Long non-coding RNAs (lncRNAs), including natural antisense transcripts (NATs), are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer's disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

Neural stem and progenitor cells (NSCs/NPCs) are distinct groups of cells found in the mammalian central nervous system (CNS). Previously we determined that members of the High Mobility Group (HMG) B family of chromatin structural proteins modulate NSC proliferation and self-renewal. Among them HMGB2 was found to be dynamically expressed in proliferating and differentiating NSCs, suggesting that it may regulate NSC maintenance. We report now that Hmgb2(-/-) mice exhibit SVZ hyperproliferation, increased numbers of SVZ NSCs, and a trend towards aberrant increases in newly born neurons in the olfactory bulb (OB) granule cell layer. Increases in the levels of the transcription factor p21 and the Neural cell adhesion molecule (NCAM), along with down-regulation of the transcription/pluripotency factor Oct4 in the Hmgb2-/- SVZ point to a possible pathway for this increased proliferation/differentiation. Our findings suggest that HMGB2 functions as a modulator of neurogenesis in young adult mice through regulation of NSC proliferation, and identify a potential target via which CNS repair could be amplified following trauma or disease-based neuronal degeneration.

Mouse cortical development relies heavily on a delicate balance between neurogenesis and gliogenesis. The lateral ventricular zone produces different classes of excitatory pyramidal cells until just before birth, when the production of astroglia begins to prevail. Epigenetic control of this fate shift is of critical importance and chromatin regulatory elements driving neuronal or astroglial development play an vital role. Different classes of chromatin binding proteins orchestrate the transcriptional repression of neuronal-specific genes, while allowing for the activation of astrocyte-specific genes. Through proteomic analysis of embryonic neural progenitor cells (NPCs) our group had previously identified high mobility group B2 (HMGB2), a chromatin protein dynamically expressed throughout embryonic development. In the current study using cultures of perinatal NPCs from HMGB2+/+ and HMGB2-/- mice we discovered that vital elements of the polycomb group (PcG) epigenetic complexes polycomb repressive complexes 1 and 2 (PRC1/2) were downregulated during the differentiation process of HMGB2-null NPCs. These epigenetic changes led to downstream changes in specific histone modification levels, specifically the trimethylation of H3K27, and a subsequent shift in the perinatal neurogenesis to gliogenesis fate transition. Collectively these results demonstrate that chromatin binding proteins, such as HMGB2, can have significant effects on the epigenetic landscape of perinatal neural stem/progenitor cells.

Gastric cancer is one of the most common malignancy with a leading mortality rate worldwide. Despite the progress in the diagnosis and therapeutic strategy, the associated mortality is still growing. It is of great significance to understand molecular mechanisms of the development of gastric cancer. Glycolysis is a main source of ATP provision for cancer cells including gastric cancer, and targeting glycolysis is a promising therapeutic strategy. Centromere protein U (CENPU) has been found to be overexpressed in many types of cancer. Downregulation of CENPU suppresses the proliferation and invasion of cancer cells. High mobility group box 2 (HMGB2) is identified as a biomarker to diagnose of gastric cancer. Knockdown of HMGB2 inhibits proliferation and glycolysis in gastric cancer cells. In this work, we identified that CENPU was upregulated in gastric cancer. Knockdown of CENPU was able to suppress the proliferation and glycolysis of gastric cancer cells. Further the results showed that the anti-cancer effect of CENPU was HMGB2-dependent. Taken together, CENPU is an upstream factor of HMGB2, which regulates proliferation and glycolysis of gastric cancer.

Hematopoietic stem cells provide life-long production of blood cells and undergo self-renewal division in order to sustain the stem cell pool. Homeostatic maintenance of hematopoietic stem cell pool and blood cell production is vital for the organism to survive. We previously reported that latexin is a negative regulator of hematopoietic stem cells in mice. Its natural variation in the expression is inversely correlated with hematopoietic stem cell number. However, the molecular mechanisms regulating latexin transcription remain largely unknown, and the genetic factors contributing to its natural variation are not clearly defined. Here we discovered a chromatin protein, high-mobility group protein B2, as a novel transcriptional suppressor of latexin by using DNA pull-down and mass spectrometry. High-mobility group protein B2 knockdown increases latexin expression at transcript and protein levels, and decreases hematopoietic stem cell number and regeneration capacity in vivo Concomitant blockage of latexin activation significantly reverses these phenotypic changes, suggesting that latexin is one of the downstream targets and functional mediators of high-mobility group protein B2. We further identified a functional single nucleotide polymorphism, rs31528793, in the latexin promoter that binds to high-mobility group protein B2 and affects the promoter activity. G allelic variation in rs31528793 associates with the higher latexin expression and lower hematopoietic stem cell number, whereas C allele indicates the lower latexin expression and higher stem cell number. This study reveals for the first time that latexin transcription is regulated by both transacting (high-mobility group protein B2) and cis-acting (single nucleotide polymorphism rs31528793) factors. It uncovers the functional role of naturally occurring genetic variants, in combination with epigenetic regulator, in determining differential gene expression and phenotypic diversity in the hematopoietic stem cell population.

Liver regeneration is an extraordinarily complex process involving a variety of factors; however, the role of chromatin protein in hepatocyte proliferation is largely unknown. In this study, we investigated the functional role of high-mobility group box 2 (HMGB2), a chromatin protein in liver regeneration using wild-type and HMGB2-knockout (KO) mice. Liver tissues were sampled after 70% partial hepatectomy (PHx), and analyzed by immunohistochemistry, western blotting and flow cytometry using various markers of cell proliferation. In WT mice, hepatocyte proliferation was strongly correlated with the spatiotemporal expression of HMGB2; however, cell proliferation was significantly delayed in hepatocytes of HMGB2-KO mice. Quantitative PCR demonstrated that cyclin D1 and cyclin B1 mRNAs were significantly decreased in HMGB2-KO mice livers. Interestingly, hepatocyte size was significantly larger in HMGB2-KO mice at 36-72 h after PHx, and these results suggest that hepatocyte hypertrophy appeared in parallel with delayed cell proliferation. In vitro experiments demonstrated that cell proliferation was significantly decreased in HMGB2-KO cells. A significant delay in cell proliferation was also found in HMGB2-siRNA transfected cells. In summary, spatiotemporal expression of HMGB2 is important for regulation of hepatocyte proliferation and cell size during liver regeneration.

Noncoding RNAs are important for regulation of cardiac hypertrophy. The function of MALAT1 (a long noncoding mRNA), miR-181a, and HMGB2; their contribution to cardiac hypertrophy; and the regulatory relationship between them during this process remain unknown. In the present study, we treated primary cardiomyocytes with angiotensin II (Ang II) to mimic cardiac hypertrophy. MALAT1 expression was significantly downregulated in Ang II-treated cardiomyocytes compared with control cardiomyocytes. Ang II-induced cardiac hypertrophy was suppressed by overexpression of MALAT1 and promoted by genetic knockdown of MALAT1. A dual-luciferase reporter assay demonstrated that MALAT1 acted as a sponge for miR-181a and inhibited its expression during cardiac hypertrophy. Cardiac hypertrophy was suppressed by overexpression of a miR-181a inhibitor and enhanced by overexpression of a miR-181a mimic. HMGB2 was downregulated during cardiac hypertrophy and was identified as a target of miR-181a by bioinformatics analysis and a dual-luciferase reporter assay. miR-181a overexpression decreased the mRNA and protein levels of HMGB2. Rescue experiments indicated that MALAT1 overexpression reversed the effect of miR-181a on HMGB2 expression. In summary, the results of the present study show that MALAT1 acts as a sponge for miR-181a and thereby regulates expression of HMGB2 and development of cardiac hypertrophy. The novel MALAT1/miR-181a/HMGB2 axis might play a crucial role in cardiac hypertrophy and serve as a new therapeutic target.

Topoisomerase IIalpha (topo IIalpha) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIalpha, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIalpha gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIalpha promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIalpha promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIalpha gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIalpha mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIalpha by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIalpha promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIalpha in tumors.

Although various surgical procedures have been developed for chronic rotator cuff tear repair, the re-tear rate remains high with severe fat infiltration. However, little is known about the molecular regulation of this process. Mesenchymal stem cells (MSCs) in the intra-muscular space are origin of ectopic fat cells in skeletal muscle. We have previously shown that high-mobility group box 2 (HMGB2), which is a nuclear protein commonly associated with mesenchymal differentiation, is involved in the early articular cartilage degeneration. In this study, we addressed the role of HMGB2 in adipogenesis of MSCs and fat infiltration into skeletal muscles. HMGB2 was highly expressed in undifferentiated MSCs and co-localized with platelet-derived growth factor receptor α (PDGFRA) known as an MSC-specific marker, while their expressions were decreased during adipocytic differentiation. Under the deficiency of HMGB2, the expressions of adipogenesis-related molecules were reduced, and adipogenic differentiation is substantially impaired in MSCs. Moreover, HMGB2+ cells were generated in the muscle belly of rat supraspinatus muscles after rotator cuff transection, and some of these cells expressed PDGFRA in intra-muscular spaces. Thus, our findings suggest that the enhance expression of HMGB2 induces the adipogenesis of MSCs and the fat infiltration into skeletal muscles through the cascade of HMGB2-PDGFRA.

High mobility group box 2 (HMGB2) is a non-histone chromosomal protein involved in various biological processes, including cellular senescence. However, its role in cellular senescence has not been evaluated extensively. To determine the regulatory role and mechanism of HMGB2 in cellular senescence, we performed gene expression analysis, senescence staining, and tube formation assays using young and senescent microvascular endothelial cells (MVECs) after small RNA treatment or HMGB2 overexpression. HMGB2 expression decreased with age and was regulated at the transcriptional level. siRNA-mediated downregulation inhibited cell proliferation and accelerated cellular senescence. In contrast, ectopic overexpression delayed senescence and maintained relatively higher tube-forming activity. To determine the HMGB2 downregulation mechanism, we screened miRNAs that were significantly upregulated in senescent MVECs and selected HMGB2-targeting miRNAs. Six miRNAs, miR-23a-3p, 23b-3p, -181a-5p, -181b-5p, -221-3p, and -222-3p, were overexpressed in senescent MVECs. Ectopic introduction of miR-23a-3p, -23b-3p, -181a-5p, -181b-5p, and -221-3p, with the exception of miR-222-3p, led to the downregulation of HMGB2, upregulation of senescence-associated markers, and decreased tube formation activity. Inhibition of miR-23a-3p, -181a-5p, -181b-5p, and -221-3p delayed cellular senescence. Restoration of HMGB2 expression using miRNA inhibitors represents a potential strategy to overcome the detrimental effects of cellular senescence in endothelial cells.

High-mobility group box 2 (HMGB2) belongs to the HMG-box family that participates in a variety of biologic processes. Recent studies have suggested that HMGB2 plays an important role in the innate immunity of fish. Cherry Valley duck is the main duck bred for meat consumption in China, but there is limited research available on the impact of duck HMGB2 (duHMGB2) in antiviral innate immunity. Here, duHMGB2 genes were first cloned and analyzed from the spleen of Cherry Valley ducks. We show that duHMGB2 is widely distributed in most tissues of healthy ducks, and duHMGB2 was differentially expressed in three organs (the spleen, brain, and lung) of ducks during different viral infections. duHMGB2 is mainly expressed in the nucleus of duck embryo fibroblast (DEF) cells. However, duHMGB2 is released into the cytoplasm after viral infection. DuHMGB2 induced expression of several genes that regulate the immune response. Moreover, duHMGB2 activated and upregulatede transcription factor NF-κB promoter activity. We also used single gene manipulations (knockout or overexpression) to confirm that duHMGB2 can inhibit the replication of duck plague virus, duck Tembusu virus, and the novel duck reovirus in DEF cells. These data show that duHMGB2 can activate the antiviral innate immunity of the host. Thus, duHMGB2 may be considered an immune adjuvant against infectious diseases in duck.

Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.

To investigate the complexity of proteomics in cervical cancer tissues, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry analysis on a panel of normal cervical tissues (N), high-grade squamous intraepithelial lesion tissues (HSIL) and cervical cancer tissues (CC). Total 72 differentially expressed proteins were identified both in CC vs N and CC vs HSIL. The expression of HMGB2 was markedly higher in CC than that in HSIL and N. High HMGB2 expression was significantly correlated with primary tumor size, invasion and tumor stage. The up-regulated HMGB2 was discovered to be associated with human cervical cancer. These findings suggest that HMGB2 may be a potentially prognostic biomarker and a target for the therapy of cervical cancer.

Due to the lack of efficiency to control malaria elicited by sub-unit vaccine preparations, vaccination with live-attenuated Plasmodium parasite as reported 70 years ago with irradiated sporozoites regained recently a significant interest. The complex life cycle of the parasite and the different stages of development between mammal host and anopheles do not help to propose an easy vaccine strategy. In order to achieve a complete long-lasting protection against Plasmodium infection and disease, we considered a genetically attenuated blood stage parasite in the hmgb2 gene coding for the high-mobility-group-box 2 (HMGB2). This Plasmodium protein belongs to the HMGB family and hold as the mammal proteins, a double life since it acts first as a nuclear factor involved in chromatin remodelling and transcription regulation and second, when secreted as an active pro-inflammatory alarmin protein. Even though the number of reports on whole living attenuated blood stage parasites is limited when compared to attenuated sporozoites, the results reported with Plasmodium KO parasites are very encouraging. In this report, we present a novel strategy based on pre-immunization with Δhmgb2PbNK65 parasitized red blood cells that confer long-lasting protection in a murine experimental cerebral malaria model against two highly pathogenic homologous and heterologous parasites.

Chemotherapy is an important treatment modality for gastric cancer (GC); however, it usually fails because of drug resistance, especially multidrug resistance (MDR). Previously, we found a novel subset of MDR-associated microRNAs (miRNAs) through high-throughput functional screening. In this report, we investigated the exact roles and mechanisms of miR-23b-3p in the MDR of GC. Using gain or loss-of-function in in vitro and in vivo experiments, we found that overexpression of miR-23b-3p reversed cancer cell resistance to multiple chemotherapeutics in vitro and sensitize tumors to chemotherapy in vivo. Reporter gene assay and western blot analysis showed that ATG12 and HMGB2 were the direct targets of miR-23b-3p. Meanwhile, ATG12 and HMGB2 were positively associated with the occurrence of autophagy. Reducing the expression of these target genes by siRNA or inhibition of autophagy both sensitized GC cells to chemotherapy. These findings suggest that a miR-23b-3p/ATG12/HMGB2/autophagy-regulatory loop has a critical role in MDR in GC. In addition, miR-23b-3p could be used as a prognostic factor for overall survival in GC. In conclusion, our data demonstrated that miR-23b-3p inhibited autophagy mediated by ATG12 and HMGB2 and sensitized GC cells to chemotherapy, and suggested the potential application of miR-23b-3p in drug resistance prediction and treatment.

High-mobility group box 2 (HMGB2) protein is a chromatin-associated nonhistone protein, involved in transcriptional regulation and nucleic-acid-mediated innate immune responses in mammalian. However, the function of piscine HMGB2 in innate immune responses is still unknown. In the present study, two HMGB2 homologue genes (CiHMGB2a, CiHMGB2b) were identified and characterized in grass carp (Ctenopharyngodon idella). Both CiHMGB2a and CiHMGB2b genes encode proteins with 213 amino acids, sharing 71.4% identities and containing two basic HMG boxes and an acidic tail. The deduced protein sequences showed the most identities to HMGB2a (93%) and HMGB2b (86.4%) of zebrafish (Danio rerio), respectively. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that CiHMGB2a and CiHMGB2b were constitutively expressed in all the 15 tested tissues. Post grass carp reovirus (GCRV) infection, mRNA levels of CiHMGB2a and CiHMGB2b were strongly up-regulated in spleen and head kidney and mildly modulated in C. idella kidney (CIK) cells. Meanwhile, mRNA expressions of CiHMGB2a and CiHMGB2b were significantly regulated by viral pathogen associated molecular patterns (PAMPs) polyinosinic-polycytidylic potassium salt (poly(I:C)) and bacterial PAMPs lipopolysaccharide (LPS), peptidoglycan (PGN) challenge in CIK cells. In CiHMGB2a and CiHMGB2b over-expression cells, expressions of CiHMGB2a and CiHMGB2b facilitated each other; transcription levels of CiTRIF, CiMyD88, CiIPS-1 and CiMx1 were remarkably enhanced, whereas CiIFN-I was inhibited, compared with those in cells transfected with pCMV (control plasmid); after GCRV challenge, all those tested genes were up-regulated with divergent expression profiles. Antiviral activities of CiHMGB2a and CiHMGB2b were manifested by the delayed appearance of cytopathic effect (CPE) and inhibition of GCRV yield. All those results demonstrate that CiHMGB2a and CiHMGB2b not only mediate antiviral immune responses but also involve in responding to viral/bacterial PAMPs challenge, which provides novel insights into the essential role of HMGB2 in innate immunity.

High mobility group proteins 1 and 2 (HMGB1 and HMGB2) are 80% conserved in amino acid sequence. The function of HMGB1 in inflammation and fibrosis has been extensively characterized. However, an unaddressed central question is the role of HMGB2 on liver fibrosis. In this study, we provided convincing evidence that the HMGB2 expression was significantly upregulated in human liver fibrosis and cirrhosis, as well as in several mouse liver fibrosis models.

High mobility group box 2 (HMGB2) is abnormally expressed in human cancers and participated in multiple biological behaviors, such as proliferation, invasion and prognosis. However, its role in hepatocellular carcinoma (HCC) is largely unknown.

High-mobility group box 2 (HMGB2) is an abundant, chromatin-associated protein that plays an essential role in the regulation of transcription, cell proliferation, differentiation, and tumorigenesis. However, the underlying mechanism of HMGB2 in adipogenesis remains poorly known. Here, we provide evidence that HMGB2 deficiency in preadipocytes impedes adipogenesis, while overexpression of HMGB2 increases the potential for adipogenic differentiation. Besides, depletion of HMGB2 in vivo caused the decrease in body weight, white adipose tissue (WAT) mass, and adipocyte size. Consistently, the stromal vascular fraction (SVF) of adipose tissue derived from hmgb2-/- mice presented impaired adipogenesis. When hmgb2-/- mice were fed with high-fat diet (HFD), the body size, and WAT mass were increased, but at a lower rate. Mechanistically, HMGB2 mediates adipogenesis via enhancing expression of C/EBPβ by binding to its promoter at "GGGTCTCAC" specifically during mitotic clonal expansion (MCE) stage, and exogenous expression of C/EBPβ can rescue adipogenic abilities of preadipocytes in response to HMGB2 inhibition. In general, our findings provide a novel mechanism of HMGB2-C/EBPβ axis in adipogenesis and a potential therapeutic target for obesity.

High mobility group protein B2 (HMGB2) is an important protein attributed to tissue function and homeostasis in osteoarthritis (OA), however, its roles in the temporomandibular joint with mechanical pressure are unclear. This study investigated the expression patterns of HMGB2 in chondrocytes of TMJ on OA-related cellular and animal models. The transcriptional level and protein expression of HMGB2 as well as Col- II, MMP-13 and β-catenin were examined in primary cultured chondrocytes from TMJs with hydrostatic pressures (HP). The immunohistochemistry of HMGB2, Col- II, MMP13 and β-catenin in rabbits with surgical anterior disc displacement (ADD) were analyzed. The results indicated that HP decreased the mRNA expression of HMGB2, MMP-13, and β-catenin. HMGB2 knockdown attenuated the sensitivity of chondrocytes response to pressure loading. Immunohistochemical results showed that the rabbits with ADD exhibited thinner condylar cartilage with disordered cell arrangement. The distribution of HMGB2 was chiefly in the fibrous layer and the proliferative layer of the condylar cartilage. In rabbit cartilage with ADD, the expression of HMGB2 and β-catenin were elevated at 1 week followed by sustained decrease at 2 and 4 weeks. Our findings suggested that HMGB2 is necessary for TMJ chondrocytes to sense pressure loading, which plays an important role in the pathogenesis of chondrogenesis and TMJOA following mechanical loading.