MiR-23b-3p suppresses epithelial-mesenchymal transition, migration, and invasion of hepatocellular carcinoma cells by targeting c-MET.

Background: Aberrant expression of c-MET is known to be associated with tumor recurrence and metastasis by promoting cell proliferation, epithelial-mesenchymal transition (EMT), and migration in hepatocellular carcinoma (HCC). Recently, miR-23b-3p has been identified as a tumor suppressor, but detailed role of miR-23b-3p in HCC is still unclear. Our study aimed to investigate how miR-23b-3p is associated with the malignant potential of HCC cells. Methods: HCC tissues and their adjacent non-tumor tissues were acquired from 30 patients with HCC. Expression of EMT- or stemness-related genes were examined in the two HCC cell lines. Migration of HCC cells was analyzed using transwell and wound healing assays. Results: c-MET was overexpressed in HCC tissues compared to the adjacent non-tumor tissues. c-MET knockdown inhibited EMT and reduced migration and invasion of HCC cells. Furthermore, c-MET was a target of miR-23b-3p, and miR-23b-3p expression was decreased in HCC tissues compared to non-tumor tissues. Treatment of miR-23b-3p inhibitor in HCC cells promoted EMT, cell migration, and invasion. In contrast, miR-23b-3p overexpression suppressed EMT, cell migration, and invasion, concomitantly reducing c-MET expression. Transfection of miR-23b-3p inhibitor with concomitant c-MET knockdown mitigated the effects of miR-23b-3p inhibitor on EMT in HCC cells. In addition, transforming growth factor beta1 (TGF-ß1) stimulation after miR-23b-3p overexpression induced neither the mesenchymal phenotype nor migratory property of HCC cells. Conclusion: In this study, we confirmed that miR-23b-3p downregulation significantly increased EMT, migration, and invasion of HCC cells. In addition, c-MET was confirmed to be a target of miR-23b-3p in HCC cells and regulated the functional effects of miR-23b-3p. These results suggest that miR-23b-3p can be used as a prognostic biomarker and candidate target for HCC treatment.

Clinical Features and Outcomes of Patients with Orbital Schwannoma.

PURPOSE: To report the clinical features, treatment, and outcome of 11 patients with orbital schwannoma in Korean patients. METHODS: The medical records of 11 orbital schwannoma patients treated between April 2007 and April 2021 were retrospectively reviewed. The demographic data, clinical characteristics, radiological features, and outcomes were reviewed. RESULTS: The mean age at the time of diagnosis was 49.00 ± 14.45 years. The most common initial symptom was ocular protrusion (n = 7), and other symptoms were decreased visual acuity ( n = 5), restriction of eye movement ( n = 4), swelling ( n = 3), and pain ( n = 2). Locations of tumors were superomedial; followed by the orbital apex and inferolateral. The most common shape seen in our patients was beads like multilobulated appearance; followed by a round, oval, fusiform, and dumbbell shape. MRI of T1-weighted revealed isointense or hypointense, whereas the T2-weighted indicated hyper or isointense lesion. Five patients had optic neuropathy at presentation, and 1 of them showed improved vision after surgery. Complete or incomplete excision was performed for all. Surgical complications include decreased vision and paraesthesia. There has been no recurrence to date. CONCLUSIONS: Orbital schwannoma is a rare disease and it is difficult to distinguish it from other orbital tumors because the initial symptoms are nonspecific. Differential diagnosis by combining the shape, location, and contrast enhancement findings seen on computed tomography and magnetic resonance imaging can be helpful in surgical treatment. Complete excision gives the best results without recurrence, but if the patient is in an inaccessible location, only surgery to reduce the volume can satisfy the patient without recurrence.

Exosomal miR-125b Exerts Anti-Metastatic Properties and Predicts Early Metastasis of Hepatocellular Carcinoma.

BACKGROUND & AIMS: Cancer metastasis is responsible for the majority of cancer-related deaths. Exosomal miRNAs have emerged as promising biomarkers for cancer, serving as signaling molecules that can regulate tumor growth and metastasis. This study examined circulating exosomal miRNAs that could predict hepatocellular carcinoma (HCC) metastasis. METHODS: Exosomal miRNA was measured by quantitative real-time PCR (qRT-PCR) in a large set of patients (n = 284). To investigate the role of exosomal miRNA in HCC, we performed a series of in vitro tests, such as exosome labeling, qRT-PCR, reverse transcription PCR, wound healing assay, transwell assay, and Western blot assay. RESULTS: Exosomal miR-125b was drastically downregulated in HCC patients with metastasis than in those without metastasis. In vitro, we observed the uptake of miR-125b by exosome in recipient cells. Exosome-mediated miR-125b significantly inhibited migration and invasion abilities and downregulated the mRNA expressions of MMP-2, MMP-9, and MMP-14 in recipient cells via intercellular communication. Further investigation revealed that miR-125b suppressed SMAD2 protein expression in recipient cells by binding to its 3' untranslated regions. Exosome-mediated miR-125b transfer also disrupted TGF-ß1-induced epithelial-mesenchymal transition and TGF-ß1/SMAD signaling pathway in recipient cells by leading to a decrease of SMAD2 protein expression. Moreover, exosomal miR-125b was downregulated after metastasis compared with that at baseline in patients with serial measurements before and after metastasis. CONCLUSIONS: The results imply that exosome-mediated miR-125b exerts anti-metastatic properties in HCC. These findings highlight that circulating exosomal miR-125b might represent a reliable biomarker with diagnostic and therapeutic implications for extrahepatic metastasis from HCC.

Analysis of Clinical Characteristics and Prognosis of Patients With Periocular Malignant Melanoma in Korea.

OBJECTIVE: To report the clinical features of 20 patients with malignant melanoma and to evaluate the survival and prognosis of patients with malignant melanoma in Korea. METHODS AND ANALYSIS: The medical records of 20 patients with malignant melanoma treated between March 2004 and March 2020 were reviewed retrospectively. The demographic data, clinical characteristics, and outcomes were also reviewed. Outcome measures included local recurrence, metastasis, and tumor-related mortality. Prognostic factors associated with recurrence, metastasis, and survival were analyzed using a Cox proportional hazards model. RESULTS: Among the 20 patients with periocular malignant melanoma, 4 (20%) showed local recurrence during follow-up (6.61 ±â€Š6.36 years). The 1-, 5-, and 10-year recurrence rates were 5%, 10.3%, and 24.1%, respectively. 10 (50%) patients developed distant metastasis, mainly involving lung, brain, parotid gland and spine. The 1-, 5-, and 10-year rates of metastasis were 5%, 10.9%, and 35.7%, respectively. Significantly fewer metastases were detected following initial extensive surgical excision (P = 0.04). 8 (40%) patients died of malignant melanoma. The main risk factor for mortality was tumor thickness (HR: 3.88, P < 0.01). Based on Kaplan-Meier survival estimates, the 1-, 5-, and 10-year tumor-related survival rates were 75.8%, 55.6%, and 55.6%, respectively. CONCLUSION: Tumor thickness is a significant prognostic factor affecting the survival rate. It is important to reduce the metastatic rate via extensive resection without leaving any residual tumor in the margin during surgery.

Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease.

Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.

Hertel exophthalmometer versus computed tomography scan in proptosis estimation in thyroid-associated orbitopathy.

OBJECTIVE: To compare exophthalmometry measured by Hertel exophthalmometer versus computed tomography (CT) scan. METHODS: For this study, 133 patients with thyroid-associated orbitopathy (TAO) were analyzed retrospectively. Two experienced clinical observers independently measured proptosis using a Hertel exophthalmometer. The CT approaches used to measure proptosis included two previous methods and one new method. Method 1 used both lateral orbital rims-corneal surface in the axial plane, method 2 used lateral to medial orbital rims-corneal surface in the axial plane, and method 3 used superior to inferior orbital rims-corneal surface in the sagittal plane (new method). Patients were separated into two groups based on 2-mm differences in proptosis between eyes. Correlation analysis was performed to find the association between Hertel and CT values. RESULTS: The Pearson's coefficient (r) was 0.727 for Hertel exophthalmometry and CT method 1, 0.712 for Hertel and CT method 2, and 0.623 for Hertel and CT method 3. For patients with eye proptosis differences larger than 2 mm between eyes, Pearson's coefficient (r) was 0.495 for Hertel exophthalmometry and CT method 1, 0.634 for Hertel and CT method 2, and 0.635 for Hertel and CT method 3. CONCLUSION: The three CT methods mentioned above had statistically significant relationships with Hertel exophthalmometry. Methods 2 and 3, which measured both eyes through different cut sections, had a significant relationship with Hertel values with eye proptosis differences larger than 2 mm. Thus, the new additive method may be effective for measuring proptosis in patients with differences greater than 2 mm between eyes.

Synergistic effects of CD44 and TGF-ß1 through AKT/GSK-3ß/ß-catenin signaling during epithelial-mesenchymal transition in liver cancer cells.

Cancer metastasis is strongly correlated with epithelial-mesenchymal transition (EMT), in which transforming growth factor-ß (TGF-ß) signaling plays a central role. CD44 has emerged as a cancer stem cell (CSC) marker that strongly induces EMT together with TGF-ß1. This study aimed to investigate the link between high CD44 and TGF-ß1 levels during EMT in HCC cell lines. FACS analysis showed high expression of CD44 in TGF-ß1-positive SNU-368 cells and TGF-ß1-negative SNU-354 cells. SNU-368 CD44(+) cells showed EMT through up-regulation of the AKT/GSK-3ß/ß-catenin pathway. By comparison, SNU-354 CD44(+) cells showed only increased N-cadherin expression, which was not accompanied by a decrease in E-cadherin expression, and also down-regulated the AKT/GSK-3ß/ß-catenin pathway. However, TGF-ß1-stimulated SNU-354 cells (CD44/TGF-ß1(+)) exhibited lower E-cadherin and higher N-cadherin expression with increased AKT/GSK-3ß/ß-catenin pathway activity. CD44/TGF-ß1(+) SNU-354 cells also showed enhanced migration and formed larger spheres, while the TGF-ß1-induced stem cell properties returned to their original state with the TGF-ß1 inhibitor SB431542. SB431542-treated SNU-368 (CD44/TGF-ß1(-)) cells also showed diminished N-cadherin and AKT/GSK-3ß/ß-catenin pathway activity and further decreased cell motility in a wound healing assay. However, CD44 knockdown in SNU-354 cells did not induce EMT even after treatment with TGF-ß1. Finally, double inhibition of both CD44 and TGF-ß1 further decreased migration and sphere formation more strongly than a single inhibition in SNU-368 cells. In conclusion, the current study demonstrated the synergistic interactions between CD44 and TGF-ß1 in EMT induction and CSC properties through the AKT/GSK-3ß/ß-catenin pathway in HCC cells.

Branched-chain amino acids ameliorate fibrosis and suppress tumor growth in a rat model of hepatocellular carcinoma with liver cirrhosis.

PURPOSE: Recent studies have revealed that branched-chain amino acids (BCAA) reduce the development of hepatocellular carcinoma (HCC) in patients with obesity and hepatitis C virus infection by improving insulin resistance (IR). The aim of this study was to examine the anti-cancer and anti-fibrotic effects of BCAA on the development of diethylnitrosamine (DEN)-induced HCC and liver cirrhosis in a rat model. METHODS: Male SD rats received weekly intraperitoneal injections of DEN (50 mg/kg of body weight) for 16 weeks to induce HCC. They were fed a diet containing 3% casein, 3% or 6% BCAA for 13 weeks beginning 6 weeks after DEN administration. DEN was used to induce HCC through stepwise development from cirrhosis to HCC. The effect of BCAA was evaluated in tumor tissues by histopathologic analyses, reverse transcription-polymerase chain reaction, and Western blotting. RESULTS: The mean area and number of dysplastic nodules (DNs) and tumors in the casein group tended to be larger than those in the BCAA group 16 weeks after DEN administration. The mean fibrotic area in the BCAA group was smaller than that in the casein group. The BCAA group showed decreased mRNA levels for markers of fibrosis, angiogenesis, and apoptosis inhibition. Compared with the casein group, the BCAA group had lower levels of α-smooth muscle actin, vascular endothelial growth factor, p-ß-catenin, p-p38 mitogen-activated protein kinase, proliferating cell nuclear antigen, and caspase-3 protein expression, as well as a higher level of cleaved caspase-3 protein expression. CONCLUSIONS: BCAA supplementation of the diet ameliorated liver fibrosis and HCC development in a DEN-induced rat model of HCC with liver cirrhosis, but not in the IR model. These results provide a rationale for anti-fibrosis and chemoprevention using BCAA treatment for HCC with liver cirrhosis, as well as decreasing the ammonia level.

Proton conductive nano-channel membranes based on polyaniline with phosphonic acid moieties for low relative humidity.

Most sulfonic acid based-membranes come to the limit because of high cost and low performance at low relative humidity (RH). Therefore, we synthesized phosphonic acid functionalized polyaniline (PA-PANI). PANI was used as polymer back bone because of excellent chemical mechanical stability and easy synthesis from relatively cheap monomer, and phosphonic acid presents the relatively high proton conducting in nano-channel without water because of its self dissociation nature. PA-PANI was synthesized by bonding chemically with phosphonic acid groups using 3-bromopropylamine as a reagent. PA-PANI nano-channel membrane showed high and stable conductivities (approximately 0.035 S/cm at 80 to approximately 180 degrees C) under low RH. And PA-PANI nano-channel membranes compared with Nafion (10(-4) S/cm at < 30% RH), have higher proton conductivities. This fact showed phosphonic acid groups were more effective at low RH than sulfonic acid groups. Because the proton conductivity of Nafion decreases considerably at elevated temperature due to dehydration, our low cost membranes using phosphonic acid moiety may be expected to substitute sulfonic acid based-membranes such as Nafion.

Isolation of glioma cancer stem cells in relation to histological grades in glioma specimens.

PURPOSE: The existence of cancer stem cells (CSCs) in glioblastoma has been proposed. However, the unknown knowledge that is yet to be revealed is the presence of glioma CSCs (gCSCs) in correlation to each WHO grades of glioma. We approached this study with a hypothesis that specimens from high-grade gliomas would have higher isolation rate of gCSCs in comparison to those of lower-grade gliomas. METHODS: The glioma specimens were obtained from patients and underwent gliomasphere assay. The gliomaspheres were chosen to be analyzed with immunocytochemisty for surface markers. Then the selected gliomaspheres were exposed to neural differentiation conditions. Lastly, we made mouse orthotopic glioma models to examine the capacity of gliomagenesis. RESULTS: The gliomaspheres were formed in WHO grade IV (13 of 21) and III (two of nine) gliomas. Among them, WHO grade IV (11 of 13) and III (two of two) gliomaspheres showed similar surface markers to gCSCs and were capable of neural differentiation. Lastly, among the chosen cells, 10 of 11 WHO grade IV and two of two WHO grade III gliomaspheres were capable of gliomagenesis. Thus, overall, the rates of existence of gCSCs were more prominent in high-grade gliomas: 47.6% (10 of 21) in WHO grade IV gliomas and 22.2% (two of nine) in WHO grade III gliomas, whereas WHO grade II and I gliomas showed virtually no gCSCs. CONCLUSIONS: This trend of stage-by-stage increase of gCSCs in gliomas showed statistical significance by chi-square test linear-by-linear association. We prove that the rates of existence of gCSCs increase proportionally as the WHO grades of gliomas rise.

Nafion/silane nanocomposite membranes for high temperature polymer electrolyte membrane fuel cell.

The polymer electrolyte membrane fuel cell (PEMFC) has been studied actively for both potable and stationary applications because it can offer high power density and be used only hydrogen and oxygen as environment-friendly fuels. Nafion which is widely used has mechanical and chemical stabilities as well as high conductivity. However, there is a drawback that it can be useless at high temperatures (> or = 90 degrees C) because proton conducting mechanism cannot work above 100 degrees C due to dehydration of membrane. Therefore, PEMFC should be operated for long-term at high temperatures continuously. In this study, we developed nanocomposite membrane using stable properties of Nafion and phosphonic acid groups which made proton conducting mechanism without water. 3-Aminopropyl triethoxysilane (APTES) was used to replace sulfonic acid groups of Nafion and then its aminopropyl group was chemically modified to phosphonic acid groups. The nanocomposite membrane showed very high conductivity (approximately 0.02 S/cm at 110 degrees C, <30% RH).

Characterization of sulfonated poly(ether ether ketone)/silane nanocomposite membrane for high temperature polymer electrolyte membrane fuel cells.

The perfluorosulfonic acid polymer membrane is most widely used in PEMFCs. However, its some major drawbacks like high cost and performance limitation at high temperature are obstacles of its commercialization. The goal of this study was to develop low cost membranes which have good conductivity in the range of PEMFCs operating temperature. We fabricated new sPEEK/3-APTES nanocomposite membrane where inorganic particles were chemically bonded to sulfuric acid group of sPEEK. PEEK is a thermally stable, mechanically tough and very cheap polymer. And the addition of 3-APTES and phosphorous acid increased the proton conductivity of composite membranes at high temperatures. This nanocomposite membranes maintained good conductivity at 110 degrees C.

Presence of glioma stroma mesenchymal stem cells in a murine orthotopic glioma model.

PURPOSE: High-grade gliomas are closely related to the mesenchymal phenotype which might be explained by unorthodox differentiation of glioma cancer stem cells (gCSCs). We reasoned that other non-neural stem cells, especially mesenchymal stem cells (MSCs), might play a role in expressing mesenchymal phenotype of high-grade gliomas. Thus we hypothesized that cells resembling MSCs exist in glioma specimens. METHODS: We created a mouse (m) orthotopic glioma model using human gCSCs. Single-cell suspensions were isolated from glioma specimens and cultured according to the methods for mMSCs or gliomaspheres. These cells were analyzed by fluorescence-activated cell sorting (FACS) for surface markers associated with mMSCs or gCSCs. Glioma stroma (GS)-MSCs were exposed to mesenchymal differentiation conditions. To decide the location of GS-MSCs, sections of orthotopic glioma models were analyzed by immunofluorescent labeling. RESULTS: GS-MSCs were isolated which were morphologically similar to mMSCs. FACS analysis showed that the GS-MSCs had similar surface markers to mMSCs (stem cell antigen-1 [Sca-1](+), CD9(+), CD45(-), CD11b(-), CD31(-), and nerve/glial antigen 2 [NG2](-)). GS-MSCs were capable of mesenchymal differentiation. Immunofluorescent labeling indicated that GS-MSCs are located around blood vessels, are distinct from endothelial cells, and have features that partially overlap with vascular pericytes. CONCLUSIONS: Our results indicate that cells similar to mMSCs exist in glioma specimens. The GS-MSCs might be located around vessels, which suggests that GS-MSCs may provide the mesenchymal elements of the vascular niche. GS-MSCs may represent non-neural stem cells that act as an important source of mesenchymal elements, particularly during the growth of gliomas.