High Mobility Group AT-hook Research Articles

Targeting of histone methyltransferase DOT1L plays a dual role in chemosensitization of retinoblastoma cells and enhances the efficacy of chemotherapy

Aberrant and exclusive expression of chromatin regulators in retinoblastoma (RB) in contrast to terminally differentiated normal retina presents a unique opportunity of selective targeting for RB. However, precise roles of these chromatin regulators in RB development and their potential as therapeutic targets have not been defined thoroughly. Here, we report that targeting of disruptor of telomeric silencing 1-like (DOT1L), a histone H3K79 methyltransferase, sensitizes RB cells to chemotherapeutic drugs by impairing the DNA damage response and thereby potentiating apoptosis while it is largely inefficacious as a single-agent therapy. Moreover, we identified high mobility group AT-hook 2 (HMGA2) as a novel DOT1L target gene in RB cells and found that its aberrant expression is dependent on DOT1L. As HMGA2 depletion reduced CHK1 phosphorylation during DNA damage response and augmented the drug sensitivity in RB cells, our results suggested that DOT1L targeting has a dual role in chemosensitization of RB cells by directly interfering with the immediate involvement of DOT1L in early DNA damage response upon genotoxic insults and also by downregulating the expression of HMGA2 as a rather late effect of DOT1L inhibition. Furthermore, we provide the first preclinical evidence demonstrating that combined therapy with a DOT1L inhibitor significantly improves the therapeutic efficacy of etoposide in murine orthotopic xenografts of RB by rendering the response to etoposide more potent and stable. Taken together, these results support the therapeutic benefits of DOT1L targeting in combination with other chemotherapeutic agents in RB, with mechanistic insights into how DOT1L targeting can improve the current chemotherapy in an RB cell-selective manner.

Long noncoding RNA CRART16 confers 5-FU resistance in colorectal cancer cells by sponging miR-193b-5p

BackgroundThe emergence of chemoresistance to 5-fluorouracil (5-FU)-based chemotherapy is the main cause of treatment failure in advanced and metastatic colorectal cancer (CRC) patients. Long noncoding RNAs (lncRNAs) have been reported to be involved in 5-FU resistance. Previously, we first detected that lncRNA cetuximab resistance-associated RNA transcript 16 (CRART16) could contribute to cetuximab resistance by upregulating V-Erb-B2 erythroblastic leukemia viral oncogene homologue 3 (ERBB3) expression by sponging miR-371a-5p in CRC cells. The current study aimed to explore the role of CRART16 in acquired 5-FU resistance in CRC cells and its possible mechanism.MethodsQuantitative real-time PCR (RT-qPCR) was used to measure the expression levels of CRART16 in a 5-FU-resistant CRC cell subline (SW620/5-FU) and the parent cell line. Lentivirus transduction was performed to establish SW620 and Caco-2 cells stably overexpressing CRART16. Cell Counting Kit-8 (CCK-8) assays and colony formation assays were applied to measure cell chemosensitivity to 5-FU. Flow cytometric and immunofluorescence staining were adopted to assess cell apoptosis induced by 5-FU. The dual-luciferase reporter assay was used to validate the direct interactions between CRART16 and miR-193b-5p and between miR-193b-5p and high-mobility group AT-hook-2 (HMGA2). The expression levels of HMGA2, apoptosis-associated proteins and p-ERK were examined by western blotting. The statistical differences within any two groups were used Student’s t test.ResultsCRART16 was upregulated in SW620/5-FU cells. Overexpression of CRART16 reduced the sensitivity of CRC cells to 5-FU by attenuating apoptosis. In addition, CRART16 promoted 5-FU resistance by suppressing the expression of miR-193b-5p. Furthermore, CRART16 modulated the expression of HMGA2 by inhibiting miR-193b-5p and activated the MAPK signaling pathway.ConclusionsCRART16 confers 5-FU resistance in CRC cells through the CRART16/miR-193b-5p/HMGA2/MAPK pathway.

Exploring Cytotoxic Potential of Ciclopirox on Colorectal Cancer Cells by In-Silico Methodology

Colorectal cancer (CRC) stands 3rd among male cancer cases and the second most prevalent disease in women, accounting for 10% of all cancer cases globally. Ciclopirox (CPX) is a broad-spectrum, synthetic, off-patent antifungal drug recommended in dermatological conditions of mycoses of the skin and nails. There are no important molecular docking studies on inhibitory aspects of CPX against CRC targets. The main objective of this study was to explore the potential of CPX as an anti-CRC agent by using in-silico approaches with the help of published literature on downregulation of overhead protein expression in CRC and combining this information in order to recognize novel drug targets [Cell division cycle 25A (Cdc25A), Protein deglycase DJ-1 (DJ-1), Retinoblastoma protein (p-Rb/Rb), Cyclin-dependent kinase-4 (CDK4), High-mobility group AT-hook-2 (HMGA2), and Catenin β-1 (Wnt/-catenin)] and to identify the perspectives for drug repurposing and comparing this with oxaliplatin; one of the standard drug used in CRC. Also, in silico drug-likeliness studies, bioavailability studies, pharmacokinetic studies, drug target prediction, and bioisosteric replacement have been performed for CPX using online SwissADME tools (SwissADME, SwissTargetPrediction, and SwissBioisostere). The in silico studies revealed that CPX successfully inhibited all the molecular targets, which suggested plausible re-utilization of CPX for treating CRC.

HMGA2 Maintains Hematopoietic Stem Cell Via Pleiotropic Regulation of the Transcription in Stress Conditions

High-mobility group AT-hook 2 (Hmga2), an epigenetic modifier, opens the chromatin and modulate the transcription. Hmga2 is highly expressed in fetal and adult hematopoietic stem cells (HSCs). Hmga2 over-expression has been shown to promote self-renewal of HSC, however the molecular mechanism of how Hmga2 enhanced the self-renewal of HSC is still unclear. In this study, we assessed the function of Hmga2 in HSCs in steady and stress conditions by utilizing new Hmga2 conditional knock-in (KI) mouse and Hmga2 conditional knock-out mouse, which were crossed with either Cre-ERT2 mouse or Vav1-iCre mouse. Hmga2 KI mice showed a mild elevation in platelet counts, but did not develop malignancies in one year observation period. We performed a competitive transplantation assay by using purified HSCs, and found that wild-type HSCs diminished the repopulating capacity at the tertiary transplantation, Hmga2 KI HSCs maintained higher chimerism in myeloid cells and platelets in the PB and HSCs in the BM. We found that Hmga2 KO cells reduced the repopulating capacity, compared to wild-type cells. Thus, the expression of Hmga2 is critical for the self-renewal of HSC upon the transplantation. By performing RNA-sequencing of HSCs in homeostatic condition, we found that Hmga2 KI HSCs showed positive enrichments in cell cycle and proliferative signature, but maintained a stem cell signature, compared to wild-type HSCs. Since Hmga2 has been shown to globally open the chromatin in neural stem cells, we performed ATAC-sequence analysis in HSCs. Notably, Hmga2 KI HSCs showed 539 opened and 387 closed chromatin in H3K27ac-marked active regulatory regions, compared to wild-type HSCs. Among these opened genes by Hmga2, we generated a virus vector for fifteen genes, which were highly expressed in Hmga2 KI HSCs, and found that ectopic expression of Igf2bp2, an RNA binding protein, increased self-renewal capacity of HSC, but did not induce the enhanced production of myeloid cells and platelets that were observed in Hmga2 KI cells, in in vitro and in vivo settings. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was directly bound to a proximal region of the Igf2bp2 gene, and CRISPR/Cas9-mediated deletion of the Igf2bp2 gene canceled the enhanced self-renewal capacity of Hmga2 KI HSCs, indicating that the Hmga2-Igf2bp2 axis is critical for the self-renewal of HSC.We next assessed function of Hmga2 in stress hematopoiesis after in vivo treatment of 5-FU. Hmga2 KI mice showed faster recoveries of reduction of platelets in the PB and increased CD41+HSCs and megakaryocyte progenitors in the BM in twelve days, in which WT mice reduced numbers of those cells in this condition. RNA-sequencing revealed that Hmga2 KI HSCs maintained expression levels of genes in stem cell- and proliferation-signatures at 3 days and 6 days post 5-FU injection, compared to WT HSCs that reduced expression of stem cell genes but activated inflammatory response genes. In contrast, Hgma2 KO HSCs enhanced expression of inflammatory response genes post the 5-FU injection, indicating Hmga2 represses expression of inflammatory response genes in the stress condition. Indeed, Hmga2-ChIP-sequencing revealed that Hmga2 was bound to larger numbers of genes involved in inflammatory responses in Kit+ cells post the 5-FU treatment from those in the control cells. Given increased expression of and a post-translational modification of Hmga2 protein in Kit+ cells post the 5-FU treatment, the remodeling of Hmga2 binding regions was appeared to depend on the modification of Hmga2 at downstream of the stress signal. Thus, Hmga2 directly activates Igf2bp2 to enhance the self-renew of HSC, but also represses the inflammatory response, leading to the enhanced megakaryopoiesis in the stress condition. DisclosuresNo relevant conflicts of interest to declare.

Systematic analysis of the long noncoding RNA (lncRNA)-miRNA-mRNA competing endogenous RNA network to identify prognostic biomarkers and the potential regulatory axis in glioblastoma multiforme.

BackgroundGlioblastoma (GBM) is an intracranial brain tumor characterized by a high final lethality rate and recurrence rate, and limited available therapies. With the development of high-throughput sequencing technology, the genomic and transcriptomic features of GBM have been fully characterized. Therefore, our study aimed to identify its underlying genetic mechanisms, thus facilitating the development of novel therapies for GBM.MethodsBased on the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases, differential expression of RNAs in GBM and control group was analyzed. After constructing the long noncoding RNA (lncRNA)-miRNA-mRNA regulatory network of GBM, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGGs) were performed to analyze related key nodes and the lncRNAs interacting with them. Further univariate Cox regression was conducted to explore independent factors, and then multivariate Cox regression was performed to construct risk prediction models.ResultsWe first constructed the lncRNA-miRNA-mRNA regulatory network of GBM and two effective prediction models that included 2 mRNAs [transcription factor 12 (TCF12) and discoidin, CUB and LCCL domain containing 2 (DCBLD2)] and 5 lncRNAs (C10orf25, LINC00343, HOXA transcript antisense RNA, myeloid-specific 1 (HOTAIRM1), FGF12 antisense RNA 2 (FGF12-AS2) and H19). Additionally, we identified several key molecules [TCF12, integrin β3 (ITGB3), high mobility group AT-hook 2 (HMGA2), C10orf25 and LINC00336] closely associated with GBM prognosis. C10orf25/miR-218/DCBLD2 may be an important regulatory pathway in GBM.ConclusionsKey molecules (TCF12, ITGB3, HMGA2, C10orf25, LINC00336 and H19) that are independent prognostic factors may be possible biomarkers to further optimize GBM prognosis. Two effective prognostic risk models that include 2 mRNAs (TCF12 and DCBLD2) and 5 lncRNAs (C10orf25, LINC00343, HOTAIRM1, FGF12-AS2 and H19) were constructed. C10orf25/miR-218/DCBLD2 may be an important regulatory pathway associated with the pathogenesis of GBM. Our findings contribute to further understanding the pathogenesis of GBM and finding possible candidate genes for prognostic and therapeutic usage with GBM.

Regulatory effect between HMGA2 and the Wnt/β-catenin signaling pathway in the carcinogenesis of sporadic colorectal tubular adenoma.

Due to the high incidence of colorectal cancer worldwide, the underlying molecular mechanisms have been extensively investigated. The Wnt/β-catenin signaling pathway plays a key role in the carcinogenesis of colorectal adenoma. In addition, the high mobility group AT-hook 2 (HMGA2) protein, which is involved in several biological processes, such as proliferation, differentiation, transformation and metastasis, is expressed at significantly high levels in colorectal cancer tissues compared with adjacent normal tissues. Currently, the role of HMGA2 in the carcinogenesis of sporadic colorectal tubular adenoma remains unclear. The downstream Wnt/β-catenin signaling molecule, T-cell factor/lymphoid enhancing factor (TCF/LEF), shares a similar domain with HMGA2, which enhances β-catenin transcriptional activity and TCF/LEF binding. Thus, the present study investigated the association between HMGA2 and the Wnt/β-catenin signaling pathway, and their role in the carcinogenesis of sporadic colorectal tubular adenoma via immunohistochemistry, siRNA, quantitative PCR and western blot analyses. The results demonstrated that the positive rate of HMGA2 expression gradually increased during tumor progression. Furthermore, HMGA2 expression was positively correlated with Wnt/β-catenin signaling protein expression [Wnt, β-catenin, cyclin-dependent kinase 4 (CDK4) and cyclin D1], suggesting its involvement in the carcinogenesis of sporadic colorectal tubular adenoma and its potential to synergistically interact with the Wnt/β-catenin signaling pathway. HMGA2 knockdown in the human colorectal cancer cell line, HCT 116 decreased β-catenin expression and its downstream targets, CDK4 and cyclin D1. Furthermore, silencing of Wnt or β-catenin decreased HMGA2 expression. Taken together, the results of the present study suggest the coordinated regulation of HMGA2 and the Wnt/β-catenin signaling pathway in the carcinogenesis of sporadic colorectal tubular adenoma.

CircRNA Circ-CCND1 Aggravates Hepatocellular Carcinoma Tumorigenesis by Regulating the miR-497-5p/HMGA2 Axis.

Circular RNAs (circRNAs) are key regulators in hepatocellular carcinoma (HCC) tumorigenesis and development, yet it is unclear whether circ-CCND1 participates in regulating HCC progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for detecting the expressions of circ-CCND1, microRNA (miR) -497-5p, and high mobility group AT-hook 2 (HMGA2) mRNA in HCC tissues and cell lines. Subcellular fractionation assay was used to analyze the localization of circ-CCND1 in HCC cell lines. Loss-of-function experiments were conducted to examine the effects of circ-CCND1 on HCC cell proliferation, migration, and invasion. Dual-luciferase reporter gene assay was employed for detecting the targeting relationships of circ-CCND1 and miR-497-5p, as well as miR-497-5p and HMGA2, respectively. Western blot was used to detect the regulatory functions of circ-CCND1 and miR-497-5p on HMGA1 expression at protein level. Circ-CCND1 and HMGA2 expressions in HCC were significantly up-regulated and miR-497-5p expression was markedly decreased. High circ-CCND1 expression was associated with relatively large tumor size and lymph node metastasis in HCC patients. In addition, circ-CCND1 was mainly distributed in the cytoplasm of HCC cells. Functionally, knockdown of circ-CCND1 remarkably suppressed HCC cell proliferation, migration, and invasion. Mechanistically, miR-497-5p was a direct target of circ-CCND1 and miR-497-5p specifically modulated HMGA2 expression. Furthermore, miR-497-5p inhibitors and or HMGA2 overexpression partially counteracted the suppressing effect induced by si-circ-CCND1 on the malignant phenotype of HCC cells. Circ-CCND1 plays a cancer-promoting role in HCC by modulating the miR-497-5p/HMGA2 axis. Therefore, targeting circ-CCND1 is likely to be a promising therapeutic strategy for HCC.

Let‑7b‑5p promotes cell apoptosis in Parkinson's disease by targeting HMGA2

Parkinson's disease (PD), a common multifactorial neurodegenerative disease, is characterized by irreversible loss of dopaminergic neurons in the substantia nigra. In-depth study of the pathogenesis of PD is of great importance. High-mobility group AT-hook 2 (HMGA2) has been proposed to be implicated with neuronal differentiation and impairment of cognitive function. However, whether HMGA2 plays a role in PD is rarely explored. In the present study, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated PD mice models and N-methyl-4- phenylpyridinium (MPP+)-treated SH-SY5Y cell models were established. Reverse transcription-quantitative PCR showed that HMGA2 displayed low levels in brain tissues of MPTP-treated mice and MPP+-treated SH-SY5Y cells. Moreover, HMGA2 overexpression suppressed SH-SY5Y cell apoptosis. Additionally, let-7b-5p bound with HMGA2 3′ untranslated region (UTR), and its expression was negatively correlated with HMGA2 level. Moreover, let-7b-5p presented high levels in brain tissues of PD mice and MPP+-treated SH-SY5Y cells, and knockdown of let-7b-5p inhibited SH-SY5Y cell apoptosis. Rescue assays illustrated that HMGA2 neutralized the promotive effects of let-7b-5p mimics on SH-SY5Y cell apoptosis. In conclusion, the present study demonstrated that let-7b-5p contributes to cell apoptosis in PD by targeting HMGA2, which offers a potential theoretical basis for the study of effective therapy in PD.

Circ-RPPH1 knockdown retards breast cancer progression via miR-328-3p-mediated suppression of HMGA2

BackgroundCircular RNA Ribonuclease P RNA Component H1 (circ-RPPH1) was confirmed to act as an oncogene in many cancers to promote cancer progression. However, the exact function and mechanism of circ-RPPH1 in breast cancer (BC) remain vague. MethodsThe expression of circ-RPPH1, microRNA (miR)-328-3p and high-mobility group AT-hook 2 (HMGA2) was detected using quantitative real-time polymerase chain reaction and western blot. Cell viability, apoptosis, migration and invasion were determined using cell counting kit-8 assay, flow cytometry and transwell assay, respectively. Glucose metabolism was calculated by detecting glucose uptake and lactate production. The target correlations between miR-328-3p and circ-RPPH1 or HMGA2 were confirmed by dual-luciferase reporter assay. The murine xenograft model was established to conduct in vivo experiments. ResultsCirc-RPPH1 expression was elevated and miR-328-3p was decreased in BC tissues and cells. Circ-RPPH1 knockdown or miR-328-3p re-expression suppressed cell proliferation, migration, invasion and glycolysis but induced apoptosis in BC in vitro. Circ-RPPH1 was a sponge of miR-328-3p, and silencing of miR-328-3p reversed the inhibitory effects of circ-RPPH1 knockdown on BC cell malignant phenotypes and glycolysis. MiR-328-3p directly targeted HMGA2, and HMGA2 overexpression abolished the action of miR-328-3p in BC cells. Besides, circ-RPPH1 could regulate HMGA2 expression by miR-328-3p in BC cells. Moreover, murine xenograft model analysis suggested circ-RPPH1 knockdown inhibited tumor growth in vivo. ConclusionCirc-RPPH1 knockdown retarded cell malignant phenotypes and glycolysis via miR-328-3p/HMGA2 axis in BC, providing a potential therapeutic target for BC treatment.

CircFBXL5 promotes the 5-FU resistance of breast cancer via modulating miR-216b/HMGA2 axis

BackgroundCircular RNAs (circRNAs) have been confirmed to be relevant to the 5-fluorouracil (5-FU) resistance of breast cancer. Nevertheless, how and whether circRNA F-box and leucine-rich repeat protein 5 (circFBXL5) regulates the 5-FU resistance of breast cancer is uncertain. This study aims to explore the function and mechanism of circFBXL5 in the 5-FU resistance of breast cancer.MethodsThirty nine paired breast cancer and normal tissues were harvested. circFBXL5, microRNA-216b (miR-216b) and high-mobility group AT-hook 2 (HMGA2) abundances were examined via quantitative reverse transcription polymerase chain reaction or western blot. Cell viability, 5-FU resistance, migration, invasion, and apoptosis were tested via cell counting kit-8 assay, wound healing analysis, transwell analysis, and flow cytometry. The relationship of miR-216b and circFBXL5 or HMGA2 was tested via dual-luciferase reporter analysis and RNA pull-down assay. The impact of circFBXL5 on breast cancer tumor growth in vivo was analyzed via xenograft model.ResultscircFBXL5 was highly expressed in breast cancer tissues and cells, and was more upregulated in 5-FU-resistant breast cancer cells. Function experiments showed that circFBXL5 knockdown inhibited the 5-FU resistance of breast cancer by inhibiting cell migration, invasion and promoting apoptosis. In the terms of mechanism, miR-216b could be sponged by circFBXL5, and its inhibitor could also reverse the influence of circFBXL5 silencing on the 5-FU resistance of breast cancer cells. In addition, HMGA2 was a target of miR-216b, and its overexpression also reversed the regulation of miR-216b overexpression on the 5-FU resistance of breast cancer. Furthermore, circFBXL5 interference declined breast cancer tumor growth in xenograft model.ConclusionOur data showed that circFBXL5 could promote the 5-FU resistance of breast cancer by regulating miR-216b/HMGA2 axis.

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.

CSNK2A1-mediated phosphorylation of HMGA2 modulates cisplatin resistance in cervical cancer.

The development of chemoresistance reduces the efficacy of anti‐cancer drugs. Cervical cancer is still one of the most common cancer types in developing countries. The oncogenic protein high mobility group AT‐hook 2 (HMGA2) is involved in the development and progression of tumors, although its role in chemoresistance of cervical cancer remains unclear. Here, we report that HMGA2 is highly expressed in cervical cancer and negatively correlated with cisplatin‐induced cell death. We performed liquid chromatography‐tandem mass spectrometry to demonstrate that HMGA2 has high potential to interact with casein kinase II A1 (CSNK2A1). Moreover, we observed that HMGA2 co‐localizes with CSNK2A1 in the nucleus by immunofluorescence. Binding of HMGA2‐CSNK2A1 was detected by immunoprecipitation assays. In addition, we identified that cisplatin induces an interaction between CSNK2A1 and HMGA2, thereby promoting the phosphorylation of HMGA2. CX‐4945, a CSNK2A1 inhibitor, could inhibit the phosphorylation of HMGA2 and sensitize tumor cells to cisplatin. Our results reveal that CSNK2A1‐dependent HMGA2 phosphorylation may partially underlie cisplatin‐resistance in cervical cancer, suggesting that HMGA2 phosphorylation may have potential as a predicative biomarker and therapeutic target to improve chemotherapeutic efficacy.

Overexpression of hsa_circ_0008274 inhibited the progression of lung adenocarcinoma by regulating HMGA2 via sponging miR-578.

BackgroundCircular RNAs (circRNAs) had been identified as a non‐coding RNA associated with many types of cancer in recent years. However, the involvement of hsa_circ_0008274 in lung adenocarcinoma (LUAD) has not been explored. The aim of our research is to explore the biological mechanism and function of hsa_circ_0008274 in LUAD.MethodsThe hsa_circ_0008274, miR‐578, and high mobility group AT‐Hook 2 (HMGA2) mRNA expression levels were detected via qRT‐PCR. Cell Counting Kit‐8 (CCK‐8) Transwell assay and wound healing assay were performed to measure the cell proliferation, invasion, and migration ability. Luciferase reporter and Western blotting experiments were performed to characterize the competing endogenous RNA (ceRNA) mechanism of hsa_circ_0008274.ResultsOur findings determined that the expression of hsa_circ_0008274 in LUAD was significantly decreased. Cell experiments showed that overexpressed hsa_circ_0008274 could reduce the proliferation and invasion ability of LUAD cells. Moreover, miRNA‐578 could identify as a miRNA sponge of hsa_circ_0008274. Overexpressed hsa_circ_0008274 reduced the proliferation and invasion of LUAD cells caused by miR‐578 mimics. Increasing the expression of miR‐578 can aggravate the proliferation and invasion of LUAD cells and block the inhibition of proliferation and invasion of LUAD cells mediated by overexpressed hsa_circ_0008274. Subsequent data indicate that HMGA2 of the tumor‐promoting gene is the target gene of miR‐578. The upregulation of HMGA2 partially reversed the tumor inhibitory effect of LUAD cells induced by overexpressed hsa_circ_0008274 or miR‐578 mimics.ConclusionsIn summary, our data show that the overexpression of hsa_circ_0008274 repressed the proliferation and invasion of LUAD through downregulating miR‐578 and activating HMGA2.

MiR-532-3p Suppresses Nucleus Pulposus Cells Proliferation and Extracellular Matrix Production, Promotes Cell Apoptosis via Targeting High Mobility Group AT-Hook 2

The present study aimed to investigate the function and mechanism of microRNA (miR)-532-3p in intervertebral disc degeneration (IDD). Further, whether miR-532-3p regulates HMGA2 in nucleus pulposus (NP) cells was explored. We collected human nucleus pulposus (NP) tissues from the patients with IDD, and detected miR-532-3p in NP tissues using RT-qPCR. MiR-532-3p mimic and inhibitor were constructed, and they were transfected into the human nucleus pulposus cells (HNPCs) by Lipofectamine 3000. MTT assay was conducted to determine cell proliferation. Cell apoptosis and extracellular matrix remodeling were examined by flow cytometric, Caspase 3/8 Assay Kits and Western blot. A dual-luciferase reporter assay was applied to investigate whether miR-532-3p targets High mobility group AT-hook 2 (HMGA2). We found miR-532-3p expression level was significantly increased in NP tissues of IDD patients, comparing with the controls. MiR-532-3p exerted an inhibitory effect on HNPCs proliferation; however, cell apoptosis and the degradation of extracellular matrix were induced by miR-532-3p. MiR-532-3p directly targets HMGA2, and HMGA2 overexpression reversed the role of miR-532-3p mimic in HNPCs proliferation, apoptosis, and extracellular matrix remodeling. Our study is the first to report that miR-532-3p might suppress NP cell proliferation, promote cell apoptosis and inhibit ECM production of NP cells via targeting HMGA2, thus facilitating the progression of IDD. MiR-532-3p was supposed to be a novel target for the treatment of IDD.

LncRNA HCP5 knockdown inhibits high glucose-induced excessive proliferation, fibrosis and inflammation of human glomerular mesangial cells by regulating the miR-93-5p/HMGA2 axis

BackgroundLong non-coding RNAs (lncRNAs) are widely reported to be involved in the development of human diseases. HLA complex P5 (HCP5) deregulation is associated with various diseases. However, the function of HCP5 in diabetic nephropathy (DN) is unclear.MethodsHuman glomerular mesangial cells (HGMCs) were treated with high glucose (HG) to establish DN cell models. The expression of HCP5, miR-93-5p and high mobility group AT-hook 2 (HMGA2) mRNA was detected using quantitative polymerase chain reaction (QPCR). Cell proliferation and cell apoptosis were assessed using cell counting kit-8 (CCK-8) assay and flow cytometry assay, respectively. The expression of apoptosis- and fibrosis-related proteins and HMGA2 protein was quantified by western blot. The release of pro-inflammatory factor was checked using enzyme-linked immunosorbent assay (ELISA). The predicted relationship between miR-93-5p and HCP5 or HMGA2 was verified using dual-luciferase reporter assay, pull-down assay or RNA immunoprecipitation (RIP) assay.ResultsThe expression of HCP5 and HMGA2 was enhanced, while the expression of miR-93-5p was declined in DN serum samples and HG-treated HGMCs. HCP5 knockdown or miR-93-5p restoration ameliorated HG-induced HGMC proliferation, fibrosis and inflammation. MiR-93-5p was a target of HCP5, and miR-93-5p inhibition reversed the effects caused by HCP5 knockdown. Moreover, HMGA2 was a target of miR-93-5p, and HMGA2 overexpression abolished the effects of miR-93-5p restoration. HCP5 knockdown inhibited the AKT/mTOR signaling pathway.ConclusionHCP5 was implicated in DN progression by modulating the miR-93-5p/HMGA2 axis, which provided new insights into the understanding of DN pathogenesis.

Circ-ACTR2 aggravates the high glucose-induced cell dysfunction of human renal mesangial cells through mediating the miR-205-5p/HMGA2 axis in diabetic nephropathy

BackgroundCircular RNAs (circRNAs) have been considered as pivotal biomarkers in Diabetic nephropathy (DN). CircRNA ARP2 actin-related protein 2 homolog (circ-ACTR2) could promote the HG-induced cell injury in DN. However, how circ-ACTR2 acts in DN is still unclear. This study aimed to explore the molecular mechanism of circ-ACTR2 in DN progression, intending to provide support for the diagnostic and therapeutic potentials of circ-ACTR2 in DN.MethodsRNA expression analysis was conducted by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Cell growth was measured via Cell Counting Kit-8 and EdU assays. Inflammatory response was assessed by Enzyme-linked immunosorbent assay. The protein detection was performed via western blot. Oxidative stress was evaluated by the commercial kits. The molecular interaction was affirmed through dual-luciferase reporter and RNA immunoprecipitation assays.ResultsCirc-ACTR2 level was upregulated in DN samples and high glucose (HG)-treated human renal mesangial cells (HRMCs). Silencing the circ-ACTR2 expression partly abolished the HG-induced cell proliferation, inflammation and extracellular matrix accumulation and oxidative stress in HRMCs. Circ-ACTR2 was confirmed as a sponge for miR-205-5p. Circ-ACTR2 regulated the effects of HG on HRMCs by targeting miR-205-5p. MiR-205-5p directly targeted high-mobility group AT-hook 2 (HMGA2), and HMGA2 downregulation also protected against cell injury in HG-treated HRMCs. HG-mediated cell dysfunction was repressed by miR-205-5p/HMGA2 axis. Moreover, circ-ACTR2 increased the expression of HMGA2 through the sponge effect on miR-205-5p in HG-treated HRMCs.ConclusionAll data have manifested that circ-ACTR2 contributed to the HG-induced DN progression in HRMCs by the mediation of miR-205-5p/HMGA2 axis.

Si-MALAT1 attenuates thymic cancer cell proliferation and promotes apoptosis via the miR-145-5p/HMGA2 pathway.

Metastasis-associated-lung-adenocarcinoma-transcript-1 (MALAT1) is a long non-coding RNA that is considered a potential tumor marker. The present study aimed to investigate the effect and mechanism of MALAT1 on cell proliferation and apoptosis in thymic cancer cells. IU-TAB-1, A549, HCT-116 and 293T cells were screened by reverse transcription-quantitative PCR to assess high-mobility group AT-hook 2 (HMGA2) expression in various types of cancer cells and were transfected with small interfering (si)RNA targeting MALAT1 (si-MALAT1). Cell proliferation was evaluated by Cell Counting Kit-8 assay. Cell apoptosis and cell cycle were examined using flow cytometry. The protein expression of cyclin D1, cyclin E, Bax, Bcl-2 and HMGA2 was determined by western blot analysis, while the associations between MALAT1 and microRNA (miR)-145-5p and between HMGA2 and miR-145-5p were determined by luciferase reporter assay. Among the four cell lines evaluated, IU-TAB-1 showed the highest expression of MALAT1; thus, IU-TAB-1 cells were selected for subsequent experiments. Compared with the findings in the control group, si-MALAT1 significantly decreased the cell proliferation of IU-TAB-1 cells, whereas the apoptosis levels and number of cells in G2 phase were increased. The protein expression levels of cyclin D1, cyclin E, Bcl-2 and HMGA2 were significantly decreased in the si-MALAT1 group compared with those in the control group, while Bax levels were significantly increased. After treatment with si-MALAT1 in combination with miR-145-5p mimics or inhibitors, cell proliferation and apoptosis were respectively enhanced and inhibited in IU-TAB-1 cells. miR-145-5p inhibited the luciferase activity of IU-TAB-1 cells transfected with the MALAT1 or HMGA2 3′ untranslated region. In conclusion, si-MALAT1 significantly attenuated cell proliferation and apoptosis via the miR-145-5p/HMGA2 pathway in thymic cancer cells.

High-mobility group AT-Hook 1 mediates the role of nuclear factor I/X in osteogenic differentiation through activating canonical Wnt signaling.

It was previously reported that the loss of the transcription factor nuclear factor I/X (NFIX) gene in mice impaired endochondral ossification and mineralization in bone. However, the cellular and molecular basis for the defect remains unexplored. In this study, we investigated if and how NFIX regulates osteoblast differentiation. Nfix mRNA was induced during osteogenic and adipogenic differentiation of progenitor cells. Loss-of-function and gain-of-function studies revealed that NFIX induced osteoblast differentiation and impaired adipocyte formation from progenitor cells. RNA-seq and promoter analysis revealed that NFIX transcriptionally stimulated the expression of high-mobility group AT-Hook 1 (HMGA1). We then demonstrated that HMGA1 stimulated osteogenic differentiation of progenitor cells at the expense of adipogenic differentiation. The effect of Nfix siRNA on the differentiation of progenitor cells could be attenuated when HMGA1 was simultaneously overexpressed. Further investigations revealed the stimulatory effect of NFIX and HMGA1 on canonical wingless-type MMTV integration site family (Wnt) signaling. HMGA1 transcriptionally activates the expression of low-density lipoprotein receptor-related protein 5. Finally, in vivo transfection of Nfix siRNA to the marrow of mice reduced osteoblasts and increased fat accumulation in the marrow, and inactivated HMGA1/β-catenin signaling in bone marrow mesenchymal stem cells. This study suggests that HMGA1 plays a role in osteoblast commitment and mediates the function of NFIX through transcriptionally activating canonical Wnt signaling.

CircARL8B Contributes to the Development of Breast Cancer Via Regulating miR-653-5p/HMGA2 Axis.

Circular RNAs (circRNAs) act as essential regulators in breast cancer (BC) progression. In this paper, we aimed to investigate the functions of circARL8B in BC. The levels of circARL8B, ADP Ribosylation Factor Like GTPase 8B (ARL8B), miR-653-5p and high-mobility group AT-hook 2 (HMGA2) mRNA were examined by qRT-PCR. The stability of circARL8B was determined by RNase R assay and Actinomycin D assay. Cell viability and metastasis were evaluated by Cell Counting Kit-8 (CCK-8) assay and transwell assay, respectively. The levels of cellular phospholipids and triglycerides were measured using relevant kits. Protein levels were measured by western blot analysis. The association between miR-653-5p and circARL8B or HMGA2 was verified by dual-luciferase reporter assay. A murine xenograft model was established to explore the function of circARL8B in vivo. CircARL8B was increased in BC tissues and cells. CircARL8B silencing inhibited cell viability, migration, invasion and fatty acid metabolism in BC cells in vitro and blocked tumor growth in vivo. MiR-653-5p was identified as the target of circARL8B and miR-653-5p was negatively modulated by circARL8B. The suppressive role of circARL8B silencing in BC cell progression was abolished by miR-653-5p downregulation. Moreover, HMGA2 was the target gene of miR-653-5p. HMGA2 overexpression abrogated the effect of miR-653-5p on BC cell development. In addition, circARL8B knockdown might block PGE2/PI3K/AKT/GSK-3β/Wnt/β-catenin pathway. Silencing of circARL8B inhibited cell viability, migration, invasion and fatty acid metabolism via miR-653-5p/HMGA2 axis in BC.

MicroRNA-296-5p inhibits cell proliferation by targeting HMGA1 in colorectal cancer.

An increasing body of evidence indicates the involvement of microRNAs (miRNAs/miRs) in the initiation and progression of colorectal cancer (CRC). miR-296-5p was recently identified as a tumor suppressor in a variety of human cancer types; however, its function in CRC remains largely unknown. The present study demonstrated that the expression of miR-296-5p was significantly downregulated in CRC tissues and cell lines. The overexpression of miR-296-5p markedly inhibited proliferation, and induced cell cycle arrest and apoptosis in CRC cells. Bioinformatics analysis suggested that high mobility group AT-hook 1 (HMGA1) may be a target of miR-296-5p in CRC cells. Further experiments showed that miR-296-5p bound the 3'-untranslated region of HMGA1 and decreased its expression in CRC cells. HMGA1 was overexpressed in CRC tissues and was inversely correlated with the expression of miR-296-5p. The restoration of HMGA1 significantly reversed the inhibitory effect of miR-296-5p on the proliferation of CRC cells. Overall, the findings of the present study indicate that miR-296-5p suppressed the progression of CRC, at least partially via targeting HMGA1. Thus, miR-296-5p is a potential target for novel therapies in CRC.