HMGB2 Release Promotes Pulmonary Hypertension and Predicts Severity and Mortality of Patients With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive and life-threatening disease characterized by pulmonary vascular remodeling, which involves aberrant proliferation and apoptosis resistance of the pulmonary arterial smooth muscle cells (PASMCs), resembling the hallmark characteristics of cancer. In cancer, the HMGB2 (high-mobility group box 2) protein promotes the pro-proliferative/antiapoptotic phenotype. However, the function of HMGB2 in PH remains uninvestigated. METHODS: Smooth muscle cell (SMC)-specific HMGB2 knockout or HMGB2-OE (HMGB2 overexpression) mice and HMGB2 silenced rats were used to establish hypoxia+Su5416 (HySu)-induced PH mouse and monocrotaline-induced PH rat models, respectively. The effects of HMGB2 and its underlying mechanisms were subsequently elucidated using RNA-sequencing and cellular and molecular biology analyses. Serum HMGB2 levels were measured in the controls and patients with pulmonary arterial (PA) hypertension. RESULTS: HMGB2 expression was markedly increased in the PAs of patients with PA hypertension and PH rodent models and was predominantly localized in PASMCs. SMC-specific HMGB2 deficiency or silencing attenuated PH development and pulmonary vascular remodeling in hypoxia+Su5416-induced mice and monocrotaline-treated rats. SMC-specific HMGB2 overexpression aggravated hypoxia+Su5416-induced PH. HMGB2 knockdown inhibited PASMC proliferation in vitro in response to PDGF-BB (platelet-derived growth factor-BB). In contrast, HMGB2 protein stimulation caused the hyperproliferation of PASMCs. In addition, HMGB2 promoted PASMC proliferation and the development of PH by RAGE (receptor for advanced glycation end products)/FAK (focal adhesion kinase)-mediated Hippo/YAP (yes-associated protein) signaling suppression. Serum HMGB2 levels were significantly increased in patients with PA hypertension, and they correlated with disease severity, predicting worse survival. CONCLUSIONS: Our findings indicate that targeting HMGB2 might be a novel therapeutic strategy for treating PH. Serum HMGB2 levels could serve as a novel biomarker for diagnosing PA hypertension and determining its prognosis.

The transcription factor HMGB2 indirectly regulates APRIL expression and Gd-IgA1 production in patients with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide. Proliferation-inducing ligand (APRIL) was identified as an important cause of glycosylation deficiency of IgA1 (Gd-IgA1), which can 'trigger' IgAN. Our previous study indicated that high migration group protein B2 (HMGB2) in peripheral blood mononuclear cells from patients with IgAN was associated with disease severity, but the underlying mechanism remains unclear. MATERIALS AND METHODS: The location of HMGB2 was identified by immunofluorescence. qRT-PCR and Western blotting were used to measure HMGB2, HMGA1, and APRIL expression. Gd-IgA1 levels were detected by enzyme-linked immunosorbent assay (ELISA). In addition, we used DNA pull-down, protein profiling, and transcription factor prediction software to identify proteins bound to the promoter region of the APRIL gene. RNA interference and coimmunoprecipitation (Co-IP) were used to verify the relationships among HMGB2, high mobility group AT-hook protein 1 (HMGA1), and APRIL. RESULTS: HMGB2 expression was greater in IgAN patients than in HCs and was positively associated with APRIL expression in B cells. DNA pull-down and protein profiling revealed that HMGB2 and HMGA1 bound to the promoter region of the APRIL gene. The expression levels of HMGA1, APRIL, and Gd-IgA1 were downregulated after HMGB2 knockdown. Co-IP indicated that HMGB2 binds to HMGA1. The Gd-IgA1 concentration in the supernatant was reduced after HMGA1 knockdown. HMGA1 binding sites were predicted in the promoter region of the APRIL gene. CONCLUSION: HMGB2 expression is greater in IgAN patients than in healthy controls; it promotes APRIL expression by interacting with HMGA1, thereby inducing Gd-IgA1 overexpression and leading to IgAN.

The Effect of HMGB1 and HMGB2 on Transcriptional Regulation Differs in Neuroendocrine and Adenocarcinoma Models of Prostate Cancer.

Human high-mobility group-B (HMGB) proteins regulate gene expression in prostate cancer (PCa), a leading cause of oncological death in men. Their role in aggressive PCa cancers, which do not respond to hormonal treatment, was analyzed. The effects of HMGB1 and HMGB2 silencing upon the expression of genes previously related to PCa were studied in the PCa cell line PC-3 (selected as a small cell neuroendocrine carcinoma, SCNC, PCa model not responding to hormonal treatment). A total of 72% of genes analyzed, using pre-designed primer panels, were affected. HMGB1 behaved mostly as a repressor, but HMGB2 as an activator. Changes in SERPINE1, CDK1, ZWINT, and FN1 expression were validated using qRT-PCR after HMGB1 silencing or overexpression in PC-3 and LNCaP (selected as an adenocarcinoma model of PCa responding to hormonal treatment) cell lines. Similarly, the regulatory role of HMGB2 upon SERPINE1, ZWINT, FN1, IGFPB3, and TYMS expression was validated, finding differences between cell lines. The correlation between the expression of HMGB1, HMGB2, and their targets was analyzed in PCa patient samples and also in PCa subgroups, classified as neuroendocrine positive or negative, in public databases. These results allow a better understanding of the role of HMGB proteins in PCa and contribute to find specific biomarkers for aggressive PCa.

Blocking the E2F transcription factor 1/high-mobility group box 2 pathway enhances the intervention effects of α-santalol on the malignant behaviors of liver cancer cells.

In view of the tumor-inhibiting effect of α-santalol in various cancers and the role of E2F transcription factor 1 (E2F1) as an important target for anticancer research, this study investigates the relation between α-santalol and E2F1, as well as the effect of α-santalol on liver cancer progression and the corresponding mechanism. Concretely, liver cancer cells were treated with different concentrations of α-santalol. The IC50 value of α-santalol was determined using Probit regression analysis. Then, transcription factors that are targeted by α-santalol and differentially expressed in liver cancer were screened out. The clinicopathological impact of E2F1 and its targets were evaluated and predicted. The expressions of E2F1 and high-mobility group box 2 (HMGB2) and their correlation in the liver cancer tissues were analyzed by bioinformatics. The effects of E2F1 and HMGB2 on the biological characteristics of liver cancer cells were examined through loss/gain-of-function and molecular assays. With the extension of treatment time, the inhibitory effects of 10 µmol/L and 20 µmol/L α-santalol on cancer cell survival rate were enhanced (P < 0.001). E2F1 and HMGB2 were highly expressed and positively correlated in liver cancer tissues (P < 0.05). High E2F1 expression was correlated with large tumors and high TNM stages (P < 0.05). E2F1 knockdown promoted the effects of α-santalol on dose-dependently inhibiting viability, colony formation, invasion and migration (P < 0.05). Moreover, E2F1 knockdown reduced the IC50 value and HMGB2 level, while HMGB2 overexpression produced opposite effects. HMGB2 overexpression and E2F1 knockdown mutually counteracted their effects on the IC50 value and on the viability and apoptosis of α-santalol-treated liver cancer cells (P < 0.01). Collectively, blocking the E2F1/HMGB2 pathway enhances the intervention effects of α-santalol on the proliferation, migration and invasion of liver cancer cells.

Ionizing radiation induces vascular smooth muscle cell senescence through activating NF-κB/CTCF/p16 pathway.

Radiation injury of blood vessels (RIBV) is a serious long-term complication of radiotherapy, characterized by the development of atherosclerosis. The involvement of vascular smooth muscle cells (VSMCs) senescence in the pathogenesis of radiation-induced atherosclerosis has been implicated, yet the precise mechanisms governing VSMCs senescence remain inadequately comprehended. In this study, the senescence of VSMCs was examined by employing SA-ß-gal staining and assessing the expression of p16 and p21, both in vivo and in vitro. Our findings revealed that ionizing radiation (IR) has the potential to augment cellular senescence. In addition, IR significantly activated the NF-κB pathway, as evidenced by increased p65 nuclear translocation, phospho-p65 expression, and enhanced binding ability of p65 (EMSA). Furthermore, a decrease in HMGB2 expression following exposure to IR was observed via Western blot analysis, while CTCF expression remained unchanged. Interestingly, the formation of CTCF spatial clustering was detected under super-resolution fluorescence microscopy. Concurrently, the ChIP technique identified the facilitation of the interaction between CTCF and p16 gene through IR. The inhibition of CTCF or the overexpression of HMGB2 through lentiviruses effectively eliminates the formation of CTCF clusters and the upregulation of p16 and p21 after IR. Inhibition of NF-κB activation induced by IR by PDTC (100 µM) led to a decrease in the staining of SA-ß-gal, a reduction in p16 expression, an increase in HMGB2 protein expression and a decrease in CTCF clusters formation. This study provided significant insights into the role and mechanism of IR in VSMCs senescence by regulating NF-κB/CTCF/p16 pathway.

HMGB2 regulates the differentiation and stemness of exhausted CD8+ T cells during chronic viral infection and cancer.

Chronic infections and cancers evade the host immune system through mechanisms that induce T cell exhaustion. The heterogeneity within the exhausted CD8+ T cell pool has revealed the importance of stem-like progenitor (Tpex) and terminal (Tex) exhausted T cells, although the mechanisms underlying their development are not fully known. Here we report High Mobility Group Box 2 (HMGB2) protein expression is upregulated and sustained in exhausted CD8+ T cells, and HMGB2 expression is critical for their differentiation. Through epigenetic and transcriptional programming, we identify HMGB2 as a cell-intrinsic regulator of the differentiation and maintenance of Tpex cells during chronic viral infection and in tumors. Despite Hmgb2-/- CD8+ T cells expressing TCF-1 and TOX, these master regulators were unable to sustain Tpex differentiation and long-term survival during persistent antigen. Furthermore, HMGB2 also had a cell-intrinsic function in the differentiation and function of memory CD8+ T cells after acute viral infection. Our findings show that HMGB2 is a key regulator of CD8+ T cells and may be an important molecular target for future T cell-based immunotherapies.

Correlation of serum HMGB1 and HMGB2 levels with clinical symptoms in allergic rhinitis children.

This research aimed to explore the serum high-mobility group box 1 (HMGB1) and high-mobility group box 2 (HMGB2) levels in allergic rhinitis (AR) children and its correlation with clinical results. This present prospective observational study enrolled 179 AR children and 100 healthy children who came to our hospital during October 2020 to August 2022. The serum HMGB1, HMGB2, interleukin (IL)-6, IL-1ß, interferon-γ, and C-reactive protein (CRP) levels were measured by enzyme-linked immunosorbent assay. Demographic and clinical statistics including age, body mass index (BMI), sex, diastolic blood pressure, SBP, family history of allergy, Visual Analogue Score (VAS) and Rhinoconjunctivitis Quality of Life Questionnaire were collected. All data used SPSS 18.0 to statistical analyses. The proportion of family history of allergy was obviously higher in the AR group than that in the healthy group. The serum levels of HMGB1, HMGB2 and cytokines were remarkably enhanced in the AR patients. Spearman analysis supported that positive correlation existed among the HMGB1, HMGB2, CRP, IL-6 and IL-1ß levels. Serum IL-6, CRP, HMGB2, IL-1ß, VAS score and Rhinoconjunctivitis Quality of Life Questionnaire score levels were significantly higher and serum interferon-γ levels were significantly lower in the HMGB1 high expression group. Similar results were found in in the HMGB2 high group compared to the HMGB2 low group. In addition, HMGB1 and HMGB2 could be potential diagnostic biomarkers of AR patients. Finally, we found that HMGB1, HMGB2, IL-6, IL-1ß, and family history of allergy were the risk factors for AR. This study showed that the serum HMGB1 and HMGB2 levels was remarkably enhanced in AR patients and closely associated with cytokines. This study may provide new targets and a comprehensive approach for the treatment of AR patients.

Targeting HMG-CoA synthase 2 suppresses tamoxifen-resistant breast cancer growth by augmenting mitochondrial oxidative stress-mediated cell death.

AIMS: In this study, we aimed to investigate previously unrecognized lipid metabolic perturbations in tamoxifen-resistant breast cancer (BC) by conducting comprehensive metabolomics and transcriptomics analysis. We identified the role of 3-hydroxy-3-methylglutary-coenzyme-A-synthase 2 (HMGCS2), a key enzyme responsible for ketogenesis, in tamoxifen-resistant BC growth. MAIN METHODS: Comprehensive metabolomics (CE-TOFMS, LC-TOFMS) and transcriptiomics analysis were performed to characterize metabolic pathways in tamoxifen-resistant BC cells. The upregulation of HMGCS2 were verified thorugh immunohistochemistry (IHC) in clinical samples obtained from patients with recurrent BC. HMGCS2 inhibitor was discovered through surface plasmon resonance analysis, enzyme assay, and additional molecular docking studies. The effect of HMGCS2 suppression on tumor growth was studied thorugh BC xenograft model, and intratumoral lipid metabolites were analyzed via MALDI-TOFMS imaging. KEY FINDINGS: We revealed that the level of HMGCS2 was highly elevated in both tamoxifen-resistant T47D sublines (T47D/TR) and clinical refractory tumor specimens from patients with ER+ breast cancer, who had been treated with adjuvant tamoxifen. Suppression of HMGCS2 in T47D/TR resulted in the accumulation of mitochondrial reactive oxygen species (mtROS) and apoptotic cell death. Further, we identified alphitolic acid, a triterpenoid natural product, as a novel HMGCS2-specific inhibitor that elevated mtROS levels and drastically retarded the growth of T47D/TR in in vitro and in vivo experiments. SIGNIFICANCE: Enhanced ketogenesis with upregulation of HMGCS2 is a potential metabolic vulnerability of tamoxifen-resistant BC that offers a new therapeutic opportunity for treating patients with ER+ BC that are refractory to tamoxifen treatment.

Anxiolytic, Analgesic and Anti-Inflammatory Effects of Peptides Hmg 1b-2 and Hmg 1b-4 from the Sea Anemone Heteractis magnifica.

Acid-sensing ion channels (ASICs) have been known as sensors of a local pH change within both physiological and pathological conditions. ASIC-targeting peptide toxins could be potent molecular tools for ASIC-manipulating in vitro, and for pathology treatment in animal test studies. Two sea anemone toxins, native Hmg 1b-2 and recombinant Hmg 1b-4, both related to APETx-like peptides, inhibited the transient current component of human ASIC3-Δ20 expressed in Xenopus laevis oocytes, but only Hmg 1b-2 inhibited the rat ASIC3 transient current. The Hmg 1b-4 action on rASIC3 as a potentiator was confirmed once again. Both peptides are non-toxic molecules for rodents. In open field and elevated plus maze tests, Hmg 1b-2 had more of an excitatory effect and Hmg 1b-4 had more of an anxiolytic effect on mouse behavior. The analgesic activity of peptides was similar and comparable to diclofenac activity in an acid-induced muscle pain model. In models of acute local inflammation induced by λ-carrageenan or complete Freund's adjuvant, Hmg 1b-4 had more pronounced and statistically significant anti-inflammatory effects than Hmg 1b-2. It exceeded the effect of diclofenac and, at a dose of 0.1 mg/kg, reduced the volume of the paw almost to the initial volume. Our data highlight the importance of a comprehensive study of novel ASIC-targeting ligands, and in particular, peptide toxins, and present the slightly different biological activity of the two similar toxins.

Induction of perineural invasion in salivary adenoid cystic carcinoma by circular RNA RNF111.

OBJECTIVE: Local recurrence, distant metastasis, and perineural invasion (PNI) viciously occur in salivary adenoid cystic carcinoma (SACC), resulting in a poor prognosis. This study aimed to explore the mechanism by which circular RNA RNF111 (circ-RNF111) regulates PNI in SACC by targeting the miR-361-5p/high mobility group box 2 (HMGB2) axis. METHOD: Circ-RNF111 and HMGB2 were highly expressed in SACC specimens, while miR-361-5p was underexpressed. Functional experiments showed that ablating circ-RNF111 or promoting miR-361-5p hindered the biological functions and PNI of SACC-LM cells. RESULTS: HMGB2 overexpression induced the reversal of SACC-LM cell biological functions and PNI caused by circ-RNF111 knockout. Furthermore, reduction of circ-RNF111 suppressed PNI in a SACC xenograft model. Circ-RNF111 regulated HMGB2 expression through targeted modulation of miR-361-5p. CONCLUSION: Taken together, circ-RNF111 stimulates PNI in SACC by miR-361-5p/HMGB2 axis and may serve as a potential therapeutic target for SACC.

Clinical and Experimental Study of High Mobility Group Box-2 and Valvular Calcification in Elderly Patients with Degenerative Heart Valve Disease.

INTRODUCTION: The aim of this study was to investigate the relationship between the high mobility group box-2 (HMGB2) and valve calcification in senile degenerative heart valve disease (SDHVD). METHODS: According to the echocardiographic results, patients with calcified heart valves were used as the experimental group and patients without calcified heart valves were used as the control group; blood was drawn for testing, and serum levels of HMGB2 were measured by an enzyme-linked immunosorbent assay. Human heart valve interstitial cells (hVICs) cultured in vitro were randomly divided into two groups. The calcification group was cultured with a medium containing calcification induction solution and cells were induced on days 1, 3, and 5, and the control group was cultured with a standard medium. Expression of bone morphogenetic protein 4 (BMP-4) and HMGB2 in both groups was detected by Western blot. RT-PCR was performed to detect the expression of the HMGB2 gene during calcification. The hVICs were cultured in vitro for 4 days with different concentrations of exogenous HMGB2 (0.01 µg/mL, 0.1 µg/mL, 1 µg/mL, 2 µg/mL), while the control group was cultured with a standard medium and the expression of BMP-4 and NF-κB P65 was detected by Western blot. RESULTS: The serum level of HMGB2 was 7.90 (5.92, 12.39) µg/L, higher than that of 7.06 (5.06, 9.73) µg/L in the valve calcification group in elderly patients with degenerative valve disease (p = 0.005); the differences were statistically significant. In in vitro experiments, the cellular calcification protein BMP-4 and the HMGB2 protein were higher in the calcification group compared to the control group (p < 0.05). Exogenous stimulation of hVICs with HMGB2 was able to upregulate the expression of BMP-4 and NF-κB P65 (p < 0.05). CONCLUSIONS: HMGB2 is correlated with valvular calcification in senile degenerative heart valve disease. The HMGB2 protein may promote the process of SDHVD valve calcification by activating the NF-κB pathway and upregulating the expression of BMP-4.

High level of LncRNA MAPKAPK5-AS1 predicts poor prognosis and contributes to the malignant proliferation and EMT of non-small cell lung cancer via sponging miR-490-3p from HMGB2.

BACKGROUND: Patients with non-small cell lung cancer (NSCLC) show a low survival rate, owing to the lack of early diagnostic method and high invasiveness. Long non-coding RNA MAPKAPK5-AS1 that regulates tumor genesis and progression through multiple signals, is upregulated and involved in the growth and apoptosis in lung adenocarcinoma (LUAD). OBJECTIVE: To investigate whether MAPKAPK5-AS1 affected the malignant progression of NSCLC. METHODS: The levels of MAPKAPK5-AS1, miR-490-3p and HMGB2 in lung cancer were first analyzed through StarBase website, and confirmed by a quantitative reverse transcriptase-PCR (qRT-PCR) assay. The biological functions of NSCLC cells were examined by CCK-8, 5-ethynyl-2'-deoxyuridine (EdU) and flow cytometry assays. The potential binding sequences lncRNA-miRNA and miRNA-mRNA were predicted by StarBase software and verified via dual luciferase reporter experiment. The effects of MAPKAPK5-AS1 on tumor growth were evaluated in a xenografted mice model. RESULTS: The expression of MAPKAPK5-AS1 was upregulated in tumor tissues from NSCLC patients. Patients with high expression of MAPKAPK5-AS1 had higher tumor size, advanced TNM stage, higher incidence of lymph node and distant metastasis, and shorter overall survival. Knockdown of MAPKAPK5-AS1 inhibited the proliferation, induced apoptosis and blocked epithelial mesenchymal transformation (EMT) of NSCLC cells. Mechanically, MAPKAPK5-AS1 could upregulate the HMGB2 level in NSCLC cells through competitively binding to miR-490-3p. MiR-490-3p inhibitor reversed the roles of MAPKAPK5-AS1 knockdown on tumor cell proliferation, apoptosis and EMT. Also, HMGB2 knockdown suppressed tumor cell malignant phenotypes. Furthermore, interference of MAPKAPK5-AS1 slowed NSCLC tumor growth in vivo. CONCLUSION: Knockdown of MAPKAPK5-AS1 inhibited the aggressive tumor phenotypes through miR-490-3p/HMGB2 axis in NSCLC. MAPKAPK5-AS1/miR-490-3p/HMGB2 might be potential biomarkers or therapeutic targets for NSCLC.

CircRNA hsa_circ_0075048 promotes the malignant progression of non-small cell lung cancer by up-regulating HMGB2 expression via targeting miR-1225-5p.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the most common forms of lung cancer. Circular RNAs (circRNAs) have been recognized that can be used as novel molecular markers for cancer therapy. Here, we attempted to identify the role of hsa_circRNA_0075048 (circ_0075048) in NSCLC. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to analyze the levels of hsa_circ_0075048, miR-1225-5p and high mobility group box-2 (HMGB2). Cell proliferation was detected by Cell Counting Kit 8 (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Flow cytometry was used to detect apoptosis. Transwell assay was used to assess cell migration and invasion. Sphere formation ability was tested by cell pellet test. The protein expression levels were detected by western blot and immunohistochemistry. Dual-luciferase reporter assay, RNA-pull down and RNA immunoprecipitation (RIP) assays were used to verify the targeting relationships between miR-1225-5p and circ_0075048 or HMGB2. Mice tumor models were constructed to ascertain the effects of circ_0075048 on tumor growth in vivo. RESULTS: Circ_0075048 was increased in NSCLC tissues and cells. NSCLC cell proliferation, migration, invasion and sphere formation ability were decreased by circ_0075048 knockdown, and cell apoptosis was induced. Downregulation of miR-1225-5p recuperated the effects of circ_0075048 knockdown on NSCLC progression. The effects of miR-1225-5p on cell proliferation, apoptosis, migration, invasion and sphere formation were attenuated by HMGB2 overexpression. CONCLUSION: This study indicated that circ_0075048 played an oncogenic role in the development of NSCLC by regulating miR-1225-5p and HMGB2. The data provide more possibilities for the treatment of NSCLC.

[microRNA let-7g-3p regulates proliferation, migration, invasion and apoptosis of bladder cancer cells by targeting HMGB2].

OBJECTIVE: To explore the molecular mechanism by which microRNA let-7g-3p regulates biological behaviors of bladder cancer cells. METHODS: The expression levels of let-7g-3p in bladder cancer and adjacent tissues, normal bladder epithelial cells (HUC cells) and bladder cancer cells (T24, 5637 and EJ cells) were detected using qRT- PCR. T24 cells were transfected with let-7g-3p mimic or inhibitor, and the changes in cell proliferation, migration, invasion, and apoptosis were examined. Transcriptome sequencing was carried out in cells overexpressing let-7g-3p, and the results of bioinformatics analysis, double luciferase reporter gene assay, qRT-PCR and Western blotting confirmed that HMGB2 gene was the target gene of let-7g-3p. The expression of HMGB2 was examined in HUC, T24, 5637 and EJ cells, and in cells with HMGB2 knockdown, the effect of let-7g-3p knockdown on the biological behaviors were observed. RESULTS: qRT-qPCR confirmed that let-7g-3p expression was significantly lower in bladder cancer tissues and cells (P < 0.01). Overexpression of let-7g-3p inhibited cell proliferation, migration and invasion, and promoted cell apoptosis, while let-7g-3p knock-down produced the opposite effects. Bioinformatics and transcriptome sequencing results showed that HMGB2 was the key molecule that mediate the effect of let-7g-3p on bladder cancer cells. Luciferase reporter gene assay, qRT-PCR and Western blotting all confirmed that HMGB2 was negatively regulated by let-7g-3p (P < 0.01). Knocking down HMGB2 could partially reverse the effect of let-7g-3p knockdown on the biological behaviors of the bladder cancer cells. CONCLUSION: The microRNA let-7g-3p can inhibit the biological behavior of bladder cancer cells by negatively regulating HMGB2 gene.

Long non-coding RNA NR2F2-AS1 regulates human osteosarcoma growth and metastasis through miR-425-5p-mediated HMGB2.

BACKGROUND: Multiple studies have revealed that long non-coding RNA (lncRNA) NR2F2-AS1 plays a role in affecting cancer cell proliferation and metastasis. Here, both in vitro and in vivo experiments were performed for investigating the function and mechanism of NR2F2-AS1 in human osteosarcoma (OS). METHODS: The NR2F2-AS1 level in human OS tissues and adjacent non-tumor tissues was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The NR2F2-AS1 overexpression model was constructed in OS cells, then cell proliferation, invasion, and apoptosis were monitored. The OS xenograft model was established in nude mice using NR2F2-AS1-overexpressed OS cells. The downstream target genes of NR2F2-AS1 were predicted. qRT-PCR and Western blot were implemented to validate the profiles of miR-425-5p and HMGB2. The targeting link between NR2F2-AS1 and miR-425-5p, miR-425-5p and HMGB2 was further probed by dual-luciferase reporter experiment. RESULTS: In comparison to adjacent non-tumor tissues, OS tissues showed upregulated NR2F2-AS1 expression. Higher NR2F2-AS1 level was predominantly correlated with worse clinical stages. In vivo and in vitro tests corroborated that NR2F2-AS1 overexpression spurred OS cell proliferation, growth, invasion, and choked apoptosis. Mechanistically, NR2F2-AS1 hampered miR-425-5p expression as its competitive endogenous RNA (ceRNA). Thus, NR2F2-AS1 facilitated the HMGB2 expression. However, miR-425-5p inhibited HMGB2 expression by targeting the latter. CONCLUSION: NR2F2-AS1 expedited the evolution of OS by elevating HMGB2 levels through sponging miR-425-5p. The NR2F2-AS1/miR-425-5p/HMGB2 regulatory axis is a promising target in treating human OS.

Identification of HMGB2 associated with proliferation, invasion and prognosis in lung adenocarcinoma via weighted gene co-expression network analysis.

BACKGROUND: High mobility group protein B2 (HMGB2) is a multifunctional protein that plays various roles in different cellular compartments. Moreover, HMGB2 serves as a potential prognostic biomarker and therapeutic target for lung adenocarcinoma (LUAD). METHODS: In this study, the expression pattern, prognostic implication, and potential role of HMGB2 in LUAD were evaluated using the integrated bioinformatics analyses based on public available mRNA expression profiles from The Cancer Genome Atlas and Gene Expression Omnibus databases, both at the single-cell level and the tissue level. Further study in the patient-derived samples was conducted to explore the correlation between HMGB2 protein expression levels with tissue specificity, (tumor size-lymph node-metastasis) TNM stage, pathological grade, Ki-67 status, and overall survival. In vitro experiments, such as CCK-8, colony-formation and Transwell assay, were performed with human LUAD cell line A549 to investigate the role of HMGB2 in LUAD progression. Furthermore, xenograft tumor model was generated with A549 in nude mice. RESULTS: The results showed that the HMGB2 expression was higher in the LUAD samples than in the adjacent normal tissues and was correlated with high degree of malignancy in different public data in this study. Besides, over-expression of HMGB2 promoted A549 cells proliferation and migration while knocking down of HMGB2 suppressed the tumor promoting effect. CONCLUSIONS: Our study indicated that HMGB2 was remarkably highly expressed in LUAD tissues, suggesting that it is a promising diagnostic and therapeutic marker for LUAD in the future.

The circular RNA circHMGB2 drives immunosuppression and anti-PD-1 resistance in lung adenocarcinomas and squamous cell carcinomas via the miR-181a-5p/CARM1 axis.

BACKGROUND: Previous studies have confirmed the oncogenic role of HMGB2 in various cancers, but the biological functions of HMGB2-derived circRNAs remain unknown. Thus, we intended to investigate the potential role of HMGB2-derived circRNAs in lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC). METHODS: The expression profiles of HMGB2-derived circRNAs in LUAD and LUSC tissues and matched normal tissues were assessed using qRT-PCR. The role of circHMGB2 in the progression of the LUAD and LUSC was determined in vitro by Transwell, CCK-8, flow cytometry and immunohistochemistry assays, as well as in vivo in an immunocompetent mouse model and a humanized mouse model. In addition, in vivo circRNA precipitation assays, luciferase reporter assays and RNA pulldown assays were performed to explore the underlying mechanism by which circHMGB2 promotes anti-PD-1 resistance in the LUAD and LUSC. RESULTS: The expression of circHMGB2 (hsa_circ_0071452) was significantly upregulated in NSCLC tissues, and survival analysis identified circHMGB2 as an independent indicator of poor prognosis in the LUAD and LUSC patients. We found that circHMGB2 exerted a mild effect on the proliferation of the LUAD and LUSC cells, but circHMGB2 substantially reshaped the tumor microenvironment by contributing to the exhaustion of antitumor immunity in an immunocompetent mouse model and a humanized mouse model. Mechanistically, circHMGB2 relieves the inhibition of downstream CARM1 by sponging miR-181a-5p, thus inactivating the type 1 interferon response in the LUAD and LUSC. Moreover, we found that the upregulation of circHMGB2 expression decreased the efficacy of anti-PD-1 therapy, and we revealed that the combination of the CARM1 inhibitor EZM2302 and an anti-PD-1 antibody exerted promising synergistic effects in a preclinical model. CONCLUSION: circHMGB2 overexpression promotes the LUAD and LUSC progression mainly by reshaping the tumor microenvironment and regulating anti-PD-1 resistance in the LUAD and LUSC patients. This study provides a new strategy for the LUAD and LUSC treatment.

Circular RNA circ_UBAP2 facilitates the progression of osteosarcoma by regulating microRNA miR-637/high-mobility group box (HMGB) 2 axis.

Circular RNA circ_UBAP2 has been reported to be closely associated with various tumors. The present work focused on exploring the roles of circ_UBAP2 and its molecular mechanism in osteosarcoma (OS). Circ_UBAP2, miR-637, and high-mobility group box (HMGB) 2 levels in OS cells and tissues were detected by quantitative real-time polymerase chain reaction. The relationship between miR-637 and circ_UBAP2, as well as between miR-637 and HMGB2, was predicted and examined through bioinformatics analysis and luciferase reporter gene experiments. Moreover, OS cell growth, invasion, migration, and apoptosis were detected using the cell counting kit-8 (CCK-8), Transwell and flow cytometry assays, respectively. HMGB2 protein levels were measured using Western blotting. Xenograft tumor formation assay was also performed. Circ_UBAP2 showed high expression levels in OS tissues and cells, which was directly proportional to metastasis and clinical stage of OS. The overexpression of circ_UBAP2 enhanced the growth, invasion, and migration of OS cells, but suppressed their apoptosis. In contrast, circ_UBAP2 silencing had opposite effects. Furthermore, miR-637 served as a downstream target of circ_UBAP2, which played opposite roles to circ_UBAP2 in OS. More importantly, HMGB2 served as miR-637's downstream target. The xenograft experiments in nude mice also proved that knockdown of circ_UBAP2 could increase miR-637 expression, but decrease HMGB2 expression, thus alleviating OS progression. Mechanistically, circ_UBAP2 exerts a cancer-promoting effect on OS by downregulating miR-637 and upregulating the expression of HMGB2. Circ_UBAP2 plays a promoting role in OS, and the circ_UBAP2/miR-637/HMGB2 axis is involved in OS progression.

Crucial role of high-mobility group box 2 in mouse ovarian follicular development through estrogen receptor beta.

High-mobility group box 2 (HMGB2) is a chromatin-associated protein that is an important regulator of gene transcription, recombination, and repair processes. The functional importance of HMGB2 has been reported in various organs, including the testis, heart, and cartilage. However, its role in the ovary is largely unknown. In this study, ovary tissues from wild-type (WT) and HMGB2-knock-out (KO) mice were examined by histopathological staining and immunohistochemistry. The ovary size and weight were significantly lower in HMGB2-KO mice than in age-matched WT littermates. Histopathological analysis revealed ovarian atrophy and progressive fibrosis in 10-month-old HMGB2-KO mouse ovaries. Compared to age-matched WT mice, the numbers of oocytes and developing follicles were significantly decreased at 2 months of age and were completely depleted at 10 months of age in HMGB2-KO mice. Immunohistochemistry revealed the expression of HMGB2 in the granulosa cells of developing follicles, oocytes, some corpora lutea, and stromal cells. Importantly, HMGB2-positive cells were co-localized with estrogen receptor beta (ERß), but not ERα. Estrogen response element-binding activity was demonstrated by southwestern histochemistry, and it was decreased in HMGB2-KO mouse ovaries. Cell proliferation activity was also decreased in HMGB2-KO mouse ovaries in parallel with the decreased folliculogenesis. These results indicated that the depletion of HMGB2 induced ovarian atrophy that was characterized by a decreased ovarian size and weight, progressive fibrosis, as well as decreased oocytes and folliculogenesis. In conclusion, we demonstrated the crucial role of HMGB2 in mouse ovarian folliculogenesis through ERß expression.

CircLPAR3 Acts as an Oncogene in Oral Squamous Cell Carcinoma Through Regulating the miR-643/HMGB2 Network.

The malignant progression of oral squamous cell carcinoma (OSCC) has been confirmed to be mediated by a variety of factors, including circular RNA (circRNA). However, the role of circLPAR3 in OSCC development is still unclear. 70 paired OSCC tissues and normal control tissues were obtained from 70 OSCC patients. Quantitative real-time PCR was used to detect the expression of circLPAR3, microRNA (miR)-643, and high-mobility group box 2 (HMGB2). Cell proliferation, apoptosis, metastasis and stemness were assessed using cell counting kit 8 assay, colony-formation assay, flow cytometry, transwell assay and sphere formation assay. Marker protein expression and HMGB2 protein expression were determined by western blot analysis. The interaction between miR-643 and circLPAR3 or HMGB2 was confirmed by RNA pull-down assay, dual-luciferase reporter and RIP assay. The role of circLPAR3 in OSCC tumorigenesis was explored by constructing the xenograft models. Our data showed that circLPAR3 was highly expressed in OSCC tissues and cells. CircLPAR3 silencing suppressed OSCC cell proliferation, metastasis and stemness, while promoted apoptosis. On the mechanism, we discovered that circLPAR3 could sponge miR-643 to positive regulate HMGB2. MiR-643 overexpression had an inhibition effect on OSCC progression, and its inhibitor could reverse the negative regulation of circLPAR3 knockdown on OSCC progression. In addition, overexpressed HMGB2 also reversed the suppressive effect of circLPAR3 silencing on OSCC progression. Animal experiments results showed that downregulated circLPAR3 repressed OSCC tumorigenesis in vivo. Taken together, our data showed that circLPAR3 contributed to OSCC malignant progression through regulating the miR-643/HMGB2 axis.