HAS2 Expression Research Articles

Age related changes in hyaluronan expression leads to Meibomian gland dysfunction

The prevalence of dry eye disease (DED) ranges from ∼5 to 50 % and its associated symptoms decrease productivity and reduce the quality of life. Approximately 85 % of all DED cases are caused by Meibomian gland dysfunction (MGD). As humans and mice age, their Meibomian glands (MGs) undergo age-related changes resulting in age related-MGD (ARMGD). The precise cause of ARMGD remains elusive, which makes developing therapies extremely challenging. We previously demonstrated that a hyaluronan (HA)-rich matrix exists surrounding the MG, regulating MG morphogenesis and homeostasis. Herein, we investigated whether changes to the HA matrix in the MG throughout life contributes towards ARMGD, and whether altering this HA matrix can prevent ARMGD. For such, HA synthase (Has) knockout mice were aged and compared to age matched wild type (wt) mice. MG morphology, lipid production, PPARγ expression, basal cell proliferation, stem cells, presence of atrophic glands and MG dropout were analyzed at 8 weeks, 6 months, 1 year and 2 years of age and correlated with the composition of the HA matrix. We found that as mice age, there is a loss of HA expression in and surrounding the MGs of wt mice, while, in contrast, Has1−/−Has3−/− mice present a significant increase in HA expression through Has2 upregulation. At 1 year, Has1−/−Has3−/− mice present significantly enlarged MGs, compared to age-matched wt mice and compared to all adult mice. Thus, Has1−/−Has3−/− mice continue to develop new glandular tissue as they age, instead of suffering MG atrophy. At 2 years, Has1−/−Has3−/− mice continue to present significantly larger MGs compared to age-matched wt mice. Has1−/−Has3−/− mice present increased lipid production, increased PPARγ expression and an increase in the number of proliferating cells when compared to wt mice at all-time points analyzed. Taken together, our data shows that a loss of the HA matrix surrounding the MG as mice age contributes towards ARMGD, and increasing Has2 expression, and consequently HA levels, prevents ARMGD in mice.

MiR-155-5p improves oocyte maturation in porcine cumulus cells through connexin 43-mediated regulation of MPF activity

In this study, we aimed to investigate the effect of the expression of miR-155-5p and its target genes on oocyte maturation. We analyzed the expression of miR-155-5p and its target genes in cumulus cells and oocytes using quantitative real-time reverse-transcription polymerase chain reaction. Using carboxyfluorescein, porcine cumulus cells were transfected with mimics and inhibitors of ssc-miR-155-5p to induce in vitro maturation, and subsequently, cumulus expansion, oocyte maturation, and cleavage rate were measured. We found that miR-155-5p expression in cumulus cells at the metaphase II stage was significantly higher than that at the germinal vesicle (GV) stage, whereas Cx43 expression was significantly lower than that at the GV stage (P < 0.05). Compared with those in the negative control group, the cumulus diffusion area of cumulus oocyte complexes; oocyte maturation rate; cleavage rate; HAS2, PTGS2, CD44, PTX3, and TNFAIP6 expression in cumulus cells; and GDF9, BMP15, CyclinB1, and CDK1 expression in oocytes were significantly increased in the miR-155-5p mimics group (P < 0.05), whereas the mRNA and protein expression of CX43 were significantly decreased (P < 0.05). Compared with that in the negative control group, the protein expression of CyclinB1 and p-CDK1 (Thr14, Tyr15) in the miR-155-5p mimics group was significantly increased (P < 0.05). These results suggest that miR-155-5p regulates maturation promoting factor activity by targeting Cx43, which improves the in vitro maturation and cleavage rate of porcine oocytes.

Exploring the anti-inflammatory potential of topical hyaluronic acid for vocal fold injury in a rat model

PurposeVocal fold injuries are associated with fibrosis and dysphonia, which is a major obstacle to surgical treatment. The aim of this study is to evaluate the effect of topical hyaluronic acid with or without diclofenac on the inflammatory phase of vocal fold wound healing.MethodsForty-one male Sprague–Dawley rats were randomly assigned to four groups: an uninjured control group, an injured control group without any treatment, and two intervention groups in which hyaluronic acid with or without diclofenac was applied to the injured vocal fold. Gene expression of inflammatory markers and ECM-related molecules were examined.ResultsVocal fold injury resulted in a significant upregulation of inflammatory parameters [Ptgs2, Il1b and Il10] and Has1. Tgfb1, Has3 and Eln gene expression were significantly downregulated by the topical application of hyaluronic acid. The combination of hyaluronic acid and diclofenac did not result in any significant changes.ConclusionsVocal fold wound healing was significantly improved by a single post-operative topical application of hyaluronic acid. The addition of diclofenac may provide no additional benefit.

CD44 Is Associated with Poor Prognosis of ccRCC and Facilitates ccRCC Cell Migration and Invasion through HAS1/MMP9.

In many solid tumors, CD44 has been identified as a cancer stem cell marker as well as an important molecular in cancer progression and metastasis, making it attractive for potential therapeutic applications. However, our knowledge of the biological function and mechanism of CD44 in clear cell renal cell carcinoma (ccRCC) is limited. In this study, the expression, prognostic values and functional enrichment analysis of CD44 in ccRCC were analyzed using public databases. Quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemical (IHC) assays were taken to detect CD44 expression in ccRCC tissues. The effects of CD44 on the proliferation, migration and invasion of ccRCC cells were investigated by gain-of-function and loss-of-function experiments. Subcutaneous models further confirmed the role of CD44 in tumor growth. The relationship between CD44, HAS1 and MMP9 was investigated to uncover the regulatory mechanism of CD44 in ccRCC. CD44 was significantly upregulated in ccRCC and associated with poor overall survival (OS). Based on the functional enrichment analysis and PPI network, we found that CD44 had associations with ECM interaction and focal adhesion pathway. Clinical ccRCC sample validation revealed that CD44 mRNA and protein expression were significantly increased in ccRCC tissues, and strong CD44 staining was observed in four metastatic ccRCC cases. In vitro experiments showed that CD44 overexpression promoted cell proliferation, migration and invasion. In vivo experiments also demonstrated that CD44 overexpression accelerated tumor formation in mice. Finally, we found that CD44 regulates the expression of HAS1 in ccRCC, which is essential for the secretion of MMP9 and cell migratory ability. The upregulation of CD44 mRNA and protein expressions in ccRCC is indicative of unfavorable clinical prognoses. The CD44/HAS1/MMP9 axis is believed to exert a significant influence on the regulation of ECM degradation and ccRCC metastasis.

Low-Molecular-Weight Hyaluronic Acid Contributes to Noise-Induced Cochlear Inflammation

Introduction: Our previous work indicated that the activation of the Toll-like receptor (TLR) 4 signaling pathway contributed to noise-induced cochlear inflammation. Previous studies have reported that low-molecular-weight hyaluronic acid (LMW-HA) accumulates during aseptic trauma and promotes inflammation by activating the TLR4 signaling pathway. We hypothesized that LMW-HA or enzymes synthesizing or degrading HA might be involved in noise-induced cochlear inflammation. Methods: The present study included two arms. The first arm was the noise exposure study, in which TLR4, proinflammatory cytokines, HA, hyaluronic acid synthases (HASs), and hyaluronidases (HYALs) in the cochlea as well as auditory brainstem response (ABR) thresholds were measured before and after noise exposure. The second arm was analysis of HA delivery-induced reactions, in which control solution, high-molecular-weight HA (HMW-HA), or LMW-HA was delivered into the cochlea by cochleostomy or intratympanic injection. Then, the ABR threshold and cochlear inflammation were measured. Results: After noise exposure, the expression of TLR4, proinflammatory cytokines, HAS1, and HAS3 in the cochlea significantly increased over the 3rd to 7th day post-noise exposure (PE3, PE7). The expression of HYAL2 and HYAL3 dramatically decreased immediately after noise exposure, gradually increased thereafter to levels significantly greater than the preexposure level on PE3, and then rapidly returned to the preexposure level on PE7. The expression of HA, HAS2, and HYAL1 in the cochlea remained unchanged after exposure. After cochleostomy or intratympanic injection, both the hearing threshold shifts and the expression of TLR4, TNF-α, and IL-1β in the cochleae of the LMW-HA group were obviously greater than those of the control group and HMW-HA group. The expression of proinflammatory cytokines in the LMW-HA and control groups on the 7th day (D7) after cochleostomy tended to increase compared to that on the 3rd day (D3), whereas levels in the HMW-HA group tended to decrease on D7 compared to D3. Conclusion: HAS1, HAS3, HYAL2, and HYAL3 in the cochlea are involved in acoustic trauma-induced cochlear inflammation through the potential proinflammatory function of LMW-HA.

Moisturizing and Antioxidant Effects of Artemisia argyi Essence Liquid in HaCaT Keratinocytes

Artemisia argyi essence liquid (AL) is an aqueous solution extracted from A. argyi using CO2 supercritical fluid extraction. There have been few investigations on the aqueous solution of A. argyi extracted via CO2 supercritical fluid extraction. This study aimed to explore the moisturizing and antioxidant effects of AL and to clarify the potential mechanism underlying those effects. Expression levels of skin moisture-related components and the H2O2-induced oxidative stress responses in human keratinocyte cells were measured via quantitative RT-qPCR, Western blot, and immunofluorescence. Our results showed that AL enhanced the expression of AQP3 and HAS2 by activating the EGFR-mediated STAT3 and MAPK signaling pathways. In addition, AL can play an antioxidant role by inhibiting the NF-κB signaling pathway and activating the Nrf2/HO-1 signaling pathway, consequently increasing the expression of antioxidant enzymes (GPX1, SOD2) and decreasing the production of reactive oxygen species (ROS). This study revealed that AL could be used as a potential moisturizing and antioxidant cosmetic ingredient.

Prognostic value of hyaluronidase-1 and CD44 immunohistological expression in gastric cancer

Abstract Introduction Although many studies have investigated molecular markers for gastric cancer, the mechanisms of carcinogenesis remain obscure. CD44 serves as the principal transmembrane hyaluronate receptor. HAS1 has been identified as a key contributor to oncogenesis and disease progression in both hematological and solid cancers HAS1 has been shown to be prognostic factor in a variety of other cancers. Aim Our aim was to evaluate the expression of the CD44 and HAS1 as prognostic indicators for gastric cancer. Methodology Seventy five cases of gastritis and gastric cancer, obtained from the archives of Pathology Departments of Theodor Bilharz Research Institute (TBRI) – Giza, Egypt, including eleven case with gastritis and sixty four cases of gastric cancer. Detailed histopathological assessment was done as well as immunohistochemical staining for CD44 and HAS1. Results Our results showed increased expression of both HAS1 &amp;CD44 with increasing grades and stages as well lymph node metastasis of gastric carcinoma. No significant difference was noticed in their expression between H.pylori associated and non-associated gastritis. Conclusion We concluded that modulation of HAS1/ CD44 expression is of significant prognostic value in gastric cancer and could be a major target for the anticancer and chemo-preventive agents.

Endogenous production of hyaluronan, PRG4, and cytokines is sensitive to cyclic loading in synoviocytes.

Synovial fluid is composed of hyaluronan and proteoglycan-4 (PRG4 or lubricin), which work synergistically to maintain joint lubrication. In diseases like osteoarthritis, hyaluronan and PRG4 concentrations can be altered, resulting in lowered synovial fluid viscosity, and pro-inflammatory cytokine concentrations within the synovial fluid increase. Synovial fibroblasts within the synovium are responsible for contributing to synovial fluid and can be targeted to improve endogenous production of hyaluronan and PRG4 and to alter the cytokine profile. We cyclically loaded SW982 synoviocytes to 0%, 5%, 10%, or 20% strain for three hours at 1 Hz. To assess the impact of substrate stiffness, we compared the 0% strain group to cells grown on tissue culture plastic. We measured the expression of hyaluronan turnover genes, hyaluronan localization within the cell layer, hyaluronan concentration, PRG4 concentration, and the cytokine profile within the media. Our results show that the addition of cyclic loading increased HAS3 expression, but not in a magnitude-dependent response. Hyaluronidase expression was impacted by strain magnitude, which is exemplified by the decrease in hyaluronan concentration due to cyclic loading. We also show that PRG4 concentration is increased at 5% strain, while higher strain magnitude decreases overall PRG4 concentration. Finally, 10% and 20% strain show a distinct, more pro-inflammatory cytokine profile when compared to the unloaded group. Multivariate analysis showed distinct separation between certain strain groups in being able to predict strain group, hyaluronan concentration, and PRG4 concentration from gene expression or cytokine concentration data, highlighting the complexity of the system. Overall, this study shows that cyclic loading can be used tool to modulate the endogenous production of hyaluronan, PRG4, and cytokines from synovial fibroblasts.

Rosa gallica and its active compound, cyanidin-3,5-O-diglucoside, improve skin hydration via the GLK signaling pathway.

Rosa gallica has been previously reported to display anti-inflammatory, anti-oxidative, and anti-skin wrinkle activities. However, the effect of Rosa gallica on skin hydration and its active components are largely unknown. Herein, we aimed to investigate the skin hydration effect of rose petal extract (RPE) in humans and elucidate the underlying molecular mechanism. A double-blinded clinical study was performed to investigate the effect of RPE on skin hydration. Stratum corneum moisture analysis demonstrated that RPE treatment significantly improved hydration levels in human skin. Furthermore, HAS2 and hyaluronic acid levels were notably increased by RPE in keratinocytes and 3D human skin equivalent model. By comparing the modulatory effect on HAS2 expression, cyanidin-3,5-O-diglucoside (CDG) was identified as the most potent compound in RPE likely responsible for skin hydration. The kinase activity of GLK, an upstream regulator of MAPK signaling, was increased by CDG in a dose-dependent manner. Importantly, silencing GLK reversed CDG-mediated HAS2 upregulation, further supporting the involvement of GLK in the CDG-mediated effects. Binding of CDG to GLK was confirmed by pull-down assay and computer modeling. These findings suggest that RPE and its active component CDG increases skin hydration by upregulating HAS2 expression through modulating the GLK-MAP2K-MAPK signaling pathway.

Glucocorticoids Directly Affect Hyaluronan Production of Orbital Fibroblasts; A Potential Pleiotropic Effect in Graves' Orbitopathy.

Orbital connective tissue expansion is a hallmark of Graves’ orbitopathy (GO). In moderate-to-severe active GO, glucocorticoids (GC) are the first line of treatment. Here we show that hydrocortisone (HC), prednisolone (P), methylprednisolone (MP), and dexamethasone (DEX) inhibit the hyaluronan (HA) production of orbital (OF) and dermal (DF) fibroblasts. HA production of GO OFs (n = 4), NON-GO OFs (n = 4) and DFs (n = 4) was measured by ELISA. mRNA expression of enzymes of HA metabolism and fibroblast proliferation was examined by RT-PCR and BrdU incorporation, respectively. After 24 h of GC treatment (1µM) HA production decreased by an average of 67.9 ± 3.11% (p < 0.0001) in all cell cultures. HAS2, HAS3 and HYAL1 expression in OFs also decreased (p = 0.009, p = 0.0005 and p = 0.015, respectively). Ten ng/mL PDGF-BB increased HA production and fibroblast proliferation in all cell lines (p < 0.0001); GC treatment remained effective and reduced HA production under PDGF-BB-stimulated conditions (p < 0.0001). MP and DEX reduced (p < 0.001, p = 0.002, respectively) PDGF-BB-induced HAS2 expression in OFs. MP and DEX treatment decreased PDGF-BB stimulated HAS3 expression (p = 0.035 and p = 0.029, respectively). None of the GCs tested reduced the PDGF-BB stimulated proliferation rate. Our results confirm that GCs directly reduce the HA production of OFs, which may contribute to the beneficial effect of GCs in GO.

FGF9 Promotes Expression of HAS2 in Palatal Elevation via the Wnt/β-Catenin/TCF7L2 Pathway.

Fgf9 mutation was found in cleft palate patients. Our previous study indicated that Fgf9 promotes timely elevation of palate by regulating hyaluronic acid (HA) accumulation at embryonic day 13.5 (E13.5). HA is synthesized by hyaluronic acid synthases (HAS) isoforms 1, 2, or 3. However, how FGF9 regulates HA in palatogenesis is still unclear. Using Ddx4-Cre mice, we generated the Fgf9-/- mouse model (with exon 2 deletion). Immunohistochemistry was used to detect the location and expression of HAS2 in WT and the Fgf9-/- palate at E13.5. We also predicted the association between Fgf9 and Has2 within the developing palate by performing a bioinformatics analysis. The expression of β-catenin, HAS2, and TCF7L2 were verified by Western blotting after knockout of Fgf9. Rescue experiments were performed by ELISA in vitro. Fgf9-/- mice exhibited 100% penetrance of the cleft palate. A knockout of Fgf9 confirmed that HAS2 and TCF7L2 expression was positively correlated with FGF9. TCF7L2 binds to the Has2 promoter, exhibiting the high specificity predicted by JASPAR. Additionally, increased HA expression by BML-284, TCF-dependent agonist, was blocked in Fgf9-/- palate because of the significant decline in TCF7L2 expression. FGF9 promotes HAS2 expression via Wnt/β-catenin/TCF7L2 pathway with TCF7L2 activating transcription of Has2 in the palate.

5-Aminolevulinic Acid-Mediated Photodynamic Therapy Improves Vocal Fold Wound Healing in Rats.

Studies showed that photodynamic therapy (PDT) might be able to prevent vocal fold scar formation when treating laryngeal lesions. We aim to investigate if PDT improves vocal wound healing and reduces scar formation in both prophylactic and remodeling procedures performed in vivo. In vivo. Vocal fold stripping was performed in Sprague-Dawley rats. PDT was performed with intraperitoneal injection of 100 mg/kg 5-Aminolevulinic Acid (5-ALA) and 635 nm laser irradiation of 20, 40, and 60 J/cm2 . PDT was performed immediately after surgery to study the prophylactic effect and 4 weeks after surgery to study the remodeling effect. Gene expression was evaluated with real-time PCR at 1 week after PDT. Histologic evaluations were performed 12 weeks after PDT, including hematoxylin-eosin, Masson, Alcian blue staining, and immunohistochemical staining of collagen I and III. PDT induced similar effects on the vocal fold wound healing outcomes in both prophylactic and remodeling procedures. Expression of MMP8, MMP13, HAS2, and TGFβ1 was significantly elevated. Histologic evaluation revealed significantly increased thickness, decreased density of collagen, and increased deposition of hyaluronic acid in the lamina propria. Immunohistochemistry also revealed better distribution and reduced density of collagen I and III. The most obvious changes were seen in the 60 J/cm2 PDT group. PDT could significantly improve vocal wound healing by providing both prophylactic effects and remodeling effects. It may be a minimally invasive treatment for vocal fold lesions with slight vocal scarring, and may be used to treat acute or chronic vocal injury to reduce vocal scarring. N/A Laryngoscope, 133:1943-1951, 2023.

Tamoxifen Alters TGF-β1/Smad Signaling in Vocal Fold Injury.

Effective treatments for vocal fold fibrosis remain elusive. Tamoxifen (TAM) is a selective estrogen receptor modulator and was recently reported to have antifibrotic actions. We hypothesized that TAM inhibits vocal fold fibrosis via altered transforming growth factor beta 1 (TGF-β1) signaling. Both in vitro and in vivo approaches were employed to address this hypothesis. In vitro, vocal fold fibroblasts were treated with TAM (10-8 or 10-9 M) ± TGF-β1 (10 ng/ml) to quantify cell proliferation. The effects of TAM on genes related to fibrosis were quantified via quantitative real-time polymerase chain reaction. In vivo, rat vocal folds were unilaterally injured, and TAM was administered by oral gavage from pre-injury day 5 to post-injury day 7. The rats were randomized into two groups: 0 mg/kg/day (sham) and 50 mg/kg/day (TAM). Histological changes were examined on day 56 to assess tissue architecture. TAM (10-8 M) did not affect Smad3, Smad7, Acta2, or genes related to extracellular matrix metabolism. TAM (10-8 or 10-9 M) + TGF-β1, however, significantly increased Smad7 and Has3 expression and decreased Col1a1 and Acta2 expression compared to TGF-β1 alone. In vivo, TAM significantly increased lamina propria area, hyaluronic acid concentration, and reduced collagen deposition compared to sham treatment. TAM has antifibrotic potential via the regulation of TGF-β1/Smad signaling in vocal fold injury. These findings provide foundational data to develop innovative therapeutic options for vocal fold fibrosis. NA Laryngoscope, 133:2248-2254, 2023.

Kuantifikasi Protein HAS2, PTX3, BCL2 dan BAX dalam Sel Kumulus Oosit Matur (MII) dan Imatur (MI) Pada Pasien Bayi Tabung

Oocyte developmental competence is one of the determining factors that in?uence the outcomes of an IVF cycle regarding the ability of a female gamete to reach maturation and be fertilized. A non-invasive approach using proteomic analysis of oocyte cumulus cells (CCs) can be used as a method to predict oocyte competence and viability that could potentially function as molecular predictors for IVF program prognosis. Our study was aimed at quantifying protein markers (HAS2, PTX3, BCL2 and BAX) that are expressed in human CCs between mature (MII) and immature (MI) oocytes. This research is an analytic observational study. Twelve samples of CCs from mature (MII) and immature (MI) oocytes were collected after ovum pick up from patients undergoing ICSI. CCs samples were isolated using an ultrasonicator and protein expressions were quantified using ELISA method. The Mann-Whitney test was used to compare the protein expressions from CCs between mature and immature oocytes. The results showed HAS2, PTX3 and BCL2 proteins expressions was increase in CCs from mature oocytes but had no significant effect compared to CCs from immature oocytes, however, BAX protein expression was significantly higher (P&lt;0.05) in CCs from immature oocytes which CCs from mature oocytes. This present study shows that BAX proteins could be used as markers for oocyte quality and maturation.&#x0D; Key words: ELISA, maturation, proteins marker, cumulus cells (CCs)

Enhanced anti-wrinkle activity of adlay bran fermented with Lactobacillus brevis MJM60390.

The aim of this study was to screen the lactic acid bacteria for fermentation of adlay bran and evaluate the anti-wrinkle effect of fermented and non-fermented adlay bran. Adlay bran was fermented with candidate LAB and extracted with 70% ethanol. The extracts from LAB-fermented adlay bran and non-fermented adlay bran were evaluated for the anti-wrinkle effects by measuring the hyaluronan, collagen, and elastin production in cells using ELISA kit. The molecular anti-wrinkle mechanism was investigated by RT-qPCR. Furthermore, the antioxidant activity, total phenolic and flavonoid content were also determined. Among the tested LAB, Lactobacillus brevis MJM60390 was selected for the highest glycosidase activity. Both extracts from adlay bran (NFAB) and L. brevis MJM60390-fermented adlay bran (LBFAB) showed anti-wrinkle effect, and LBFAB showed higher activity. Compared with control, hyaluronan production was increased by 24.73% and 59.38%, collagen production was increased by -13.08% and 34.19%, and the elastin production was increased by 29.78% and 53.73% by NFAB and LBFAB treatment, respectively. Investigation on the mRNA expression showed that LBFAB upregulated the expression of Has 2 and Has 3 and downregulate HYAL1 and HYAL2. LBFAB also upregulated the mRNA expression of COL1A1, COL1A2, ELN and inhibited the expression of collagenase and elastase. However, not all of these genes were regulated by NFAB. Furthermore, the antioxidant activity was significantly increased after fermentation, and the content of the phenolic and flavonoid compounds also increased in the LBFAB. In this study, we demonstrated that fermentation of adlay bran with L. brevis MJM60390 enhanced the anti-wrinkle activity through increasing the hyaluronan synthesis in keratinocytes and improving collagen and elastin production in dermal fibroblasts.

The Upregulation of HAS2-AS1 Relates to the Granulosa Cell Dysfunction by Repressing TGF-β Signaling and Upregulating HAS2.

HAS2 antisense RNA 1 (HAS2-AS1) is a long noncoding RNA that has increased expression in mature granulosa cells (GCs) and contributes to cumulus expansion by regulating HAS2 expression. However, the roles of HAS2-AS1 during the pathological process of polycystic ovary syndrome (PCOS) are still unclear. This study investigated the roles of HAS2-AS1 in patients with PCOS. Here, a significant upregulation of HAS2-AS1 was found in the primary GCs from patients with PCOS, which was positively correlated with the level of the protein HAS2. The knockdown of HAS2 restored the upregulation of HAS2-AS1 in promoting migration but could not restore the effects of HAS2-AS1 overexpression in promoting proliferation and repressing apoptosis. Transforming growth factor β (TGF-β) upregulated HAS2-AS1 levels, while HAS2-AS1 functioned as a feedback inhibition factor repressing TGF-β signaling by inhibiting TGF-β receptor type 2 (TGFBR2) expression. HAS2-AS1 bonded with EZH2 and guided the polycomb complex 2 to the TGFBR2 promoter region. HAS2-AS1 overexpression induced H3K27 hypermethylation in the TGFBR2 promoter region and then repressed TGFBR2 transcription in KGN cells and primary GCs. In conclusion, we identified for the first time that HAS2-AS1 is upregulated in patients with PCOS and represses TGF-β signaling via inducing TGFBR2 promoter region hypermethylation, which allowed us to explore the pathological processes of PCOS.

Teprotumumab Divergently Alters Fibrocyte Gene Expression: Implications for Thyroid-associated Ophthalmopathy.

Teprotumumab, an IGF-I receptor (IGF-IR) inhibitor, is effective in thyroid-associated ophthalmopathy (TAO). The drug can modulate induction by TSH of IL-6 and IL-8 in CD34+ fibrocytes and their putative derivatives, CD34+ orbital fibroblasts (CD34+ OF). Fibrocytes express multiple thyroid autoantigens and cytokines implicated in TAO, which are downregulated by Slit2. Inflammation and disordered hyaluronan (HA) accumulation occur in TAO. Whether teprotumumab alters these processes directly in fibrocytes/CD34+ OF remains uncertain. Determine teprotumumab effects on expression/synthesis of several TAO-relevant molecules in fibrocytes and GD-OF. Patients with TAO and healthy donors were recruited from an academic endocrine and oculoplastic practice. Real-time PCR, specific immunoassays. Teprotumumab attenuates basal and TSH-inducible autoimmune regulator protein, thyroglobulin, sodium iodide symporter, thyroperoxidase, IL-10, and B-cell activating factor levels in fibrocytes. It downregulates IL-23p19 expression/induction while enhancing IL-12p35, intracellular and secreted IL-1 receptor antagonists, and Slit2. These effects are mirrored by linsitinib. HA production is marginally enhanced by teprotumumab, the consequence of enhanced HAS2 expression. Teprotumumab affects specific gene expression in fibrocytes and GD-OF in a target-specific, nonmonolithic manner, whereas IGF-IR control of these cells appears complex. The current results suggest that the drug may act on cytokine expression and HA production systemically and locally, within the TAO orbit. These findings extend our insights into the mechanisms through which IGF-IR inhibition might elicit clinical responses in TAO, including a potential role of Slit2 in attenuating inflammation and tissue remodeling.

The hyaluronan-related genes HAS2, HYAL1-4, PH20 and HYALP1 are associated with prognosis, cell viability and spheroid formation capacity in ovarian cancer

PurposeHyaluronan modulates tumour progression, including cell adhesion, cohesion, proliferation and invasion, and the cancer stem cell phenotype. In ovarian cancer, high levels of stromal hyaluronan are associated with poor prognosis. In this work, hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-4, PH-20, HYALP1) were examined with regard to different levels of gene expression and its influence on ovarian cancer patients’ survival. The impact of a siRNA depletion of HAS2 was investigated in vitro.MethodsUsing the Kaplan–Meier Plotter tool, we investigated the influence of hyaluronic synthases and hyaluronidases on the survival of a collective of 1435 ovarian cancer patients. Differences in gene expression between normal (n = 46) and cancerous (n = 744) ovarian tissue were examined using the TNMplot database. Following an evaluation of hyaluronan-related gene expression in the ATCC ovarian cancer panel, we studied SKOV3 and SW 626 ovarian cancer cells subjected to HAS2 siRNA or control siRNA treatment in terms of HAS1-3, HYAL2 and HYAL3 mRNA expression. We investigated the ability to form spheroids using the Hanging Drop method and the response to chemotherapy at different concentrations using the MTT Assay. By STRING analysis, interactions within the enzymes of the hyaluronic acid system and with binding partners were visualized.ResultsHAS1, HYAL1 and HYAL4 mRNA expression is significantly upregulated, whereas HAS2, HYAL2 and HYAL3 mRNA expression is significantly downregulated in ovarian cancer tissue compared to controls. HAS2 improves cell viability, the capability to form tumour spheroids and has a negative prognostic value regarding overall survival. Lower HAS2 expression and high expression of HYAL2 and HYAL3 favours the survival of ovarian cancer patients. HAS2 knockdown cells and control cells showed a moderate response to combinatorial in vitro chemotherapy with taxol and cisplatin.ConclusionIn conclusion, our study shows that the hyaluronic acid system has a relevant influence on the survival of ovarian cancer patients and could therefore be considered as a possible prognostic factor.

Abstract 2387: The impact of beta 1,6-GlcNAc branched N-glycan synthesis on hexosamine pathway and UDP-GlcNAc demands in colorectal cancer cells

Abstract Metabolic changes are commonly seen in cancer. Tumor cells present a high rate of glucose and glutamine uptake and part of these nutrients supplies the hexosamine biosynthesis pathway (HBP), whose exacerbation has been related to solid tumors progression, including colorectal cancer. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the end product of HBP, serves as the donor for several enzymes involved in glycan biosynthesis. Aberrant glycosylation is known to affect cellular and molecular mechanisms related to the malignant phenotype. Beta 1,6-GlcNAc branches on N-glycans (products of the MGAT5 enzyme) are associated with greater stability of transmembrane glycoproteins, as glutamine and glucose transporters, and growth factor receptors. Furthermore, changes in the availability of UDP-GlcNAc can affect the levels of hyaluronic acid (HA) and O-GlcNAc-modified proteins. The interplay between different direct demands for UDP-GlcNAc is not well understood and may help to better understand tumor metabolism. Therefore, we intend to evaluate in colorectal cancer cells how changes in levels of branched beta1,6-GlcNAc N-glycans can affect both the hexosamine pathway and the different processes which demand UDP-GlcNAc (such as O-GlcNAcylation and hyaluronic acid synthesis). For this, two experimental strategies were performed. The first focused on the inhibition of complex N-glycans using swainsonine, and the second focused on the MGAT5 inactivation using CRISPR-Cas9. The levels of UDP-GlcNAc were also manipulated using DON, an inhibitor of the GFAT enzyme. Initially, it was found that both the pharmacological inhibition of complex N-glycan biosynthesis and the specific inactivation of MGAT5 lead to significantly reduced levels of hexosamine pathway intermediates, activated monosaccharides, and proteins modified by O-GlcNAc. Furthermore, it was also observed that MGAT5 inactivation enhances the expression of genes encoding HA synthases (HAS2 and HAS3). Upon detection of metabolites by LC-MS, a multivariate analysis was performed using the statistical method of Partial Least-Squares Discriminant Analysis (PLS-DA), and it was observed that KO MGAT5 cells formed a metabolically distinct population from MOCK cells. Interestingly, after pharmacological inhibition of GFAT, an increase in the levels of beta 1,6-GlcNAc branched N-glycans and a reduction in O-GlcNAcylation were observed. Treatment with DON was also able to reduce HAS2 expression however an increased in HAS3 expression was observed. These data suggest that there is a relationship among different demands for UDP-GlcNAc in tumor cells and a possible regulatory role played by beta 1,6-GlcNAc branched N-glycans in this issue. This work contributes to a better understanding of interconnections between biochemical processes that demand UDP-GlcNAc in the context of colorectal cancer. Citation Format: Érika Elias Ferreira, Isadora de Araújo Oliveira, Adriane Regina Todeschini, Julio Cesar de Freitas-Junior. The impact of beta 1,6-GlcNAc branched N-glycan synthesis on hexosamine pathway and UDP-GlcNAc demands in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2387.

Abstract 5447: Enhanced antitumoral effect of the glycosylated 4-methylumbelliferone in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death worldwide, and is particularly refractory to the available therapeutic drugs. Unfortunately, curative treatments such as surgery, liver transplantation, or ablation are reserved for early stages and can only be applied in less than 30% of the patients with HCC. Glycosylation can be a potent and interesting strategy for drug delivery to a specific target. We enzymatically synthesized a glycosylated derivative of the coumarin 4-methylumbelliferone (4MU), namely 4MUR.The antitumoral effect of 4MUR in comparison with the aglycone 4MU was evaluated onto liver cancer cells. Cellular toxicity was determined on murine liver tumor cell lines as well as non-hepatic tumoral cells. Tumor cell lines were significantly more sensitive than non-tumoral cells to the glycosylated molecule in a dose-dependent manner (p&amp;lt;0.05). 4MUR was incorporated mostly into tumoral cells by the interaction with the asialoglycoprotein receptor (ASGPR), which is overexpressed in liver tumor cells. Hyaluronic acid (HA) is the major component of the extracellular matrix (ECM). It is produced at the plasma membrane by three hyaluronan synthases (HAS1, 2 and 3), which couple glucuronic acid and N-acetylglucosamine into a linear polymer using the corresponding UDP-sugars (UDP-GlcUA and UDP-GlcNAc) as substrates. Unlike 4MUR, the glucuronidation of 4MU competitively inhibits chain elongation of HA. Furthermore, 4MUR treatment regulates the levels of HAS2 and HAS3 expression in the tumoral cell line. Finally, 4MUR treatment induced apoptosis cell death in tumoral cells. Our results suggest that the 4MUR is safe and effective against liver tumor cells via a specific targeting to ASGPR receptor and decreasing HA synthesis. Citation Format: Gisela Weiz, Alina L. Gonzalez, Flavia Piccioni, Mariel Fusco, Marco Diaz Gutiérrez, Guillermo Mazzolini, Javier D. Breccia, Mariana Malvicini, Maria I. Molejon. Enhanced antitumoral effect of the glycosylated 4-methylumbelliferone in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5447.