Circular RNA 100146 Promotes Colorectal Cancer Progression by the MicroRNA 149/HMGA2 Axis.

Colorectal cancer (CRC) has developed into the third leading cause of cancer-associated death worldwide. Studies have confirmed that circular RNAs (circRNAs) absorb microRNAs (miRNAs) to regulate the function of downstream genes. This study aimed to explore the underlying mechanism of circRNA 100146 in CRC. The expression of circRNA 100146, miRNA 149 (miR-149), and high mobility group AT-Hook 2 (HMGA2) was detected by quantitative real-time PCR (RT-qPCR). A series of biofunctional effects (cell viability, apoptosis, migration/invasion) were evaluated by the use of methyl thiazolyl tetrazolium (MTT), flow cytometry, and transwell assays. Protein levels were measured by Western blot assay. A xenograft model was established for in vivo experiments. The interactions among circRNA 100146, miR-149, and HMGA2 were evaluated by dual-luciferase reporter assay, RNA immunoprecipitation assays, or RNA pulldown assay. circRNA 100146 was upregulated in CRC tissues and cells. circRNA 100146 knockdown inhibited cell proliferation, promoted apoptosis, and suppressed migration and invasion in vitro and impeded tumor growth in vivo Also, miR-149 was negatively regulated by circRNA 100146 and was targeted to HMGA2 and mediated its expression. Moreover, miR-149 interference abrogated the activities of silenced circRNA 100146 in proliferation, apoptosis, migration, and invasion. Furthermore, HMGA2 overexpression abated the effects described above caused by circRNA 100146 silencing, while the mutations on miR-149 binding sites in the 3' untranslated region (3'-UTR) of HMGA2 led to its loss of this ability. circRNA 100146 knockdown repressed proliferation, enhanced apoptosis, and hindered migration and invasion in SW620 and SW480 cells through targeting the miR-149/HMGA2 axis.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

Identification of the Effects of Aspirin and Sulindac Sulfide on the Inhibition of HMGA2-Mediated Oncogenic Capacities in Colorectal Cancer.

Distant metastatic colorectal cancer (CRC) is present in approximately 25% of patients at initial diagnosis, and eventually half of CRC patients will develop metastatic disease. The 5-year survival rate for patients with metastatic CRC is a mere 12.5%; thus, there is an urgent need to investigate the molecular mechanisms of cancer progression in CRC. High expression of human high-mobility group A2 (HMGA2) is related to tumor progression, a poor prognosis, and a poor response to therapy for CRC. Therefore, HMGA2 is an attractive target for cancer therapy. In this study, we identified aspirin and sulindac sulfide as novel potential inhibitors of HMGA2 using a genome-wide mRNA signature-based approach. In addition, aspirin and sulindac sulfide induced cytotoxicity of CRC cells stably expressing HMGA2 by inhibiting cell proliferation and migration. Moreover, a gene set enrichment analysis (GSEA) revealed that gene sets related to inflammation were positively correlated with HMGA2 and that the main molecular function of these genes was categorized as a G-protein-coupled receptor (GPCR) activity event. Collectively, this is the first study to report that aspirin and sulindac sulfide are novel potential inhibitors of HMGA2, which can induce cytotoxicity of CRC cells stably expressing HMGA2 by inhibiting cell proliferation and migration through influencing inflammatory-response genes, the majority of which are involved in GPCR signaling.

MiR-219-5p inhibits prostate cancer cell growth and metastasis by targeting HMGA2.

OBJECTIVE: To investigate the expression of micro ribonucleic acid (miR)-219-5p in prostate cancer (PCa), its influences on the biological functions of PCa, and its mechanism. PATIENTS AND METHODS: The expression differences of miR-219-5p and high mobility group protein A2 (HMGA2) in 30 pairs of PCa tissues and para-carcinoma tissues were detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and the difference in miR-219-5p expression in PCa cell lines and normal prostatic epithelial cells was also determined via qRT-PCR. The human PC-3 cells were divided into negative control group and miR-219-5p overexpression group. Methyl thiazolyl tetrazolium (MTT) and colony formation assays were adopted to detect the cell proliferative ability, and flow cytometry was applied to determine the cell apoptosis. The expression of apoptosis-related proteins was measured via Western blotting, and the invasive and migratory abilities of the cells were examined through wound-healing and transwell assays. Bioinformatics prediction software and luciferase reporter assay were employed to verify the targets that might be controlled by miR-219-5p. Rescue experiment was conducted to clarify whether the inhibitory effects of miR-219-5p on the growth and metastasis of PC-3 cells depend on the inhibition of HMGA2. RESULTS: It was shown in qRT-PCR results that the expression level of miR-219-5p was downregulated remarkably in PCa tissues and cell lines, but overexpressed miR-219-5p could repress the proliferation and promote the apoptosis of PC-3 cells notably. The results of wound-healing and transwell assays indicated that overexpressed miR-219-5p was able to suppress the invasion and metastasis of PC-3 cells. According to Western blotting results, overexpressed miR-219-5p could up-regulate the expressions of pro-apoptotic proteins [Bax, cleaved-caspase-3 and cleaved-poly-ADP-ribose-polymerase (PARP)] and reverse the epithelial-mesenchymal transition (EMT) of PCa cells. It was predicted via the bioinformatics software that HMGA2 gene might be a target gene of miR-219-5p. The Dual-Luciferase reporter assay confirmed that there was a direct regulatory relationship between miR-219-5p and HMGA2. The rescue experiment manifested that overexpressed HMGA2 could reverse the inhibition of miR-219-5p on the growth and metastasis of PC-3 cells. CONCLUSIONS: MiR-219-5p suppresses the growth and metastasis abilities of prostate cancer cells by directly repressing the expression of HMGA2.

Gene Expression Signature-Based Approach Identifies Antifungal Drug Ciclopirox As a Novel Inhibitor of HMGA2 in Colorectal Cancer.

Human high-mobility group A2 (HMGA2) encodes for a non-histone chromatin protein which influences a variety of biological processes, including the cell cycle process, apoptosis, the DNA damage repair process, and epithelial-mesenchymal transition. The accumulated evidence suggests that high expression of HMGA2 is related to tumor progression, poor prognosis, and a poor response to therapy. Thus, HMGA2 is an important molecular target for many types of malignancies. Our recent studies revealed the positive connections between heat shock protein 90 (Hsp90) and HMGA2 and that the Hsp90 inhibitor has therapeutic potential to inhibit HMGA2-triggered tumorigenesis. However, 43% of patients suffered visual disturbances in a phase I trial of the second-generation Hsp90 inhibitor, NVP-AUY922. To identify a specific inhibitor to target HMGA2, the Gene Expression Omnibus (GEO) database and the Library of Integrated Network-based Cellular Signatures (LINCS) L1000platform were both analyzed. We identified the approved small-molecule antifungal agent ciclopirox (CPX) as a novel potential inhibitor of HMGA2. In addition, CPX induces cytotoxicity of colorectal cancer (CRC) cells by induction of cell cycle arrest and apoptosis in vitro and in vivo through direct interaction with the AT-hook motif (a small DNA-binding protein motif) of HMGA2. In conclusion, this study is the first to report that CPX is a novel potential inhibitor of HMGA2 using a drug-repurposing approach, which can provide a potential therapeutic intervention in CRC patients.

MicroRNA-9 exerts antitumor effects on hepatocellular carcinoma progression by targeting HMGA2.

Accumulating evidence has demonstrated that the aberrant expression of microRNAs (miRs or miRNAs) may contribute to the initiation and progression of various types of human cancer and may also constitute biomarkers for cancer diagnosis and therapy. However, the specific function of miR-9 in hepatocellular carcinoma (HCC) remains unclear, and the mechanisms that underlie HCC are incompletely understood. Here, we report that miR-9 expression was significantly decreased in clinical tumor tissue samples, as well as in a cohort of HCC cell lines. In addition, it was demonstrated that overexpression of miR-9 suppressed the proliferative and migratory capacity of HCC cells and impaired cell cycle progression. Furthermore, high mobility group AT-hook 2 (HMGA2) was verified as a downstream target gene of miR-9 using a luciferase reporter assay. Quantitative RT-PCR and western blotting implicated HMGA2 in the miR-9-mediated reduction of HCC cell growth. In vivo, transfection with miR-9 mimics down-regulated the expression of HMGA2, thus leading to a dramatic reduction in tumor growth in a mouse xenograft model. These results suggest that miR-9 may exert critical antitumor effects on HCC by directly targeting HMGA2, and the miR9/HMGA2 signaling pathway may be of use for the diagnosis and prognosis of patients with HCC.

Knockdown of HMGA2 regulates the level of autophagy via interactions between MSI2 and Beclin1 to inhibit NF1-associated malignant peripheral nerve sheath tumour growth.

BACKGROUND: Malignant peripheral nerve sheath tumours (MPNSTs) are sarcomas of Schwann cell lineage origin that occur sporadically or in association with the inherited syndrome, neurofibromatosis type 1 (NF1). This study aimed to examine the function of High mobility group protein A2 (HMGA2) in NF1 MPNST progression and the underlying molecular mechanism. METHODS: Immunohistochemistry (IHC) was used to detect HMGA2 expression in MPNST and neurofibroma patient samples. Cell Cycle Kit-8 (CCK-8) and 5-ethynyl-20-deoxyuridine (EdU) assays, terminal deoxynucleotidyl transferase-mediated nick end labelling, and transmission electron microscopy were performed to reveal HMGA2 functions in NF1 MPNST cells in vitro and in vivo. Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) were used to detect HMGA2-modulated genes regulating autophagy and growth in NF1 MPNSTs in vitro and in vivo. RESULTS: NF1 MPNST samples exhibit higher HMGA2 positivity rates (13/16) than sporadic MPNST (16/41) and neurofibroma (0/7) patient samples. High HMGA2 expression is correlated with poor prognosis. Neurofibromin 1 (NF1)-deficient MPNST cells display elevated HMGA2 expression. Functional experiments revealed that HMGA2 knockdown inhibits NF1 MPNST cell growth in vitro and in vivo. In addition to promoting cell cycle arrest and apoptosis, HMGA2 knockdown inhibits autophagy, favouring cell death. RNA-Seq and ChIP-Seq revealed that HMGA2 directly activates the Musashi-2 (MSI2) promoter region, and MSI2 overexpression reverses autophagy and growth in shHMGA2-transfected cells. MSI2 interacts with Beclin1, and Beclin1 blockade inhibits autophagy, thereby inhibiting cell proliferation. CONCLUSIONS: HMGA2 knockdown regulates autophagy via MSI2-Beclin1 interactions to inhibit NF1 MPNST growth, revealing potential therapeutic targets for these untreatable tumours.

miR-98 acts as an inhibitor in chronic constriction injury-induced neuropathic pain via downregulation of high-mobility group AT-hook 2.

Neuropathic pain, resulting from somatosensory nervous system dysfunction, remains a serious public health problem worldwide. microRNAs are involved in the physiological processes of neuropathic pain. However, the biological roles of miR-98 in neuropathic pain development have not been investigated. Therefore, in our current study, we focused on the effects of miR-98 in neuropathic pain. It was shown that miR-98 was significantly downregulated in chronic sciatic nerve injury (CCI) rat models. In addition, high mobility group A2 (HMGA2) was obviously upregulated in CCI rats. Overexpression of miR-98 inhibited neuropathic pain progression, including mechanical and thermal hyperalgesia. By a bioinformatics analysis, HMGA2 was predicted as a direct target of miR-98. The negative correlation between miR-98 and HMGA2 was validated in our present study. Furthermore, overexpression of miR-98 dramatically repressed HMGA2 protein and messenger RNA (mRNA) expression. Neuroinflammation participates in neural-immune interactions, which can contribute to the neuropathic pain development. Meanwhile, we found that inflammatory cytokine (interleukin [IL]-6, IL-1ß, and COX-2) protein expression in rats infected with LV-miR-98 was greatly suppressed. Taking these results together, we concluded that miR-98 might depress neuropathic pain development through modulating HMGA2.

SiRNA/DOX lodeded chitosan based nanoparticles: Development, Characterization and in vitro evaluation on A549 lung cancer cell line.

High-mobility group AT-hook2 (HMGA2), involved in epithelial mesenchymal transition (EMT) process, has a pivotal role in lung cancer metastasis. Lung cancer therapy with HMGA2 suppressing small interfering RNA (siRNA) has been introduced recently while doxorubicin (DOX) has been used as a frequent cancer chemotherapy agent. Both reagents have been faced with obstacles in clinic which make them ineffective. NanoParticles (NPs) provided a platform for efficient co delivery of the anticancer drugs. The aim of this study was production and in vitro characterization of different pharmacological groups (siRNA, DOX or siRNA-DOX) of carboxymethyl dextran thrimethyl chitosan nanoparticles (CMDTMChiNPs) on cytotoxicity, gene expression, apoptosis and migration of metastatic lung cancer cell line (A-549). CMDTMChiNPs were synthesized and encapsulated with siRNA, DOX or siRNA-DOX. Then the effects of HMGA2 siRNA and DOX co delivery was assessed in A549 viability and target genes (HMGA2, Ecadherin, vimentin and MMP9) by MTT and real time PCR, respectively. In addition capability of apoptosis induction and anti-migratory features of formulated NPs were analyzed by flowcytometry and wound healing assays. SiRNA-DOX-CMDTM ChiNPs approximate size were 207±5 with poly dispersity index (PDI) and zeta potential of 0.4 and 16.3±0.3, respectively. NPs loaded with DOX and siRNA were the most efficient drug formulations in A549 cell cytotoxicity, altering of EMT markers, apoptosis induction and migration inhibition. Generally our results showed that co delivery of HMGA2 siRNA and DOX by novel designed CMDTMChiNPs is a new therapeutic approach with great potential efficiency for lung cancer treatment.

Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma.

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor prognosis. Here, we studied the effects of phenformin, a mitochondrial complex I inhibitor and more potent chemical analog of the diabetes drug metformin on the inhibition of cell growth and induction of apoptosis of glioma stem cells (GSCs) using both in vitro and in vivo models. Phenformin inhibited the self-renewal of GSCs, decreased the expression of stemness and mesenchymal markers and increased the expression of miR-124, 137 and let-7. Silencing of let-7 abrogated phenformin effects on the self-renewal of GSCs via a pathway associated with inhibition of H19 and HMGA2 expression. Moreover, we demonstrate that phenformin inhibited tumor growth and prolonged the overall survival of mice orthotopically transplanted with GSCs. Combined treatments of phenformin and temozolomide exerted an increased antitumor effect on GSCs in vitro and in vivo. In addition, dichloroacetate, an inhibitor of the glycolysis enzyme pyruvate dehydrogenase kinase, that decreases lactic acidosis induced by biguanides, enhanced phenformin effects on the induction of cell death in GSCs and prolonged the survival of xenograft-bearing mice. Our results demonstrate for the first time that phenformin targets GSCs and can be efficiently combined with current therapies for GBM treatment and GSC eradication.

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.

The oncogenic triangle of HMGA2, LIN28B and IGF2BP1 antagonizes tumor-suppressive actions of the let-7 family.

The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment.

Silencing of HMGA2 suppresses cellular proliferation, migration, invasion, and epithelial-mesenchymal transition in bladder cancer.

The high-mobility group protein A2 (HMGA2) is an architectural transcription factor that plays a crucial role in the development and progression of various malignant cancers. However, the function of HMGA2 in bladder cancer remains largely unknown. Therefore, we aim to investigate the effect of HMGA2 on the proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of bladder cancer cells. The expression of HMGA2 in human bladder cancer cells was downregulated by small interfering RNA (siRNA). The protein levels of HMGA2 and other related proteins were detected by Western blotting. The cell proliferation and apoptosis were examined by Cell Counting Kit-8 and flow cytometry, respectively. Transwell migration and invasion assays were performed to assess the effect of HMGA2 on the migration and invasion ability of cells. In conclusion, we found that HMGA2 knockdown markedly inhibited cell proliferation; this reduced cell growth was due to the high apoptosis rate of cells, as Bcl-xl was diminished, whereas Bax was upregulated. Moreover, our results showed that silencing of HMGA2 in cancer cells greatly inhibited the cell migration and invasion, decreased the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), and affected the occurrence of EMT. We further found that decreased HMGA2 expression suppressed the transforming growth factor-ß (TGF-ß)/Smad and Wnt/ß-catenin signaling pathway in bladder cancer cells. These results revealed that HMGA2 played an important role in the progression of bladder cancer and might be a novel target for therapy in human bladder cancer.

HMGA2 as a potential molecular target in KMT2A-AFF1-positive infant acute lymphoblastic leukaemia.

Acute lymphoblastic leukaemia (ALL) in infants is an intractable cancer in childhood. Although recent intensive chemotherapy progress has considerably improved ALL treatment outcome, disease cure is often accompanied by undesirable long-term side effects, and efficient, less toxic molecular targeting therapies have been anticipated. In infant ALL cells with KMT2A (MLL) fusion, the microRNA let-7b (MIRLET7B) is significantly downregulated by DNA hypermethylation of its promoter region. We show here that the expression of HMGA2, one of the oncogenes repressed by MIRLET7B, is reversely upregulated in infant ALL leukaemic cells, particularly in KMT2A-AFF1 (MLL-AF4) positive ALL. In addition to the suppression of MIRLET7B, KMT2A fusion proteins positively regulate the expression of HMGA2. HMGA2 is one of the negative regulators of CDKN2A gene, which encodes the cyclin-dependent kinase inhibitor p16(INK4A) . The HMGA2 inhibitor netropsin, when combined with demethylating agent 5-azacytidine, upregulated and sustained the expression of CDKN2A, which resulted in growth suppression of KMT2A-AFF1-expressing cell lines. This effect was more apparent compared to treatment with 5-azacytidine alone. These results indicate that the MIRLET7B-HMGA2-CDKN2A axis plays an important role in cell proliferation of leukaemic cells and could be a possible molecular target for the therapy of infant ALL with KMT2A-AFF1.

A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions.

DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein-nucleic acids interactions based on protein-DNA or protein-RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2-DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2-DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2-DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

Nuclear Localization Signal-Enhanced Polyurethane-Short Branch Polyethylenimine-Mediated Delivery of Let-7a Inhibited Cancer Stem-Like Properties by Targeting the 3'-UTR of HMGA2 in Anaplastic Astrocytoma.

Anaplastic astrocytoma (AA) is a grade III glioma that often occurs in middle-aged patients and presents a uniformly poor prognosis. A small subpopulation of cancer stem cells (CSCs) possessing a self-renewing capacity is reported to be responsible for tumor recurrence and therapeutic resistance. An accumulating amount of microRNAs (miRNA) were found aberrantly expressed in human cancers and regulate CSCs. Efforts have been made to couple miRNAs with nonviral gene delivery approaches to target specific genes in cancer cells. However, the efficiency of delivery of miRNAs to AA-derived CSCs is still an applicability hurdle. The present study aimed to investigate the effectiveness and applicability of nonviral vector-mediated delivery of Let-7a with regard to eradication of AA and AA-derived CSC cells. Herein, our miRNA/mRNA microarray and RT-PCR analysis showed that the expression of Let-7a, a tumor-suppressive miRNA, is inversely correlated with the levels of HMGA2 and Sox2 in the AA side population (SP(+)) cells. Luciferase reporter assay showed that Let-7a directly targets the 3'-UTRs of HMGA2 in AA-SP(+) cells. Knockdown of HMGA2 significantly suppressed the protein expression of Sox2 in AA-SP(+) cells, whereas overexpression of HMGA2 upregulated Sox2 expression in AA-SP(-). Nuclear localization signal (NLS) peptides can facilitate nuclear targeting of DNA and are used to improve gene delivery. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic delivery vehicle, we conjugated NLS with Let-7 and successfully delivered it to AA-SP(+) cells, resulting in significantly suppressed expression of HMGA2 and Sox2, tumorigenicity, and CSC-like abilities. This treatment facilitated the differentiation of AA-SP(+) cells into non-SP CSCs. Furthermore, PU-PEI-mediated delivery of NLS-conjugated Let-7a in AA-SP(+) cells suppressed the expression of drug-resistant and antiapoptotic genes, and increased cell sensitivity to radiation. Finally, the in vivo delivery of PU-PEI-NLS-Let-7a significantly suppressed the tumorigenesis of AA-SP(+) cells and synergistically improved the survival rate of orthotopically AA-SP(+)-transplanted immunocompromised mice when combined with radiotherapy. Therefore, PU-PEI-NLS-Let-7a is a potential novel therapeutic approach for AA.

Let-7 g microRNA sensitizes fluorouracil-resistant human hepatoma cells.

Let-7 microRNA is expressed in lower lever in a variety of human tumors and is involved in tumorigenesis. This study investigated the inhibitory effect of the let-7g microRNA on the expression of the HMGA2 gene in the fluorouracil (5-Fu)-resistant human hepatoma cell line Bel-7402/5-Fu, and the effect of let-7 g microRNA on drug sensitization in Bel-7402/5-Fu cells. Let-7 g microRNA and negative microRNA plasmids were constructed and transient transfected into Bel-7402/5-Fu cells. Expression levels of HMGA2 mRNA and protein in microRNA transient transfectants were clearly reduced as compared with negative microRNA transfectants and untreated cells. Flow cytometry revealed increased in S phase in let-7 g microRNA cells. dimethylthiazol-diphenyltetrazolium bromide (MTT) results indicated that microRNA transfectants had a higher cell inhibition rate than the negative vector or untreated cells after treatment with 0.13-13 microg/ml 5-Fu. In addition, cyclin A was down-regulated in the let-7 g transfectants cells.The results showed that let-7 g microRNA contributed to an increase of 5-Fu-induced cell cycle inhibit in human hepatoma cell and sensitized cells to 5-Fu, leading to increased the effectiveness of the drug in treating hepatoma cancer.

HMGA2 regulates the in vitro aging and proliferation of human umbilical cord blood-derived stromal cells through the mTOR/p70S6K signaling pathway.

The human high-mobility group protein A2 (HMGA2) protein is an architectural transcription factor that transforms chromatin structure by binding to DNA. Recently, it has been reported that HMGA2 is highly expressed in fetal neural stem cells and has the capacity to promote stemness. However, there is currently no information available on the functional significance and molecular mechanisms of the cellular in vitro aging and proliferation of human umbilical cord blood-derived stromal cells (hUCBSCs). In the present study, we evaluated the direct effects of HMGA2 on the cellular aging and proliferation of hUCBSCs and investigated potential regulatory mechanisms responsible for the corresponding functions. We found that the overexpression of HMGA2 enhanced proliferation and reduced or even reversed the in vitro aging process of hUCBSCs. This effect was accompanied by the increased expression of cyclin E and CDC25A and the significantly decreased expression of cyclin-dependent kinase inhibitors. Furthermore, HMGA2 inhibition compromised cell proliferation and adipogenic differentiation in early-stage hUCBSCs. From the molecular/cellular functional analysis of microarray data, we found that HMGA2 overexpression induced a PI3K/Akt/mTOR/p70S6K cascade, which in turn suppressed the expression of p16(INK4A) and p21(CIP1/WAF1) in hUCBSCs. These results provide novel insights into the mechanism by which HMGA2 regulates the in vitro aging and proliferation of hUCBSCs.

Molecular deregulation induced by silencing of the high mobility group protein A2 gene in retinoblastoma cells.

AIM: To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2) gene silencing in retinoblastoma (RB) cells. METHODS: Synthetic anti-HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in post-silenced RB cell lines (Y79 and WERI Rb1). These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10). Zymographic determination of matrix metalloproteinase (MMP) activity was performed in RB cells. A cell cycle assay and a proliferation assay were performed in post-transfected RB cells. RESULTS: HMGA2 gene silencing in cultured RB cells results in reduced cell proliferation and transition in the G1/S phase. The whole genome microarray analysis of HMGA2 silenced Y79 cells revealed overall upregulation of 1,132 genes (≥ 1.0 fold) and downregulation of 1,562 genes (≤ -1.0 fold). Specific quantitative pathway analysis of the deregulated genes (using Biointerpreter) revealed 150 upregulated genes and 77 downregulated genes (≥ 1.0 fold) involved in vital pathways, namely, mitogen-activated protein kinase, Janus kinase/signal transducers and activators of transcription, Ras pathway, Ras-induced extracellular signal-regulated protein kinases 1 and 2, and tumor protein p53. The differential expression of genes obtained from microarray analysis (Homo sapiens ELK1, member of ETS oncogene family [ELK1], Homo sapiens cyclin-dependent kinase 6 [CDK6], Homo sapiens E2F transcription factor 4, p107/p130-binding [E2F4], Homo sapiens G-2 and S-phase expressed 1 [GTSE1], Damage-regulated autophagy modulator [DRAM], Homo sapiens cadherin 1, type 1,E-cadherin (epithelial) [CDH1], Homo sapiens snail homolog 1 (Drosophila) [SNAI1], Homo sapiens matrix metallopeptidase 2 [MMP2], and Homo sapiens matrix metallopeptidase 9 [MMP9]) was confirmed with quantitative reverse-transcriptase polymerase chain reaction in post-silenced RB cells. Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity. CONCLUSIONS: Our study revealed molecular regulatory changes induced by HMGA2 silencing in RB cancer cells, offering mechanistic insights into the anticancer potential. HMGA2 may be considered a promising candidate for gene silencing therapy in RB.

Reverse engineering a hierarchical regulatory network downstream of oncogenic KRAS.

RAS mutations are highly relevant for progression and therapy response of human tumours, but the genetic network that ultimately executes the oncogenic effects is poorly understood. Here, we used a reverse-engineering approach in an ovarian cancer model to reconstruct KRAS oncogene-dependent cytoplasmic and transcriptional networks from perturbation experiments based on gene silencing and pathway inhibitor treatments. We measured mRNA and protein levels in manipulated cells by microarray, RT-PCR and western blot analysis, respectively. The reconstructed model revealed complex interactions among the transcriptional and cytoplasmic components, some of which were confirmed by double pertubation experiments. Interestingly, the transcription factors decomposed into two hierarchically arranged groups. To validate the model predictions, we analysed growth parameters and transcriptional deregulation in the KRAS-transformed epithelial cells. As predicted by the model, we found two functional groups among the selected transcription factors. The experiments thus confirmed the predicted hierarchical transcription factor regulation and showed that the hierarchy manifests itself in downstream gene expression patterns and phenotype.