Mobility Group AT-hook Research Articles

MiR-143-5p inhibits proliferation, invasion, and epithelial to mesenchymal transition of colorectal cancer cells by downregulation of HMGA2


 
 
 
 Purpose: To investigate the regulatory effect and molecular mechanism of miR-143-5p in colorectal cancer (CRC) progression.
 Methods: Expression of miR-143-5p in CRC cell lines SW620 and HCT116 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Stable miR-143-5p overexpression was mediated by lentivirus. The effects of miR-143-5p on proliferation, migration, invasion, and epithelial- mesenchymal transition (EMT) of SW620 and HCT116 cells were assessed by colony formation assay, CCK-8, Transwell assay, wound healing assay, and western blot. Target prediction was performed for miR-143-5p, and a dual luciferase assay was used to verify the targeting relationship.
 Results: Compared to CRC cells transfected with negative controls, cell proliferation, migration and invasion, and EMT were inhibited in miR-143-5p-overexpressing cells. Expression of HMGA2 (high- mobility Group AT-Hook 2), a target gene of miR-143-5p, was repressed by miR-143-5p. Rescue experiments confirmed that upregulation of HMGA2 due to mIR-143-5p overexpression reversed inhibition of CRC cell proliferation, invasion and EMT.
 Conclusion: MiR-143-5p inhibits the malignant progression of CRC by regulating HMGA2 expression and is expected to provide new therapeutic approaches for clinical treatment of CRC.
 
 
 

Induction of microRNA hsa-let-7d-5p, and repression of HMGA2, contribute protection against lipid accumulation in macrophage ‘foam’ cells

Accumulation of excess cholesterol and cholesteryl ester in macrophage ‘foam’ cells within the arterial intima characterises early ‘fatty streak’ atherosclerotic lesions, and is accompanied by epigenetic changes, including altered expression of microRNA sequences which determine of gene and protein expression. This study established that exposure to lipoproteins, including acetylated LDL, induced macrophage expression of microRNA hsa-let-7d-5p, a sequence previously linked with tumour suppression, and repressed expression of one of its target genes, high mobility group AT hook 2 (HMGA2). A let-7d-5p mimic repressed expression of HMGA2 (18%; p < 0.05) while a marked increase (2.9-fold; p < 0.05) in expression of HMGA2 was noted in the presence of let-7d-5p inhibitor. Under these conditions, let-7d-5p mimic significantly (p < 0.05) decreased total (10%), free (8%) and cholesteryl ester (21%) mass, while the inhibitor significantly (p < 0.05) increased total (29%) and free cholesterol (29%) mass, compared with the relevant controls. Let-7d-5p inhibition significantly (p < 0.05) increased endogenous biosynthesis of cholesterol (38%) and cholesteryl ester (39%) pools in macrophage ‘foam’ cells, without altering the cholesterol efflux pathway, or esterification of exogenous radiolabelled oleate. Let-7d-5p inhibition in sterol-loaded cells increased the level of HMGA2 protein (32%; p < 0.05), while SiRNA knockdown of this protein (29%; p < 0.05) resulted in a (21%, p < 0.05) reduction in free cholesterol mass. Thus, induction of let-7d-5p, and repression of its target HMGA2, in macrophages is a protective response to the challenge of increased cholesterol influx into these cells; dysregulation of this response may contribute to atherosclerosis and other disorders such as cancer.

RETRACTED: LNCRNA CDKN2B-AS1 regulates mesangial cell proliferation and extracellular matrix accumulation via miR-424-5p/HMGA2 axis

Previous study has demonstrated that long noncoding RNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) was abnormally expressed in diabetic nephropathy (DN). However, the underlying mechanism that allows CDKN2B-AS1 in the progression of DN remains to be further elucidated. Peripheral blood cells of 24 diabetes patients with DN and 20 without DN were collected. Human glomerular mesangial cells (HGMC) were cultured in high glucose or low glucose medium. The expression levels of CDKN2B-AS1, microRNA (miR)-424-5p and high mobility group AT hook 2 (HMGA2) were detected by quantitative real-time polymerase chain reaction or western blot. The target association between miR-424-5p and CDKN2B-AS1 or HMGA2 was confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. Cell proliferation, extracellular matrix (ECM) accumulation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and western blot, respectively. CDKN2B-AS1 expression was up-regulated and miR-424-5p level was down-regulated in peripheral blood of DN patients and high glucose-treated HGMC cells. CDKN2B-AS1 was validated as a sponge of miR-424-5p. Silence of CDKN2B-AS1 repressed proliferation and ECM accumulation by increasing miR-424-5p. HMGA2 was a target of miR-424-5p and miR-424-5p overexpression inhibited proliferation, ECM accumulation and PI3K/AKT pathway by targeting HMGA2. Moreover, knockdown of CDKN2B-AS1 inhibited HMGA2 expression and PI3K/AKT pathway by increasing miR-424-5p. Knockdown of CDKN2B-AS1 suppressed proliferation, ECM accumulation and PI3K/AKT signaling by increasing miR-424-5p and decreasing HMGA2 in high glucose-treated HMGC cells.

The HMGA2-IMP2 Pathway Promotes Granulosa Cell Proliferation in Polycystic Ovary Syndrome.

The high mobility group AT hook 2 (HMGA2) gene was previously identified in a genome-wide association study as a candidate risk gene that might be related to polycystic ovary syndrome (PCOS). Whether HMGA2 contributes to promoting granulosa cell (GC) proliferation in PCOS remains unknown. We sought to determine whether HMGA2 is involved in the ovarian dysfunction of PCOS and in the mechanism of increased GC proliferation. mRNA expression was analyzed in ovarian GCs from 96 women with PCOS and 58 healthy controls. Immortalized human GCs (KGN and SVOG cells) were used for the mechanism study. mRNA expression in ovarian GCs was measured using quantitative RT-PCR, and KGN cells were cultured for proliferation assays after overexpression or knockdown of target genes. Protein expression analysis, luciferase assays, and RNA binding protein immunoprecipitation assays were used to confirm the mechanism study. HMGA2 and IGF2 mRNA binding protein 2 (IMP2) were highly expressed in the GCs of women with PCOS, and the HMGA2/IMP2 pathway promoted GC proliferation. Cyclin D2 and SERPINE1 mRNA binding protein 1 were regulated by IMP2 and were highly expressed in women with PCOS. The HMGA2/IMP2 pathway was activated in women with PCOS and promoted the proliferation of GCs. This might provide new insights into the dysfunction of GCs in PCOS.

Expression of HMGA2 Cooperates with Jak2V617F in the Development of Myelofibrosis

High Mobility Group AT Hook 2 (HMGA2) is a non-histone chromatin protein that regulates gene transcription and controls cell proliferation, survival and self-renewal of stem cells. HMGA2 is expressed at a low level in normal adult hematopoietic progenitors but is highly expressed in hematopoietic progenitors of patients with Myelofibrosis (MF). However, the contribution of HMGA2 to the pathogenesis of MF remains unknown.MF is the deadliest form of myeloprolifearative neoplasm (MPN) characterized by deposition of fibrous tissues in the bone marrow, increased megakaryopoiesis, ineffective erythropoiesis and extramedullary hematopoiesis. Median survival of patients with MF is less than 6 years. The JAK2V617F mutation has been found in 50-60% patients with MF. However, it is not clear whether JAK2V617F mutation alone is sufficient to cause MF. Interestingly, up-regulation of HMGA2 expression has been found in association with the JAK2V617F mutation in a significant percentage of patients with MF. To understand the role of JAK2V617F mutation in the pathogenesis of MPN, we previously generated a conditional Jak2V617F knock-in mouse. We observed that expression of heterozygous Jak2V617F in mouse hematopoietic compartments is sufficient to induce a polycythemia vera (PV)-like MPN. Recently, we have shown that deletion of EZH2 promotes the development of MF in Jak2V617F knock-in mice and EZH2 deletion increases the expression of HMGA2 in hematopoietic progenitors of EZH2-deleted Jak2V617F mice. To directly assess the effects of concomitant expression of HMGA2 and heterozygous Jak2V617F in mice hematopoietic compartments, we expressed control vector or HMGA2 in wild type and heterozygous Jak2V617F knock-in mice BM by lentiviral transduction and performed bone marrow transplantation into lethally irradiated C57BL/6 recipient mice.Whereas recipients of vector-transduced Jak2V617F knock-in BM cells exhibited a PV-like MPN characterized by increased red blood cells (RBC), hemoglobin, hematocrit and platelets in their peripheral blood, recipients of HMGA2-transduced Jak2V617F knock-in BM showed reduced hemoglobin and hematocrit parameters compared with recipients of vector-expressing Jak2V617F BM cells. Interestingly, peripheral blood neutrophil and platelet counts were further increased in transplanted animals receiving HMGA2-transduced Jak2V617F BM cells. Expression of HMGA2 also resulted in significantly larger spleen size in the transplanted animals receiving HMGA2-expressing Jak2V617F BM cells. Flow cytometric analysis showed significant increase in megakaryocytic precursors (CD41+) but decrease in erythroid precursors (CD71+/Ter119+) in the BM and spleens of transplanted animals receiving HMGA2-expressing Jak2V617F BM compared with control vector-expressing Jak2V617F BM. Furthermore, the frequency of hematopoietic stem/progenitor cells (LSK; Lin-Sca-1+c-kit+) was significantly increased in recipients of HMGA2-transduced Jak2V617F knock-in BM compared with control vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM. Histopathologic analysis revealed extensive fibrosis in the BM and spleens from recipients of HMGA2-expressing Jak2V617F mice at 32 weeks after transplantation while BM and spleens from recipients of vector-transduced Jak2V617F knock-in BM or HMGA2-transduced wild type BM showed very little or no fibrosis at this age. Together, these data suggest that expression of HMGA2 promotes megakaryopoiesis and accelerates the development of MF in mice expressing Jak2V617F.To gain insights into the mechanisms by which expression of HMGA2 accelerates the development of MF in Jak2V617F mice, we performed RNA-sequencing analysis on purified LSK (Lin-Sca-1+c-kit+) cells. Gene set enrichment and pathway analyses revealed that the genes related to chemokine, TGF-β, MAP Kinase, PI3 kinase-Akt, mTOR and WNT signaling pathways were up-regulated in HMGA2-expressing Jak2V617F mice LSK compared with vector-expressing Jak2V617F LSK cells. We also found that HMGA2 directly binds to the promoter regions of some of these target genes and regulate their expression. Further studies will validate the targets of HMGA2 and determine their contribution in MF mediated by Jak2V617F. In conclusion, our studies show that expression of HMGA2 cooperates with Jak2V617F in the development of MF. DisclosuresNo relevant conflicts of interest to declare.

Role of HMGA2 in human hematopoietic stem and progenitor cells

Hematopoietic stem cells maintain life long blood generation during homeostasis and stress. Factors controlling the key characteristics of these cells such as self-renewal, quiescence and the potential to form various blood lineages are poorly understood. We reasoned that genes found to be located near common sites of vector insertion in clinical gene therapy studies might have important functions in this regard. The DNA binding protein, high mobility Group AT hook 2 (HMGA2) is one such factor. HMGA2 activation coupled with a myeloid biased clonal dominance of stem/progenitor cells was observed in a clinical gene therapy trial for β-thalassemia.

HMGA2 protein expression in ovarian serous carcinoma effusions, primary tumors, and solid metastases

The objective of this study was to analyze the expression and clinical role of the high mobility group AT hook (HMGA) protein in advanced-stage serous ovarian carcinoma. HMGA2 protein expression was investigated in 199 effusions and in 50 patient-matched primary tumors and solid metastases using immunohistochemistry. Results were analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival. HMGA2 was expressed in tumor cells in 94.5 %, 96 %, and 90 % of specimens, respectively. There was no difference in HMGA2 expression between patient-matched samples from different anatomic sites (p > 0.3). HMGA2 expression in chemo-naïve samples was significantly higher in older patients (p = 0.006, p = 0.01, and p = 0.005 for effusions, primary tumors, and solid metastases, respectively). No association was found with residual disease volume. Furthermore, HMGA2 expression was not associated with FIGO stage (p > 0.2), except in chemo-naïve effusions (n = 106, p = 0.016). There was no difference in HMGA2 expression between chemo-naïve samples and samples obtained post-chemotherapy in effusions (p = 0.2) or primary tumors (p = 0.1). However, solid metastases obtained after chemotherapy exposure had higher HMGA2 expression compared with chemo-naïve samples (p = 0.032). HMGA2 expression was unrelated to chemotherapy response or survival. However, it was directly related to protein expression of the previously studied cancer stem cell marker Nestin (p = 0.01) and the gap junction protein claudin-7 (p = 0.02) and inversely related to the mRNA level of the E-cadherin repressor SIP1 (p = 0.02). This study provides evidence that HMGA2 is universally expressed in advanced-stage ovarian serous carcinoma irrespective of anatomic site, suggesting that HMGA2 may have a clinical role as therapeutic target.

Abstract 1344: HMGA2-targeted drug discovery for breast cancer brain metastasis

Abstract Breast cancer is a major health concern for women in the United States, with nearly 200,000 women diagnosed annually. An estimated 10-16% of women with breast cancer will develop symptomatic brain metastases and the incidence of brain metastasis has increased in recent years mainly due to the inability of current therapies to cross the blood-brain barrier (BBB). It is suggested that metastatic tumors contain a unique subpopulation of cancer cells with stem cell properties (CSCs), and these CSCs are enriched in metastatic tumors and express genes that enhance self-renewal and prevent differentiation. We used the MDA-MB-231 breast cancer cell line (MD-231) and its variant brain metastasis (231-BR) isolated from a xenograft model to characterize signaling molecules uniquely active in brain metastatic cells and to identify drugs that may target these molecules. 231-BR cells expressed lower levels of Let-7 microRNA, which is known to suppress levels of H-ras and high mobility group AT hook 2 (HMGA2) proteins, compared to parental MD-231 cells. H-ras is associated with self-renewal, whereas HMGA2 blocks differentiation of CSCs and effects the epithelial-to-mesenchymal transformation (EMT). HMGA2 binds to the minor grove of DNA and alters the expression of target genes, which include EMT-associated and NF-κB-driven genes. Reduced levels of Let-7 correlated with elevated HMGA2 in 231-BR cells, as evidenced by quantitative RT-PCR and western blot analysis. Although both MD-231 and 231-BR cells displayed CSC phenotype based on their CD44 and CD24 cell marker expression pattern (CD44+/CD24-), 231-BR cells contained higher numbers of a unique subset of CSCs compared to MD-231 cells based on CFSE-1 dye retention assay. Given these unique characteristics of 231-BR cells, we hypothesized that netropsin, a Streptomyces-derived antimicrobial agent with anti-tumoral properties, capable of crossing the BBB, and that interferes with the binding of proteins to DNA's minor groove, might be effective in targeting HMGA2 over-expressing cells. 231-BR cells, but not the MD-231 cells, were highly sensitive to netropsin, as evidenced by BrdU cell proliferation assay. Given that NF-κB is a downstream effector of HMGA2, we also tested LC-1, a parthenolide derived compound developed by this laboratory as an inhibitor of NF-κB, for its effects on 231-BR cells. As predicted, the 231-BR cells were more sensitive to LC-1 than the MD-231 cells. In contrast, salinomycin, a recently discovered drug that targets CD44+/CD24- CSCs, was effective in reducing proliferation of both cell types. Based on these results, we propose netropsin or an NF-κB inhibitor as potential new therapies for the growing problem of breast cancer brain metastases. Further testing is currently underway to establish the efficacy of netropsin and LC-1 in addressing the two main issues with brain metastases; cancer cell specificity, particularly CSCs, and ability to cross the BBB. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1344. doi:10.1158/1538-7445.AM2011-1344