML) eventually develop drug resistance, leading to a poor prognosis. Dysregulated long gene non coding RNAs (lincRNAs) have been implicated in chemoresistance in AML. Unfortunately, the effects of lincRNAs which participate in regulating the Adriamycin (ADR) resistance in AML cells remain unclear. Thus, the purpose of this study is to determine LINC00987 function in ADR-resistant AML.MethodsIn this study, ADR-resistant cells were constructed. LINC00987, miRNAs, and HMGA2 mRNA expression were measured by qRT-PCR. P-GP, BCRP, and HMGA2 protein were measured by Western blot. The proliferation was analyzed by MTS and calculated IC50. Soft agar colony formation assay and TUNEL staining were used to analyze cell colony formation and apoptosis. Xenograft tumor experiment was used to analyze the xenograft tumor growth of ADR-resistant AML.ResultsWe found that higher expression of LINC00987 was observed in AML patients and associated with poor overall survival in AML patients. LINC00987 expression was increased in ADR-resistant AML cells, including ADR/MOLM13 and ADR/HL-60 cells. LINC00987 downregulation reduces ADR resistance in ADR/MOLM13 and ADR/HL-60 cells in vitro and in vivo, while LINC00987 overexpression enhanced ADR resistance in MOLM13 and HL-60 cells. Additionally, LINC00987 functions as a competing endogenous RNA for miR-4458 to affect ADR resistance in ADR/MOLM13 and ADR/HL-60 cells. HMGA2 is a target of miR-4458. LINC00987 knockdown and miR-4458 overexpression reduced HMGA2 expression. HMGA2 overexpression enhanced ADR resistance, which reversed the function of LINC00987 silencing in suppressing ADR resistance of ADR/MOLM13 and ADR/HL-60 cells.ConclusionsDownregulation of LINC00987 weakens ADR resistance by releasing miR-4458 to deplete HMGA2 in ADR/MOLM13 and ADR/HL-60. Therefore, LINC00987 may act as the therapeutic target for treating chemoresistant AML.

Insulin-like growth factor 2 mRNA-binding protein 3 enhanced melanoma migration through regulation of AKT1 and RELA expression.

IMP-3 expression is a poor prognostic factor of melanomas and it promotes melanoma cell migration and invasion by a pathway modulating HMGA2 mRNA expression. We tried to identify other putative targets of IMP-3. We identified putative IMP-3-binding RNAs, including AKT1, MAPK3, RB1 and RELA, by RNA immunoprecipitation coupled with next-generation sequencing. IMP-3 overexpression increased AKT and RELA levels in MeWo cells. siRNAs against AKT1 and RELA inhibited MeWo/Full-length IMP-3 cell migration. IMP-3 knockdown of A2058 cells decreased AKT1 and RELA expression and lowered migration ability. Co-transfection of A2058 cells with AKT1- or RELA-expressing plasmids with IMP-3 siRNA restored the inhibitory effects of IMP-3 knockdown on migration. HMGA2 did not influence AKT1 and RELA expression in melanoma cells. Human melanoma samples with high IMP-3 levels also showed high HMGA2, AKT1 and RELA expression. Our results show that IMP-3 enhances melanoma cell migration through the regulation of the AKT1 and RELA axis.

Study of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples.

Simultaneous quantitative measurement of mRNA of the WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples reflects the specific pathological proliferative activity in acute leukemia and their ratio is promising as a diagnostic marker. The transcriptome profile of acute leukemia cells is usually assessed using NGS or microarray techniques after a preliminary procedure for isolation of mononuclear cells. However, the results of using the multiplex PCR reaction for the simultaneous determination of all above mRNAs in whole blood samples have not been published so far. Determination of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in venous blood level samples by multiplex RT-PCR. The study included 127 blood samples from patients who diagnosis of acute leukemia was subsequently confirmed. In the comparison group, 87 samples of patients without oncohematological diagnosis were selected, including 31 samples (K1) with a normal blood formula and 56 samples (K2) with a violation of the cellular composition - anemia, leukocytosis and thrombocytopenia. RNA isolation and reverse transcription were performed using the Ribozol-D and Reverta-L kits (TsNIIE, Russia). Determination of the mRNA expression level of the WT1, BAALC, EVI1, PRAME and HMGA2 genes by multiplex real-time PCR using a homemade multiplex PCR kit. The mRNA level was characterized by high interindividual variation and did not correlate with the rate of circulating leukocytes or blood blasts. Expression of WT1 mRNA was observed in whole blood only in one patient from the control group and in 112 (88%) patients with leukemia and was combined with a decrease in the level of HMGA2 mRNA expression and BAALC mRNA values. In contrast to the control groups, patients with leukemia had higher levels of BAALC mRNA in AML and ALL, increased PRAME mRNA in AML and APL, but lower levels of HMGA2 in APL.

Abstract IA041: Basic science mechanisms associated with bone tumor microenvironment and health disparities in breast and prostate cancer

Abstract Prostate cancer (PCa) and breast cancer (BCa) are leading causes of cancer related deaths in American men and women, and morbidity is due to metastasis preferentially to bone. Compared to Caucasian American (CA), African American (AA) men and women have a higher bone density, as well as mortality rate due to bone metastasis. Recent studies have shown that high mobility group two (HMGA2), a non-histone chromatin binding protein, plays a critical role in promoting epithelial-mesenchymal transition (EMT) and metastasis. HMGA2 full-length/wild-type and truncated (without the 3’UTR) isoforms have been shown to be overexpressed in several cancers, however their distinct roles in metastasis have not been reported. Our laboratory focuses on tumor-micronenvironmental interactions, particularly, how PCa cells interact with bone at the metastatic site. We have previously published that PCa cells may degrade hydroxyapatite, the major inorganic component of bone, to release calcium which promotes paracrine cell proliferation and migration, and this signaling increases with increased bone density. We hypothesize that EMT is associated with prostate health disparities and that HMGA2 isoforms may play differential roles at the bone metastatic site. We examined EMT marker expression in breast and prostate patient tissue and cell lines by immunohistochemistry (IHC), RNA In Situ Hybridization (RISH), western blot, and real-time PCR. Next, LNCaP PCa cell line stably overexpressing either full length or truncated HMGA2 was co-cultured with low (100 mg) or high (200 mg) hydroxyapatite, demineralized bone matrix or human ground bone matrix, for 6 days followed by collection of condition media (CM). CM was added to parental LNCaP cells for various time points followed by analysis of paracrine cell proliferation using MTS assay, cell migration across collagen using boyden chamber, and the effect on various pathways using western blot analysis. EMT marker (Snail and Cathepsin L) protein expression was higher in AA normal and cancer tissue compared to CA, while it was intermediate in Bahamian tissue. Wild-type HMGA2 mRNA expression was higher in metastatic patient tissue compared to truncated HMGA2, and higher in AA compared to CA tissue. CM taken from LNCaP cells overexpressing wild-type and truncated HMGA2 co-cultured with hydroxyapatite, demineralized bone matrix or ground bone matrix when added to parental LNCaP cells led to higher paracrine cell proliferation and paracrine cell migration. This was associated with MAPK activation that could be abrogated by MAPK inhibitor, UO126. These results indicate that EMT is higher in AA compared to CA. Additionally, both wild-type and truncated HMGA2 interaction with bone matrix promotes paracrine cell proliferation and migration via ERK signaling. Our study shows that wild-type and truncated isoforms of HMGA2 mediate PCa/bone interactions which may lead to increased bone metastasis, particularly in African American men that have higher bone density. Citation Format: Taaliah Campbell, Ohuod Hawsawi, Bor-Jang Hwang, Liza J. Burton, Quentin Loyd, Veronica Henderson, Simone Howard, Camille Ragine, Robin Roberts, Andrew Gachii, Valerie Odero-Marah. Basic science mechanisms associated with bone tumor microenvironment and health disparities in breast and prostate cancer [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr IA041.

RNA Binding Protein RBM47 Inhibits the K562 Cell Proliferation by Regulating HMGA2 mRNA Expression

To investigate the regulatory effects of RBM47 on HMGA2 and the function of RBM47 in human chronic myeloid leukemia cell K562. K562 cells were transduction by the overexpressed and knockdown RBM47 lentiviral vector. CCK-8 assay was used to detect the effect of RBM47 on the proliferation of K562 cells. Flow cytometry assay was used to detect the effect of RBM47 on the cell cycle progression of K562 cells. RNA immunoprecipitation assay was used to detect the association between RBM47 and HMGA2 mRNA. RT-qPCR was used to detect the effects of RBM47 on the stability of HMGA2 mRNA. Western blot was used to evaluate the effect of RBM47 on HMGA2 protein expression. The overexpressed RBM47 could inhibit the proliferation and cell cycle progression of K562 cells. However, the inhibitation of RBM47 could improve the proliferation and cell cycle progression of K562 cells. RBM47 combined with HMGA2 mRNA could promote the degradation of HMGA2 mRNA. Thus, the overexpressed RBM47 could decrease the expression of HMGA2 protein in K562 cells. RNA binding protein RBM47 can inhibit the proliferation of K562 cells by regulating HMGA2 expression.

Expression of erythropoietin messenger ribonucleic acid in wild-type MED12 uterine leiomyomas under estrogenic influence: new insights into related growth disparities

To determine factors that impact erythropoietin (EPO) production in leiomyomas. We have previously implicated EPO production in promoting the growth of some leiomyomas. The relationship between EPO messenger RNA (mRNA) expression and MED12 gene mutations or mRNA expression levels of high-mobility group AT-hook (HMGA) 1 and HMGA2 were analyzed. Effects of 10-8M 17β-E2 on EPO mRNA expression were evaluated using leiomyoma cells grown in primary cultures. Graduate school of medicine. Patients with leiomyoma. We used tissue samples and clinical data of 108 patients with leiomyomas to analyze the relation between EPO mRNA expression and MED12 mutation. Tissue samples from another 10 patients with leiomyomas were collected for invitro experimentation using primary cultures of leiomyoma and myometrial cells. Relations between EPO mRNA expression, MED12 exon 2 mutation, and HMGA1/HMGA2 mRNA expression levels in leiomyoma samplings, in addition to effects of estrogen (E) on EPO mRNA expression in cultures of leiomyoma cells. The EPO mRNA level was threefold higher in leiomyomas with wild-type (vs. mutated) MED12 genes. There was no correlation between EPO and HMGA1 or HMGA2 mRNA expression levels. In wild-type MED12 leiomyomas only, E2 treatment produced a twofold increase in EPO mRNA expression, whereas mutated MED12 leiomyomas were unaffected. The EPO mRNA expression increased significantly after E2 treatment only in leiomyomas lacking MED12 mutations. In conjunction with prior evidence linking EPO mRNA expression levels and tumor size, E2-stimulated EPO mRNA expression may explain the marked growth disparities seen in these tumors.

HMGA2 mRNA Expression in Patients with Myelodysplastic/Myeloproliferative Neoplasms (MDS/MPN)

MDS/MPN comprise a group of uncommon myeloid neoplasms with clinical and hematological features overlapping MDS and MPN. Pathogenesis remain elusive. HMGA2 on chromosome 12q plays a crucial role in self-renewal and proliferation of fetal hematopoietic stem cells (HSCs), but not adult HSCs because of repression by binding of MIRLET-7 with the 3'-untranslated region (3'UTR) of HMGA2 (Copley et al, Nat Cell Biol, 2013). Trimethylation of histone H3 at lysine 27, which is catalyzed by EZH2 - a member of polycomb recessive complex 2 (PRC2) - also suppresses HMGA2 (Sashida et al, JEM, 2016). We have demonstrated that expression of Hmga2 cDNA lacking 3'UTR caused proliferative hematopoiesis mimicking MPN in young mice and led to anemia with ineffective erythropoiesis and leukocytosis like MDS/MPN in old mice (Ikeda et al, Blood, 2011; Ueda et al, Blood Adv, 2017), suggesting that HMGA2 may contribute to the pathogenesis of MDS/MPN. In fact, we found high HMGA2 mRNA levels in granulocytes of MPN including >90% of patients with myelofibrosis (MF) and about 30% with essential thrombocythemia (ET), and either reduced MIRLET-7c levels or PRC2-related mutations were correlated with high HMGA2 mRNA in MF, while reduced MIRLET-7c with no particular mutation was correlated with high HMGA2 mRNA in ET (Harada-Shirado et al, BJH, 2015; Blood Adv, 2017). However, in MDS/MPN, only one case series has reported high HMGA2 mRNA due to the 12q rearrangement that removed the 3'UTR of HMGA2 in a few patients (Odero et al, Leukemia, 2005). So far, expression of HMGA2 has not been studied in MDS/MPN without the 12q rearrangement.To clarify the expression of HMGA2 in MDS/MPN in relation to clinical and other genetic findings, we investigated HMGA2 mRNA and MIRLET-7c levels by real-time RT-PCR and sought PRC2-related mutations by targeted deep sequencing in peripheral leukocytes of 11 patients with MDS/MPN [5 chronic myelomonocytic leukemia (CMML), 2 atypical chronic myeloid leukemia (aCML), and 4 unclassifiable MDS/MPN (MDS/MPN-u)]. In addition, to identify the features specific for MDS/MPN, we also evaluated 10 patients with low-risk MDS [MDS with multilineage dysplasia (MDS-MLD)]. No MDS/MPN or MDS-MLD patients had 12q rearrangement.In MDS/MPN, high HMGA2 mRNA [>1.0 relative to HPRT1 mRNA above mean + 2SD of healthy volunteers, per our previous study (Blood Adv, 2017)] was found in each subtype: 2 of 5 (40%) in CMML, 1 of 2 (50%) in aCML, and 3 of 4 (75%) in MDS/MPN-u. Therefore, more than half of patients with MDS/MPN (6 of 11, 54.5%) showed high HMGA2 mRNA levels. In contrast, high HMGA2 mRNA was found in only 1 of 10 (10%) patients with MDS-MLD (P = 0.06). Reduction in MIRLET-7c was seen in 5 of 11 (45.5%) MDS/MPN and 3 of 10 (30.0%) MDS-MLD (P = 0.16). Among these 5 MDS/MPN patients with low MIRLET-7c, only 2 patients had high HMGA2 mRNA. Thus, MIRLET-7c reduction did not correlate with deregulation of HMGA2 mRNA in MDS/MPN as frequently as in MPN. In contrast, HMGA2 mRNA levels were high in all 3 patients with MDS/MPN carrying EZH2 mutations, suggesting that loss of EZH2 might augment HMGA2 in MDS/MPN. Laboratory findings of MDS/MPN patients with high HMGA2 mRNA were not different from those without high HMGA2 mRNA. However, MDS/MPN patients with both high HMGA2 mRNA and an EZH2 mutation showed more severe anemia compared with those with high HMGA2 mRNA without an EZH2 mutation (hemoglobin level: 9.6±2.0 vs. 11.0±2.3 g/dL, P = 0.05).In conclusion, we demonstrated that MDS/MPN cases highly express HMGA2 mRNA even without 12q rearrangement, more often than MDS-MLD. Unlike MPN, HMGA2 mRNA levels did not correlate with reduced MIRLET-7c, but probably with mutations in EZH2, which may also contribute to anemia in MDS/MPN. DisclosuresNo relevant conflicts of interest to declare.

Overexpression mRNA of HMGA2 and WT1 Genes in Whole Blood Samples As Potential Diagnostic Markers in MPN

BackgroundHigh-mobility group AT-hook protein 2 (HMGA2) gene codes for a non-histone protein which expressed during embryogenesis and early hematopoiesis but suppressed in differentiated cells. Overexpression of HMGA2 as well as transcription factor Wilms' tumor 1 (WT1) gene has been found in a variety of human cancers, but only a few reports has included myeloproliferative neoplasms (MPNs) (Guglielmelli P. et al., 2007; Chen C-C. et al., 2017; Tasdemir S. et al, 2015). Furthermore, there were no any data about co-expression of mRNA both genes in blood in patients with MPNs.Aims: evaluate of the diagnostic potential of the WT1 and HMGA2 mRNA level in MPN patients.MethodsIn our retrospective study was analysed 115 archived frozen peripheral blood samples with RNAse inhibitor from 62 MPN patients: 29 with polycythemia vera (PV), 16 with essential thrombocythemia (ET), and 17 with myelofibrosis (MF) were included. Samples of 28 healthy volunteers were tested as control group. Total RNA was isolated using “Ribo-zol-D” (Aplisens) and then were transcribed using “Reverta-L” (Aplisens). Synthesized cDNA was analysed by real-time PCR to detect of WT1 and HMGA2 expression levels on the CFX96 (Bio-Rad) housekeeping kits using. The results were calculated utilizing the delta Ct method in the software package of “R”. The threshold cycles (Ct) WT1 and HMGA2 genes and reference gene ABL determined using Cy0 method. Results are presented as median and range (25% and 75%). Comparisons between the patient and control group were made using the Mann-Whitney U test. Statistical analysis was performed using Statistica 12.0 software.ResultsThe level of HMGA2 mRNA in five samples of venous blood in PV patients and 12 samples in MF patients was higher than in healthy subjects (Figure 1). Expression of WT1 mRNA (≥ 0.01%) was observed only in samples among patients with MF (in 19 of 28). In 12 blood samples of patients with MF, transcripts of both genes were detected together (Table 1). The increased level of HMGA2 mRNA expression was associated with a higher level of erythrocytosis in patients with PV (p<0.05). The co-expression of HMGA2 mRNA and WT1 mRNA in patients with MF was associated with a low level of white and red blood cell (p<0.05). Interestingly that in MF patients the most pronounced thrombocytopenia was observed if only WT1 mRNA was detected. In all cases of repeated testing of patients, the growth of HMGA2 or WT1 mRNA level was accompanied by decline of the cellular blood composition. The level of HMGA2 mRNA and WT1 mRNA in our study did not depend on the driver mutations presence or their allele burden. It should be noted that 4 out of 12 patients with MF died within 5 months after the first detection of WT1 mRNA and one patient died more after 15 months. At that time only one MF patient without WT1 or HGMA2 transcripts died during of 20 months' observation.Summary and ConclusionsExpression of the WT1 and HMGA2 genes is involved in the pathogenesis and progression of the disease and can be used as diagnostic markers. The question of the independent prognostic significance of these transcripts in blood samples for various phenotypes of MPN is promising for further study. DisclosuresNo relevant conflicts of interest to declare.

HMGA2 and MED12 alterations frequently co-occur in uterine leiomyomas

ObjectiveAround 70% of uterine leiomyomas show MED12 mutations while overexpression of HMGA2 mRNA is also highly frequent in fibroids. However, previous studies suggested that alterations in both genes are mutually exclusive. In the present study, we searched for mutation in MED12 and analyzed the expression of HMGA2 in 20 uterine leiomyomas and their matched myometrium. MethodsNormal and tumor tissue obtained from premenopausal women who underwent hysterectomy were collected after surgery and DNA, RNA and proteins were isolated and analyzed for MED12 mutations using Sanger sequencing, HMGA2 mRNA expression by quantitative PCR and HMGA2 protein detection by western blot and immunohistochemistry. Results75% of the tumors displayed MED12 mutation while 65% of them showed overexpression of HMGA2 mRNA in leiomyomata compared to myometrial tissues (p = 0,0008). Interestingly, 50% of the tumors showed mutations in MED12 and overexpression of HMGA2 mRNA simultaneously, suggesting that alterations in both genes are relatively frequent in uterine leiomyomas. ConclusionsContrary to the present findings, former studies showed that mutations in MED12 and overexpression of HMGA2 are mutually exclusive. Here, we observed that overexpression of HMGA2 mRNA in tumors measured by quantitative PCR and compared to myometrium is a common phenomenon in fibroids and is frequently associated with MED12 mutations. In addition, the common clonal origin of tumors overexpressing HMGA2 mRNA and its expression in few myometrial tissue points to HMGA2 up-regulation as an early event in leiomyoma tumorigenesis.

A study of the WT1 and HMGA2 mRNA expression in myeloproliferative neoplasms

A study of the WT1 and HMGA2 mRNA expression in myeloproliferative neoplasms

HMGA2 overexpression plays a critical role in the progression of esophageal squamous carcinoma.

Esophageal Squamous Cell Carcinoma (ESCC) is the most common esophageal tumor worldwide. However, there is still a lack of deeper knowledge about biological alterations involved in ESCC development. High Mobility Group A (HMGA) protein family has been related with poor outcome and malignant cell transformation in several tumor types. In this way, the aim of this study was to analyze the expression of HMGA1 and HMGA2 expression in ESCC and their role in crucial cellular features. We evaluated HMGA1 and HMGA2 mRNA expression in 52 paired ESCC and normal surrounding tissue samples by qRT-PCR. Here, we show that HMGA2, but not HMGA1, is overexpressed in ESCC samples. This result was further confirmed by the immunohistochemical analysis. Indeed, accordingly to mRNA expression data, HMGA2, but not HMGA1, was overexpressed in approximately 90% of ESCC samples, while it was barely expressed in the respective control. Conversely, HMGA1, but not HMGA2, was overexpressed in esophageal adenocarcinoma samples. Interestingly, HMGA2 abrogation attenuated the malignant phenotype of two ESCC cell lines, suggesting that HMGA2 overexpression is involved in ESCC progression.

The MYCN-HMGA2-CDKN2A pathway in non-small cell lung carcinoma—differences in histological subtypes

BackgroundExtensive research has increased our understanding of the molecular alterations needed for non-small cell lung cancer (NSCLC) development. Deregulation of a pathway including MYCN, HMGA2 and CDKN2A, with the participation of DICER1, is of importance in several solid tumours, and may also be of significance in the pathogenesis of NSCLC.MethodsGene expression of MYCN, HMGA2, CDKN2A and DICER1 were investigated with RT-qPCR in surgically resected NSCLC tumour tissue from 175 patients. Expression of the let-7 microRNA family was performed in 78 adenocarcinomas and 16 matching normal lung tissue samples using microarrays. The protein levels of HMGA2 were determined by immunohistochemistry in 156 tumour samples and the protein expression was correlated with gene expression. Associations between clinical data, including time to recurrence, and expression of mRNA, protein and microRNAs were analysed.ResultsCompared to adenocarcinomas, squamous cell carcinomas had a median 5-fold increase in mRNA expression of HMGA2 (p = 0.003). A positive correlation (r = 0.513, p < 0.010) between HMGA2 mRNA expression and HMGA2 protein expression was seen. At the protein level, 90 % of the squamous cell carcinomas expressed high levels of the HMGA2 protein compared to 47 % of the adenocarcinomas (p < 0.0001). MYCN was positively correlated with HMGA2 (p < 0.010) and DICER1 mRNA expression (p < 0.010), and the expression of the let-7 microRNAs seemed to be correlated with the genes studied. MYCN expression was associated with time to recurrence in multivariate survival analyses (p = 0.020).ConclusionsA significant difference in HMGA2 mRNA expression between the histological subtypes of NSCLC was seen with a higher expression in the squamous cell carcinomas. This was also found at the protein level, and we found a good correlation between the mRNA and the protein expression of HMGA2. Moreover, the expression of MYCN, HMGA2, and DICER1 seems to be correlated to each other and the expression of the let7-genes impacted by their expression. MYCN gene expression seems to be of importance in time to recurrence in this patient cohort with resected NSCLC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2104-9) contains supplementary material, which is available to authorized users.

MicroRNA-490-3p regulates cell proliferation and apoptosis by targeting HMGA2 in osteosarcoma

MicroRNA-490-3p (miR-490-3p) has been implicated in several human malignancies; however, its potential functions and the underlying molecular mechanisms in osteosarcoma progression remain largely unclear. Here, we showed that miR-490-3p was down-regulated in osteosarcoma cell lines. Ectopic expression of miR-490-3p decreased cell proliferation, induced G1 arrest and apoptosis in vitro and inhibited tumorigenicity in a mouse xenograft model. Furthermore, miR-490-3p bound directly to HMGA2 mRNA 3′UTR and mediated a decrease in HMGA2 mRNA and protein expression. Re-expression of HMGA2 reversed the inhibitory effects of miR-490-3p. Further investigations showed an inverse correlation between low miR-490-3p and high HMGA2 expression in osteosarcoma tissues. Taken together, our results suggest that miR-490-3p functions as a potential tumor suppressor by down-regulating HMGA2 expression directly, and it may represent a potential therapeutic target for patients with osteosarcoma.

Dysregulation of the MIRLET7/HMGA2 axis with methylation of the CDKN2A promoter in myeloproliferative neoplasms.

Overexpression of high mobility group AT-hook 2 (Hmga2), which is negatively regulated by MIRLET7 micro RNAs through 3'-untranslated region (3'UTR), causes proliferative haematopoiesis mimicking myeloproliferative neoplasms (MPNs) and contributes to progression of myelofibrosis in mice. Thus, we investigated HMGA2 mRNA expression in 66 patients with MPNs including 23 polycythaemia vera (PV), 33 essential thrombocythaemia (ET) and 10 primary myelofibrosis (PMF). HMGA2 mRNA expression, especially variant 1 with 3'UTR that contains MIRLET7-specific sites, rather than variant 2 lacking 3'UTR, is frequently deregulated due to decreased MIRLET7 expression in granulocytes from over 20% of PV and ET, and in either granulocytes or CD34(+) cells from 100% of PMF. Patients with deregulated HMGA2 mRNA expression were significantly more likely to show splenomegaly, high serum lactate dehydrogenase values, and methylation of the CDKN2A promoter compared with other patients without deregulation of HMGA2. A histone deacetylase inhibitor, panobinostat, significantly increased MIRLET7 expression and reduced variant 1 of HMGA2 mRNA expression, but not variant 2, in both U937 cells and PMF-derived CD34(+) cells. Moreover, both panobinostat and small interfering RNA of HMGA2 demethylated the CDKN2A promoter in U937 cells. In conclusion, the frequently dysregulated MIRLET7/HMGA2 axis could be a therapeutic target in MPNs.

IMP3 RNP Safe Houses Prevent miRNA-Directed HMGA2 mRNA Decay in Cancer and Development

The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated by let-7, and let-7 antagomiRs make HMGA2 refractory to IMP3. Removal of let-7 target sites eliminates IMP3-dependent stabilization, and IMP3-containing bodies are depleted of Ago1-4 and miRNAs. The relationship between Hmga2 mRNA and IMPs also exists in the developing limb bud, where IMP1-deficient embryos show dose-dependent Hmga2 mRNA downregulation. Finally, IMP3 ribonucleoproteins (RNPs) contain other let-7 target mRNAs, including LIN28B, and a global gene set enrichment analysis demonstrates that miRNA-regulated transcripts in general are upregulated following IMP3 induction. We conclude that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression.

Effect of high mobility protein A2 overexpression on cell cycle and proliferation of rat pituitary tumor cells

Objective To observe the effects of high mobility protein A2 (HMGA2) overexpression on proliferation and cell cycle of rat pituitary tumor cells.Methods The mRNA and protein expression of HMGA2 in rat GH3 cells transduced by pIRES2-dsRed-HMGA2cDNA were examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.The effects of the HMGA2 overexpression on GH3 cell proliferation and cell cycle were analyzed by cell counting kit-8 (CCK-8) assay and flow cytometry.Results pIRES2-dsRed-HMGA2cDNA transfected rat GH3 cells after 24 h the HMGA2 mRNA and protein expression levels were respectively higher than the control group 26.40％ and 36.60％,the difference was statistically significant (P ＜ 0.05).To compare with control group the number of pIRES2-dsRed-HMGA2cDNA group cells increased at 24,48,72,96 h (P ＜ 0.05).Flow cytometry analysis showed that the experimental group,the percentage of cells in G0/G1 phase was significantly reduced compared with the control group,(43.15 ±0.63)％ vs.(65.33 ±0.37)％,while the S phase was significantly increased compared with the control group (39.57 ± 0.65) ％ vs.(18.26 ± 0.54) ％ (P ＜ 0.05).Conclusion HMGA2 overexpression increases rat GH3 cell proliferation and changes normal cell cycle. Key words: High mobility protein A2; Over expression; Pituitary tumor; Proliferation; Cell cycle

BMP4 increases expression of HMGA2 in mesenchymal stem cells

BMP4 has been linked to early steps of adipocyte lineage differentiation but only little is known about its corresponding downstream pathways. Herein, we have investigated whether or not the expression of high mobility group protein HMGA2, another protein linked to proliferation and differentiation within the process of adipogenesis, may be influenced by BMP4 signaling in adipose tissue derived stem cells. Compared to FGF1, a strong inducer of HMGA2 in immortalized pre-adipocytes, BMP4 was found moderately to induce the HMGA2 mRNA expression in serum starved adipose tissue derived stem cells and myometrial cells. In contrast, no such activity was noted in canine bone marrow derived mesenchymal stem cells. As to adipocyte lineage differentiation the functions of BMP4 and HMGA2 mechanistically overlap. Thus, we propose that in adipose tissue BMP4 acts in part by activating HMGA2 making this architectural transcription factor one of the major downstream players in that system.

Overexpression of HMGA2 in bladder cancer and its association with clinicopathologic features and prognosis: HMGA2 as a prognostic marker of bladder cancer

PurposeTo examine HMGA2 expression and investigate its clinical and prognostic significance in human urothelial bladder cancer (BUC). MethodsWe detected HMGA2 mRNA and protein expression by quantitative reverse-transcription polymerase chain reaction and western blotting, respectively in 44 frozen bladder cancer tissues and 18 adjacent normal bladder tissues. HMGA2 protein expression was assessed by immunohistochemical analysis of 148 paraffin-embedded specimens of human BUC and 30 specimens of adjacent normal bladder tissue. Correlations between HMGA2 and clinicopathologic features and prognosis were tested by statistical analyses. ResultsHMGA2 mRNA and protein levels in bladder cancer samples were significantly increased compared with adjacent normal bladder tissues (P < 0.001). mRNA overexpression correlated with high stage and grade of the bladder cancer (P < 0.001 and P = 0.002 respectively). HMGA2 protein expression was negative in all normal urothelial tissue samples, but positive in 52% (77/148) of bladder cancers (P < 0.001). HMGA2 expression correlated with tumor grade and stage (P < 0.001 and P = 0.003 respectively), Overexpression of HMGA2 protein in non-muscle-invasive bladder cancer was significantly associated with shorter recurrence-free survival (P < 0.001), and progression-free survival (P = 0.0004). Multivariate analysis showed that HMGA2 expression was an independent prognostic factor for both tumor recurrence (P < 0.001) and tumor progression (P = 0.006). ConclusionsHMGA2 is up-regulated in bladder cancer at both the transcriptional and translational levels compared with normal bladder tissue, HMGA2 protein is thus a potential prognostic marker for predicting tumor recurrence and progression.

Association of a high mobility group gene (HMGA2) variant with bone mineral density

High mobility group (HMG) proteins regulate chromatin architecture and gene expression. Constitutional rearrangement of an HMG family member, HMGA2, in an 8-year old boy resulted in extreme overgrowth and advanced bone development. Moreover, a recent genome-wide association study documented an association between a variant in the 3′ untranslated region of HMGA2 (rs1042725) and height in otherwise healthy individuals. We attempted to extend these findings by testing if this HMGA2 polymorphism is associated with other skeletal measures in two large population cohorts of diverse race/ethnicity. Genotyping was completed in 1680 Afro-Caribbean men aged ≥40 years and 1548 Caucasian American men aged ≥69 years. Bone mineral density (BMD) was assessed with peripheral quantitative computed tomography. The minor allele frequency of rs1042725 was 32% among Afro-Caribbeans and 48% among Caucasians (p<0.0001). No association was observed with height in either study cohort. However, presence of the minor allele of this SNP was associated with decreased tibia trabecular volumetric BMD in both populations (p=0.007 Afro-Caribbean; p=0.0007 Caucasian). Real-time quantitative RT-PCR and Western blot analysis demonstrated HMGA2 mRNA and protein expression in the human fetal osteoblast cell line, hFOB. Our analyses suggest a novel association between a common genetic variant in HMGA2 and trabecular BMD in ethnically diverse older men. Additional research is needed to better understand the role of HMGA2 in the regulation of bone metabolism.

HMGA2 mRNA expression correlates with the malignant phenotype in human thyroid neoplasias

We have analysed the expression of the HMGA2 gene in a panel of normal and neoplastic thyroid tissues by immunohistochemistry and quantitative RT-PCR. HMGA2 protein was detectable in four out of 21 follicular carcinomas, 30 out of 45 papillary carcinomas, and 11 out of 12 undifferentiated carcinomas. As far as follicular thyroid adenomas are concerned, only three cases of the 31 analysed showed HMGA2 protein expression, whereas it was absent in seven normal thyroid tissues and in 12 hyperplastic nodules. Quantitative RT-PCR showed that almost all the papillary thyroid carcinomas and 13 out of 16 follicular thyroid carcinomas express much higher HMGA2 specific mRNA levels in comparison to normal thyroids and adenomas. Therefore, our data support the quantitative RT-PCR analysis of HMGA2 expression, rather than immunohistochemistry, as a powerful tool for the diagnosis of thyroid neoplasias.