High Mobility Group Box 2 Research Articles

Oncogenic KSHV-encoded interferon regulatory factor upregulates HMGB2 and CMPK1 expression to promote cell invasion by disrupting a complex lncRNA-OIP5-AS1/miR-218-5p network.

Kaposi’s sarcoma (KS), a highly disseminated tumor of hyperproliferative spindle endothelial cells, is the most common AIDS-associated malignancy caused by infection of Kaposi’s sarcoma-associated herpesvirus (KSHV). KSHV-encoded viral interferon regulatory factor 1 (vIRF1) is a viral oncogene but its role in KSHV-induced tumor invasiveness and motility remains unknown. Here, we report that vIRF1 promotes endothelial cell migration, invasion and proliferation by down-regulating miR-218-5p to relieve its suppression of downstream targets high mobility group box 2 (HMGB2) and cytidine/uridine monophosphate kinase 1 (CMPK1). Mechanistically, vIRF1 inhibits p53 function to increase the expression of DNA methyltransferase 1 (DNMT1) and DNA methylation of the promoter of pre-miR-218-1, a precursor of miR-218-5p, and increases the expression of a long non-coding RNA OIP5 antisense RNA 1 (lnc-OIP5-AS1), which acts as a competing endogenous RNA (ceRNA) of miR-218-5p to inhibit its function and reduce its stability. Moreover, lnc-OIP5-AS1 increases DNA methylation of the pre-miR-218-1 promoter. Finally, deletion of vIRF1 from the KSHV genome reduces the level of lnc-OIP5-AS1, increases the level of miR-218-5p, and inhibits KSHV-induced invasion. Together, these results define a novel complex lnc-OIP5-AS1/miR-218-5p network hijacked by vIRF1 to promote invasiveness and motility of KSHV-induced tumors.

Knockdown of high mobility group box 3 impairs cell viability and colony formation but increases apoptosis in A549 human non-small cell lung cancer cells.

Previous research has linked high mobility group box 3 (HMGB3) overexpression to the malignant progression and poor prognosis of non-small cell lung cancer (NSCLC). The present study investigated the role of HMGB3 in cell survival and colony formation of NSCLC cells. Stable knockdown of HMGB3 in A549 cells was achieved by lentiviral-based shRNA interference and verified by detection of the transcriptional and translational level of HMGB3 with reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The influence of HMGB3 knockdown on A549 cell viability and apoptotic rate was evaluated by Cell Counting Kit-8 assay and flow cytometry following annexin V staining, respectively. The proliferative capacity of A549 cells with or without HMGB3 knockdown was compared by measuring their colony forming efficiency. The results of the current study revealed that HMGB3 knockdown significantly reduced cell viability and colony forming efficiency while promoting apoptosis in A549 cells, indicating that HMGB3 may be pivotal for the survival and colony formation of A549 cells, serving a notable role in NSCLC progression.

Long noncoding RNA CRCMSL suppresses tumor invasive and metastasis in colorectal carcinoma through nucleocytoplasmic shuttling of HMGB2.

Long noncoding RNAs (lncRNAs) are pervasive transcripts that play pivotal roles in regulating chromatin dynamics, gene and protein expression. Aberrant expression and mutations of lncRNAs represent a driving force behind tumor invasion and metastasis, making them attractive cancer targets. However, most of the lncRNAs are still being discovered and conclusive experimental evidence for their functional relevance is still lacking for most malignancies. In this study, a differentially expressed lncRNA, designated as lnc-CRCMSL, is identified by microarray-based screenings on non-metastatic and metastatic CRC specimens. Lnc-CRCMSL is verified as an anti-metastatic gene and negatively correlated with the poor prognosis of CRC patients. Lnc-CRCMSL overexpression restricts tumor growth and metastasis in vivo and in vitro. Instead, lnc-CRCMSL silencing accelerates CRC cell proliferation and migration. RNA-pulldown assay identifies high mobility group box 2 (HMGB2) as a downstream protein of lnc-CRCMSL. Mechanically, lnc-CRCMSL physically binds to HMGB2 and stabilizes the localization of HMGB2 in the cytoplasm. Notably, lnc-CRCMSL knockdown lead to the shift of HMGB2 into nuclear, in which it triggers epithelial to mesenchymal transition (EMT) programming. Importantly, lnc-CRCMSL controls the cytoplasmic retention of HMGB2 and attenuates the interaction between HMGB2 and OCT4 to suppress EMT. Treatment of leptomycin B (LMB), a potent and specific nuclear export inhibitor, counteracts lnc-CRCMSL-mediated suppression of aggressive phenotypes and EMT process by accumulating the nuclear HMGB2. CONCLUSION: Our data highlight the anti-metastatic role of lnc-CRCMSL in stabilizing HMGB2 through lncRNA-protein interactions in the cytoplasm, and suggest that targeting lnc-CRCMSL may represent a therapeutic opportunity for managing metastatic CRC.

Down-regulated expressed protein HMGB3 inhibits proliferation and migration, promotes apoptosis in the placentas of fetal growth restriction.

Fetal growth restriction (FGR) is one of the major complications of pregnancy, which can lead to serious short-term and long-term diseases. High-mobility group box 3 (HMGB3) has been found to contribute to the development of many cancers. However, the role of HMGB3 in the pathogenesis of FGR is blank. Here, we measured the expression level of HMGB3 in the placenta tissues of six normal pregnancies and five FGR patients by western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). CCK8 assay, transwell assay and flow cytometry were used to detect the functional effects of overexpression and silencing of HMGB3 on the HTR8/SVneo trophoblast cell line. The results showed that the protein levels of HMGB3 were significantly decreased in FGR placentas compared to normal controls, while mRNA levels of HMGB3 were not significantly altered. Furthermore, when overexpressed of protein HMGB3 of the trophoblast cells, the proliferation and migration abilities were significantly promoted, and the apoptosis abilities of these cells were statistically inhibited. Cell functional experiments showed the opposite results when the expression of HMGB3 was silent. And the expression of cell function-related genes PCNA, Ki67, Tp53, Bax, MMP-2 and E-cadherin was observed to show corresponding changes by qRT-PCR. The results of mass spectrometry showed that HMGB3 may directly or indirectly interact with 71 proteins. In summary, our results indicated that HMGB3 might be of very great significance to the pathogenesis of FGR and might play the role by leading the dysfunction of placental villous trophoblast cells and through the interaction with some other proteins.

High mobility group box 2 of black rockfish Sebastes schlegelii: Gene cloning, immunoregulatory properties and antibacterial effect

High-mobility group box 2 (HMGB2) is a non-histone chromosomal protein that involved diverse functions such as transcriptional regulation and innate immune responses in mammalian. In teleost, very limited studies on HMGB2 proteins have been documented. Black rockfish (Sebastes schlegelii) is an economic fish species and cultured worldwide. However, the study of black rockfish about immunology is very scarce. In the present study, a HMGB2 homologue gene (SsHMGB2) was identified and characterized in black rockfish. The open reading frame of SsHMGB2 is 648 bp, and the deduced amino acid sequence of SsHMGB2 shares 74.4%–91.2% overall sequence identities with the HMGB2 proteins of several fish species. In silico analysis identified several conserved features, including two basic HMG boxes and an acidic C-terminal tail composed of 24 Asp/Glu residues. Expression of SsHMGB2 occurred in multiple tissues and was upregulated during pathogens infection. Recombinant SsHMGB2 (rSsHMGB2) exhibited apparent binding activities against DNA. In vivo studies showed that the expressions of multiple immune-related genes in head kidney were significantly enhanced when black rockfish were treated with rSsHMGB2. Furthermore, rSsHMGB2 reduced pathogen dissemination and replication in fish kidney and spleen. Taken together, these results suggest that SsHMGB2 possesses apparent immunoregulatory properties and played a role in fighting bacterial infection.

Periconceptional undernutrition affects the levels of DNA methylation in the peri-implantation pig endometrium and in embryos

Maternal undernutrition during the periconceptional period alters the transcriptomic profile of pig endometrium and embryos. Herein, we tested the hypothesis that restricted maternal consumption by females during the periconceptional period impairs the pattern of DNA methylation in both the endometrium and embryos during the peri-implantation period (Day 15–16 of gestation). Affected genes in restricted-diet-fed pig endometrium and embryos were identified using quantitative methylation-specific PCR and comprised those genes which are known to be important in reproductive, metabolic and epigenetic function, thereby exhibiting altered transcriptomic expression in endometrium and embryos of restricted-diet-fed gilts. Specifically, levels of DNA methylation of selected genes with altered expression in the endometrium included acid phosphatase type 2C (PPAP2C), salivary lipocalin (SAL1), endothelin receptor type B (EDNRB), regulator of G-protein signalling 12 (RGS12), type 4 17β-hydroxysteroid dehydrogenase (HSD17B4), toll-like receptor 3 (TLR3), and adiponectin receptor 1 (ADIPOR1). In embryos, adiponectin receptor 2 (ADIPOR2), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), progestin and adipoQ receptor family member 7 (PAQR7), progesterone receptor membrane component 2 (PGRMC2), steroidogenic acute regulatory protein (STAR), and serpin family A member 1 (SERPINA1) were altered. Finally, 5 acid phosphatase tartrate resistant (ACP5), high mobility group box 2 (HMGB2), and DNA (cytosine-5)-methyltransferase 1 (DNMT1) were altered in both the endometrium and in embryos. In the endometrium, the methylation levels of ACP5 (regulation of endometrial-conceptus iron transport), RGS12 (protein-coupled receptor signalling), and TLR3 (immune response) were increased, while that of EDNRB (corpus luteum maintenance) was decreased. In embryos, the methylation levels of ADIPOR2 (metabolic homeostasis) and DNMT1 (DNA methylation maintenance) were increased. The levels of methylation in other studied endometrial and embryonic genes were unchanged. DNA methylation levels in both the peri-implantation pig endometrium and embryos may be altered in response to female nutritional restriction.

HMGB3 promotes the proliferation and metastasis of glioblastoma and is negatively regulated by miR-200b-3p and miR-200c-3p.

High mobility group box 3 (HMGB3) is strongly involved in oncogenesis in a variety of cancers. In the present study, we have explored the role of HMGB3 in glioblastoma multiforme (GBM) tumorigenesis and have identified the microRNAs (miRNAs) miR-200b-3p and miR-200c-3p as negative regulators of HMGB3 expression. We began by determining that endogenous HMGB3 expression levels were significantly elevated in GBM tissues and in 3 GBM cell lines. To study the function of HMGB3 in GBM, we transfected a small-interfering RNA (siRNA) against HMGB3 into GBM cell lines U251 and LN229. MTT and tumour sphere assays demonstrated that HMGB3 knockdown significantly inhibited cell proliferation. Wound healing and transwell assays determined that HMGB3 knockdown substantially suppressed GBM cell migration and invasion. We evaluated the effects of HMGB3 knockdown on MAPK phosphorylation and target gene expression, finding that HMGB3 knockdown significantly reduced MAPK phosphorylation (p-ERK (1/2), p-p38, and p-JNK). We then used the biologic prediction algorithm TargetScan to identify the 3' untranslated region (3'-UTR) of HMGB3 as a target of miR-200b-3p and miR-200c-3p. Luciferase, qRT-PCR, and western blot assays confirmed that miR-200b-3p and miR-200c-3p bind and inhibit HMGB3 expression. Finally, Pearson correlation analyses demonstrated a negative correlation between relative HMGB3 mRNA and miR-200b/c-3p expression levels in GBM tissue samples. Overall, the present study strongly suggests that HMGB3 promotes GBM oncogenesis through the MAPK signalling pathway while miR-200b-3p and miR-200c-3p inhibit its expression.

Peri-conceptional nutritional restriction alters transcriptomic profile in the peri-implantation pig embryos

Restricted nutritional consumption during the peri-conceptional period affects the potential for DNA methylation and alters endometrial transcriptomic profile during the peri-implantation period. The restricted diet fed to females during the peri-conceptional period may affect the transcriptomic profile in peri-implantation embryos. In the present study, the transcriptome of embryos of normal-diet-fed gilts was determined and compared with that in embryos of restricted-diet-fed gilts during the peri-implantation period. The restricted-diet-fed gilts were fed forage, in which the dose of proteins and energy had been reduced by 30% compared to the normal diet (Polish Norms of Nutrition). To clarify the issue Agilent’s Porcine (V2) Two-Color Gene Expression Microarray 4 × 44 was used. Analysis of the microarray data revealed that the expression of 787 genes with known biological function were consistently altered (496 up- and 291 down-regulated) in embryos. The accurately annotated genes were organized into five categories and 18 subcategories containing 62 biological pathways. The qPCR analysis of ten selected genes [i.e., 5 acid phosphatase, tartrate resistant (ACP5), high mobility group box 2 (HMGB2), prostaglandin-endoperoxide synthase 2 (PTGS2), arachidonate 12-lipoxygenase (ALOX12), adiponectin receptor 2 (ADIPOR2), DNA (cytosine-5)-methyltransferase 1 (DNMT1), steroidogenic acute regulatory protein (STAR), progesterone receptor membrane component 2 (PGRMC2), progestin and adipoQ receptor family member 7 (PAQR7) and serpin family A member 1 (SERPINA1)] confirmed altered gene expression in embryos of restricted-diet-fed gilts. The insight into embryonic transcriptome indicates that female under-nutrition during the peri-conceptional period may create alterations in the pattern of genes expressed in the peri-implantation embryos.

Abnormal expression of HMGB-3 is significantly associated with malignant transformation of hepatocytes.

AIMTo explore the relationship between dynamic expression of high mobility group box-3 (HMGB3) and malignant transformation of hepatocytes.METHODSExpression of HMGB family proteins were observed in rat hepatocarcinogenesis models induced with 2-acetylaminofluorene. Alterations of HMGB3 were analyzed at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and at the protein level by immunohistochemistry or Western blotting. HMGB3 in human liver cancer tissues were evaluated using bioinformatics databases from GEO, TCGA, and Oncomine. A specific HMGB3-shRNA was used to knock down HMGB3 expression in order to investigate its effects on proliferation and cell cycle in vitro and in vivo.RESULTSElevated HMGB3 levels were first reported in hepatocarcinogenesis, with increasing expression from normal liver to cancer. Bioinformatic databases showed that HMGB3 expression in hepatocellular carcinoma tissues was significantly higher than that in normal liver tissues. Higher HMGB3 expression was discovered in liver cancer cells compared with LO2 cells in vitro. According to gene set enrichment analysis, HMGB3 mRNA levels were correlated with cell cycle and DNA replication pathways. Knocking down HMGB3 by specific shRNA significantly inhibited proliferation of HepG2 cells by cell cycle arrest and downregulating DNA replication related genes (cyclin B1, FEN1, and PCNA) at the mRNA and protein level. Furthermore, silencing HMGB3 significantly inhibited xenograft tumor growth (measured by Ki67) in vivo.CONCLUSIONHMGB3 is involved in malignant transformation of hepatocytes and could be a useful biomarker for diagnosis and a potential target for therapy of liver cancer.

HMGB2 is a novel adipogenic factor that regulates ectopic fat infiltration in skeletal muscles

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.

Transcriptional Repression of High-Mobility Group Box 2 by p21 in Radiation-Induced Senescence.

High mobility group box 2 (HMGB2) is an abundant, chromatin-associated, non-histone protein involved in transcription, chromatin remodeling, and recombination. Recently, the HMGB2 gene was found to be significantly downregulated during senescence and shown to regulate the expression of senescent-associated secretory proteins. Here, we demonstrate that HMGB2 transcription is repressed by p21 during radiation-induced senescence through the ATM-p53-p21 DNA damage signaling cascade. The loss of p21 abolished the downregulation of HMGB2 caused by ionizing radiation, and the conditional induction of p21 was sufficient to repress the transcription of HMGB2. We also showed that the p21 protein binds to the HMGB2 promoter region, leading to sequestration of RNA polymerase and transcription factors E2F1, Sp1, and p300. In contrast, NF-Y, a CCAAT box-binding protein complex, is required for the expression of HMGB2, but NF-Y binding to the HMGB2 promoter was unaffected by either radiation or p21 induction. A proximity ligation assay results confirmed that the chromosome binding of E2F1 and Sp1 was inhibited by p21 induction. As HMGB2 have been shown to regulate premature senescence by IR, targeting the p21-mediated repression of HMGB2 could be a strategy to overcome the detrimental effects of radiation-induced senescence.

Regulation of HMGB3 by antitumor miR-205-5p inhibits cancer cell aggressiveness and is involved in prostate cancer pathogenesis.

Our recent determination of a microRNA (miRNA) expression signature in prostate cancer (PCa) revealed that miR-205-5p was significantly reduced in PCa tissues and that it acted as an antitumor miRNA. The aim of this study was to identify oncogenic genes and pathways in PCa cells that were regulated by antitumor miR-205-5p. Genome-wide gene expression analyses and in silico miRNA database searches showed that 37 genes were putative targets of miR-205-5p regulation. Among those genes, elevated expression levels of seven in particular (HMGB3, SPARC, MKI67, CENPF, CDK1, RHOU, and POLR2D) were associated with a shorter disease-free survival in a large number of patients in the The Cancer Genome Atlas (TCGA) database. We focused on high-mobility group box 3 (HMGB3) because it was the most downregulated by ectopic expression of miR-205-5p in PC3 cells and its expression was involved in PCa pathogenesis. Luciferase reporter assays showed that HMGB3 was directly regulated by miR-205-5p in PCa cells. Knockdown studies using si-HMGB3 showed that expression of HMGB3 enhanced PCa cell aggressiveness. Overexpression of HMGB3/HMGB3 was confirmed in naive PCa and castration-resistant PCa (CRPC) clinical specimens. Novel approaches to analysis of antitumor miRNA-regulated RNA networks in PCa cells may provide new insights into the pathogenic mechanisms of the disease.

HMGB3 promotes growth and migration in colorectal cancer by regulating WNT/β-catenin pathway.

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths and a major health problem. High mobility group box 3 (HMGB3), a member of the high-mobility group box (HMGB) family, was reported to be over-expressed in gastric carcinoma and bladder cancer. However, the function of HMGB3 in CRC remains unclear. Here, we found that HMGB3 was up-regulated in CRC at both mRNA and protein levels. qRT-PCR results showed that high expression of HMGB3 had positive correlation with serosal invasion, lymph metastasis, and tumor–node–metastasis (TNM) stage in CRC patient. Functional experiments showed that HMGB3 can promote CRC cells proliferation and migration in vitro. Moreover, we found HMGB3 can active WNT/β-catenin pathway to increase the expression level of c-Myc and MMP7. These results may be the reason for HMGB3 oncogene role in CRC. In summary, our data indicated that HMGB3 may serve as an oncoprotein and could be used as a potential prognostic marker in CRC.

HMGB2 orchestrates the chromatin landscape of senescence-associated secretory phenotype gene loci.

Cellular senescence is a stable cell growth arrest that is characterized by the silencing of proliferation-promoting genes through compaction of chromosomes into senescence-associated heterochromatin foci (SAHF). Paradoxically, senescence is also accompanied by increased transcription of certain genes encoding for secreted factors such as cytokines and chemokines, known as the senescence-associated secretory phenotype (SASP). How SASP genes are excluded from SAHF-mediated global gene silencing remains unclear. In this study, we report that high mobility group box 2 (HMGB2) orchestrates the chromatin landscape of SASP gene loci. HMGB2 preferentially localizes to SASP gene loci during senescence. Loss of HMGB2 during senescence blunts SASP gene expression by allowing for spreading of repressive heterochromatin into SASP gene loci. This correlates with incorporation of SASP gene loci into SAHF. Our results establish HMGB2 as a novel master regulator that orchestrates SASP through prevention of heterochromatin spreading to allow for exclusion of SASP gene loci from a global heterochromatin environment during senescence.

Knockdown of High Mobility Group-Box 3 (HMGB3) Expression Inhibits Proliferation, Reduces Migration, and Affects Chemosensitivity in Gastric Cancer Cells

BackgroundHigh mobility group-box 3 (HMGB3) has been shown to affect tumor initiation and progression. This research aimed to investigate the role of HMGB3 in gastric cancer (GC) cell proliferation, migration, invasion, chemoresistance, and its potential molecular mechanisms.Material/MethodsGC MGC803 and BGC823 cells were transfected with siRNA targeting the HMGB3 gene. The expressions of HMGB3 protein in MGC803 and BGC823 cells after transfection were detected by Western blot assays. We detected cell proliferation and cell cycle by MTT and flow cytometry assay. Cell migration and invasion were determined by wound scratch and transwell assay. MGC803 and BGC823 cells were treated with various concentrations of oxaliplatin, cisplatin, and paclitaxel. After 24 hours of drug exposure, we performed MTT assays to investigate chemoresistance in both groups. Western blot assays were used to detect related proteins expression.ResultsSilencing of HMGB3 inhibited cell proliferation and induced G0/G1 phase arrest of GC cells partly via modulating p53 and p21 pathways, and downregulating Bcl-2/Bax ratio. RNA interference of HMGB3 inhibited cell invasion and migration by downregulating MMP2 and MMP9. Silencing of HMGB3 enhanced sensitive to cisplatin and paclitaxel, and reduced sensitive to oxaliplatin.ConclusionsThese findings suggest the importance of HMGB3 in the regulation of growth, migration, and apoptosis of GC, improve our understanding of the mechanisms of GC pathogenesis, and may promote the development of novel targeted therapies.

Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid.

Damage-associated molecular pattern molecules (DAMPs) signal the presence of tissue damage to induce immune responses in plants and animals. Here, we report that High Mobility Group Box 3 (HMGB3) is a novel plant DAMP. Extracellular HMGB3, through receptor-like kinases BAK1 and BKK1, induced hallmark innate immune responses, including i) MAPK activation, ii) defense-related gene expression, iii) callose deposition, and iv) enhanced resistance to Botrytis cinerea. Infection by necrotrophic B. cinerea released HMGB3 into the extracellular space (apoplast). Silencing HMGBs enhanced susceptibility to B. cinerea, while HMGB3 injection into apoplast restored resistance. Like its human counterpart, HMGB3 binds salicylic acid (SA), which results in inhibition of its DAMP activity. An SA-binding site mutant of HMGB3 retained its DAMP activity, which was no longer inhibited by SA, consistent with its reduced SA-binding activity. These results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

Overexpression of HMGB3 protein promotes cell proliferation, migration and is associated with poor prognosis in urinary bladder cancer patients.

Human urinary bladder cancer (UBC) is the fourth most common cancer and the eighth most common cause of cancer death in the USA. High mobility group box 3 (HMGB3), a member of a family of proteins containing one or more high mobility group DNA binding motifs, was reported to be overexpressed in a variety of human cancers. However, the expression and role of HMGB3 in human UBC remains unclear. Here, we found that UBC patients had upregulated HMGB at both mRNA and protein levels. Immunochemistry (IHC) evaluation of HMGB3 expression in 113 UBC clinical specimens showed that high expression of HMGB3 had positive correlation with UBC tumor size (P = 0.019), tumor WHO grade (P = 0.031), stage (P = 0.028), and lymph node metastasis (P = 0.017). Moreover, patients with higher HMGB3 expression showed a poorer overall survival rate than those with relatively low HMGB3 (P = 0.0079, log-rank test). Multivariate analysis revealed that HMGB3 expression is an independent prognostic marker. The UBC cancer cell proliferation and migration ability were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and wound healing assays, respectively. RNA interference of HMGB3 in UBC cell lines inhibited cancer cell growth and migration, along with the downregulation of PCNA and MMP2 protein levels. In sum, our data suggests HMGB3 may serve as an important oncoprotein and indicate that overexpression of HMGB3 in UBC could be used as a potential prognostic marker.

Silencing of high-mobility group box 2 (HMGB2) modulates cisplatin and 5-fluorouracil sensitivity in head and neck squamous cell carcinoma.

Dysregulation of protein expression is associated with most diseases including cancer. MS-based proteomic analysis is widely employed as a tool to study protein dysregulation in cancers. Proteins that are differentially expressed in head and neck squamous cell carcinoma (HNSCC) cell lines compared to the normal oral cell line could serve as biomarkers for patient stratification. To understand the proteomic complexity in HNSCC, we carried out iTRAQ-based MS analysis on a panel of HNSCC cell lines in addition to a normal oral keratinocyte cell line. LC-MS/MS analysis of total proteome of the HNSCC cell lines led to the identification of 3263 proteins, of which 185 proteins were overexpressed and 190 proteins were downregulated more than twofold in at least two of the three HNSCC cell lines studied. Among the overexpressed proteins, 23 proteins were related to DNA replication and repair. These included high-mobility group box 2 (HMGB2) protein, which was overexpressed in all three HNSCC lines studied. Overexpression of HMGB2 has been reported in various cancers, yet its role in HNSCC remains unclear. Immunohistochemical labeling of HMGB2 in a panel of HNSCC tumors using tissue microarrays revealed overexpression in 77% (54 of 70) of tumors. The HMGB proteins are known to bind to DNA structure resulting from cisplatin-DNA adducts and affect the chemosensitivity of cells. We observed that siRNA-mediated silencing of HMGB2 increased the sensitivity of the HNSCC cell lines to cisplatin and 5-FU. We hypothesize that targeting HMGB2 could enhance the efficacy of existing chemotherapeutic regimens for treatment of HNSCC. All MS data have been deposited in the ProteomeXchange with identifier PXD000737 (http://proteomecentral.proteomexchange.org/dataset/PXD000737).

Genomic interaction between ER and HMGB2 identifies DDX18 as a novel driver of endocrine resistance in breast cancer cells.

Breast cancer resistance to endocrine therapies such as tamoxifen and aromatase inhibitors is a significant clinical problem. Steroid receptor coactivator-1 (SRC-1), a coregulatory protein of the oestrogen receptor (ER), has previously been shown to have a significant role in the progression of breast cancer. The chromatin protein high mobility group box 2 (HMGB2) was identified as an SRC-1 interacting protein in the endocrine-resistant setting. We investigated the expression of HMGB2 in a cohort of 1068 breast cancer patients and found an association with increased disease-free survival time in patients treated with endocrine therapy. However, it was also verified that HMGB2 expression could be switched on in endocrine-resistant tumours from breast cancer patients. To explore the function of this poorly characterized protein, we performed HMGB2 ChIPseq and found distinct binding patterns between the two contexts. In the resistant setting, the HMGB2, SRC-1 and ER complex are enriched at promoter regions of target genes, with bioinformatic analysis indicating a switch in binding partners between the sensitive and resistant phenotypes. Integration of binding and gene expression data reveals a concise set of target genes of this complex including the RNA helicase DDX18. Modulation of DDX18 directly affects growth of tamoxifen-resistant cells, suggesting that it may be a critical downstream effector of the HMGB2:ER complex. This study defines HMGB2 interactions with the ER complex at specific target genes in the tamoxifen-resistant setting.

Upregulation of miR-513b inhibits cell proliferation, migration, and promotes apoptosis by targeting high mobility group-box 3 protein in gastric cancer.

The high mobility group-box 3 (HMGB3) protein belongs to the high mobility group box (HMG-box) subfamily, and recent studies have shown that HMGB3 is an oncogene for leukemia. HMGB3 is also expressed at a high level in the progression phase of breast and gastric cancer (GC). Using bioinformatic analyses, we found that HMGB3 is a potential target for miR-513b. However, the pathophysiological role of miR-513b and its relevance to the growth and development of GC have yet to be investigated. This study focuses on whether miR-513b acts as a tumor suppressor in GC. Compared with non-malignant adjacent tissues samples, qRT-PCR data showed significant downregulation of miR-513b in 74 GC tissue samples (P < 0.01). Furthermore, western blotting revealed that HMGB3 protein was overexpressed in tumor samples relative to matched, non-malignant adjacent tissues. Western blotting and qRT-PCR results showed that high expression of HMGB3 and low expression of miR-513b were both significantly associated with primary tumors, lymph node metastases, and the clinical stage (P < 0.01). MiR-513b was shown to not only inhibit the proliferation and migration of gastric cancer cells (MKN45 and SGC7901) in the CCK-8 and transwell assays, but also to promote cell apoptosis in a flow-cytometric apoptosis assay. In western blot and luciferase assays, HMGB3 was identified as a major target of miR-513b. Moreover, we also found that the expression of HMGB3 lacking in 3' UTR could abrogate the anti-migration and pro-apoptosis function of miR-513b. These findings suggest the importance of miR-513b targeting of HMGB3 in the regulation of growth, migration and apoptosis of GC, improve our understanding of the mechanisms of GC pathogenesis, and may promote the development of novel targeted therapies.