Immunofluorescence Staining Assay Research Articles

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis

PurposeTo explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. ResultsCS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. ConclusionCS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

Plin5 inhibits proliferation and migration of vascular smooth muscle cell through interacting with PGC-1α following vascular injury

ABSTRACT Abnormal proliferation and migration of vascular smooth muscle cell (VSMC) is a hallmark of vascular neointima hyperplasia. Perilipin 5 (Plin5), a regulator of lipid metabolism, is also confirmed to be involved in vascular disorders, such as microvascular endothelial dysfunction and atherosclerosis. To investigate the regulation and function of plin5 in the phenotypic alteration of VSMC, -an animal model of vascular intima hyperplasia was established in C57BL/6 J and Plin5 knockdown (Plin5 ±) mice by wire injure. Immunohistochemical staining was used to analyze neointima hyperplasia in artery. Ki-67, dihydroethidium immunofluorescence staining and wound healing assay were used to measure proliferation, reactive oxygen species (ROS) generation and migration of VSMC, respectively. Plin5 was downregulated in artery subjected to vascular injury and in VSMC subjected to platelet-derived growth factor (PDGF)-BB. Plin5 knockdown led to accelerated neointima hyperplasia, excessive proliferation and migration of VSMC after injury. In vitro, we observed increased ROS content in VSMC isolated from Plin5 ± mice. Antioxidative N-acetylcysteine (NAC) inhibited VSMC proliferation and migration induced by PDGF-BB or plin5 knockdown. More importantly, plin5-peroxlsome proliferator-activated receptor-γ coactivator (PGC)-1α interaction was also attenuated in VSMC after knockdown of plin5. Overexpression of PGC-1α suppressed PDGF-BB-induced ROS generation, proliferation, and migration in VSMC isolated from Plin5 ± mice. These data suggest that plin5 serves as a potent regulator of VSMC proliferation, migration, and neointima hyperplasia by interacting with PGC-1α and affecting ROS generation.

Ginsenoside Compound K Enhances Fracture Healing via Promoting Osteogenesis and Angiogenesis.

Fractures have an extraordinarily negative impact on an individual’s quality of life and functional status, particularly delayed or non-union fractures. Osteogenesis and angiogenesis are closely related to bone growth and regeneration, and bone modeling and remodeling. Recently Chinese medicine has been extensively studied to promote osteogenic differentiation in MSCs. Studies have found that Ginseng can be used as an alternative for tissue regeneration and engineering. Ginseng is a commonly used herbal medicine in clinical practice, and one of its components, Ginsenoside Compound K (CK), has received much attention. Evidence indicates that CK has health-promoting effects in inflammation, atherosclerosis, diabetics, aging, etc. But relatively little is known about its effect on bone regeneration and the underlying cellular and molecular mechanisms. In this study, CK was found to promote osteogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs) by RT-PCR and Alizarin Red S staining in vitro. Mechanistically, we found CK could promote osteogenesis through activating Wnt/β-catenin signaling pathway by immunofluorescence staining and luciferase reporter assay. And we also showed that the tube formation capacity of human umbilical vein endothelial cells (HUVECs) was increased by CK. Furthermore, using the rat open femoral fracture model, we found that CK could improve fracture repair as demonstrated by Micro-CT, biomechanical and histology staining analysis. The formation of H type vessel in the fracture callus was also increased by CK. These findings provide a scientific basis for treating fractures with CK, which may expand its application in clinical practice.

Ginsenoside Rh1 Inhibits Angiotensin II-Induced Vascular Smooth Muscle Cell Migration and Proliferation through Suppression of the ROS-Mediated ERK1/2/p90RSK/KLF4 Signaling Pathway.

Vascular smooth muscle cell (VSMC) proliferation and migration play key roles in the progression of atherosclerosis and restenosis. A variety of ginsenosides exert various cardiovascular benefits. However, whether and how ginsenoside Rh1 (Rh1) inhibits VSMC dysfunction remain unclear. Here, we investigated the inhibitory effects of Rh1 on rat aortic smooth muscle cell (RASMC) migration and proliferation induced by angiotensin II (Ang II) and the underlying mechanisms. Cell proliferation and migration were evaluated using sulforhodamine B and wound-healing assay. The molecular mechanisms were investigated using Western blotting, quantitative reverse-transcription polymerase chain reaction analysis, immunofluorescence staining, and luciferase assay. Reactive oxygen species (ROS) production was measured using dihydroethidium and MitoSOX staining. We found that Rh1 dose-dependently suppressed Ang II-induced cell proliferation and migration. Concomitantly, Ang II increased protein levels of osteopontin, vimentin, MMP2, MMP9, PCNA, and cyclin D1, while these were reduced by Rh1 pretreatment. Notably, Ang II enhanced both the protein expression and promoter activity of KLF4, a key regulator of phenotypic switching, whereas pretreatment with Rh1 reversed these effects. Mechanistically, the effects of Rh1 on VSMC proliferation and migration were found to be associated with inhibition of ERK1/2/p90RSK signaling. Furthermore, the inhibitory effects of Rh1 were accompanied by inhibition of ROS production. In conclusion, Rh1 inhibited the Ang II-induced migration and proliferation of RASMCs by suppressing the ROS-mediated ERK1/2/p90RSK signaling pathway.

UBE2B promotes ovarian cancer growth via promoting RAD18 mediated ZMYM2 monoubiquitination and stabilization

ABSTRACT Ubiquitin-conjugating enzyme E2 B (UBE2B) can form a heterodimer with ubiquitin E3 ligase RAD18. In this study, we aimed to explore new substrates of the UBE2B/RAD18 complex and their regulatory effects in ovarian cancer. Protein physical interactions were predicted using GeneMANIA. Serial sections of commercial ovarian cancer tissue arrays were used to check the protein expression of UBE2B, RAD18, and ZMYM2. Immunofluorescence staining and co-immunoprecipitation assays were performed to check their location and interactions. Cycloheximide chase assay was applied to explore the influence of UBE2B and RAD18 on ZMYM2 degradation. Xenograft tumor models were constructed to assess the influence of the UBE2B-ZMYM2 axis on in vivo tumor growth. A strong positive correlation between UBE2B and ZMYM2 and a moderate positive correlation between RAD18 and ZMYM2 were observed in 23 ovarian cancer cases. In CAOV4 and OVCAR3 cells, myc-ZMYM2 interacted with UBE2B and RAD18. UBE2B and ZMYM2 could be detected in the samples immunoprecipitated by anti-RAD18. UBE2B overexpression or knockdown did not alter ZMYM2 mRNA expression. UBE2B overexpression increased ZMYM2 monoubiquitination but reduced its polyubiquitination. RAD18 knockdown impaired UBE2B-induced ZMYM2 monoubiquitination. UBE2B overexpression significantly enhanced the stability of ZMYM2 protein, the effect of which was weakened by RAD18 knockdown. UBE2B overexpression significantly enhanced the growth of xenograft tumors derived from CAOV4 cells. ZMYM2 knockdown remarkedly suppressed tumor growth and impaired the growth-promoting effect of UBE2B overexpression. In conclusion, this study revealed a novel regulatory effect of the UBE2B/RAD18 complex on ZMYM2 monoubiquitination and stability in ovarian cancer.

HIV-1 but not SIVmac239 induces higher interferon-α antiviral state in chronic infected northern pig-tailed macaques (Macaca leonina)

Studies have shown that interferon (IFN)-α has an inhibitory effect on human immunodeficiency virus type 1 (HIV-1) replication in the acute infection stage, but its role in chronic infection is still unclear. We previously established a nonpathogenic HIV-1 and pathogenic simian immunodeficiency virus (SIV) model in northern pig-tailed macaques (NPMs, Macaca leonina). In the current study, we detected viral RNA and DNA in various tissues (axillary lymph nodes (LNs), inguinal LNs, and spleen) in HIV-1NL4-3- and SIVmac239-infected NPM during the chronic stage of infection. Results indicated that the levels of viral DNA and RNA were higher in the tested tissues (LNs and spleen) of the SIVmac239-infected NPMs than in the HIV-1NL4-3 infected NPMs. Furthermore, IFN-α expression was higher in the HIV-infected tissues than in the SIV-infected controls. The HIV restriction factors induced by IFN-α (i.e., tetherin and MX2), as well as inflammatory factors IFN-γ, tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6), were analyzed using real-time polymerase chain reaction (PCR) and immunofluorescence staining assays. Results showed that their expression levels were much higher in the HIV-infected tissues than in the SIV-infected controls. These findings were confirmed by in vitro experiments on healthy NPM peripheral blood mononuclear cells infected with HIV-1NL4-3, which showed lower viral replication, higher IFN-α expression, and an antiviral status. This study demonstrated that HIV-1 infection, but not SIVmac239 infection, in NPMs caused higher expression of IFN-α and induced a higher antiviral status. This may be one of the reasons why HIV-1 cannot replicate at a high level or develop into AIDS in NPMs.

EID1 plays a protective role in early-onset pre-eclampsia via promoting proliferation and invasion in trophoblast cells

Pre-eclampsia is a pregnancy-specific syndrome, which is partly due to abnormal proliferation and invasion of trophoblast cells. EP300 interacting inhibitor of differentiation 1 (EID1) participates in cell proliferation and invasion. This study aims to investigate the roles of EID1 in trophoblast cells and pre-eclampsia. The expression of EID1 in placental tissues from 60 women with pre-eclampsia and 60 health pregnancies was detected by real-time PCR and immunohistochemical staining. EID1 was overexpressed or silenced by transfection of plasmid or siRNA in HTR-8/SVneo trophoblast cells, and then cell proliferation, cell cycle transition, migration, and invasion were determined by CCK-8 assay, flow cytometry, immunofluorescent staining, immunoblotting, and transwell assays. In addition, the activity of Akt/b-catenin signaling was measured by immunofluorescent staining and Western blot. EID1 mRNA level was decreased in placental tissues of pre-eclampsia patients, especially early-onset pre-eclampsia, accompanied by more severe clinical manifestation and a higher rate of fetal growth restriction (FGR). Gain- and loss-of-function experiments demonstrated that EID1 promoted proliferation and cell cycle transition, migration, and invasion in HTR-8/SVneo cells and its knockdown played opposite roles, suggesting that EID1 may be required for normal gestation. Akt/b-catenin signaling was activated after EID1 forced expression and deactivated after its silencing. EID1 promoted proliferation and invasion of cultured trophoblast cells with possible involvement of Akt/b-catenin signaling. These findings may provide novel insights for the diagnosis and treatment of early-onset pre-eclampsia in a clinic.

Characterization of Theabrownins Prepared From Tea Polyphenols by Enzymatic and Chemical Oxidation and Their Inhibitory Effect on Colon Cancer Cells.

Theabrownins (TBs) are prepared from dark tea and contain a large number of complex heterogeneous components, such as carbohydrates, proteins, and flavonoids, which are difficult to remove. In addition, some toxic and harmful extraction solvents are used to purify TBs. These obstacles hinder the utilization and industrialization of TBs. In this study, tea polyphenols were used as substrates and polyphenol oxidase and sodium bicarbonate (NaHCO3) were used successively to prepare theabrownins (TBs-E). The UV-visible characteristic absorption peaks of the TBs-E were located at 203 and 270 nm and Fourier-transform IR analysis showed that they were polymerized phenolic substances containing the hydroxy and carboxyl groups. The TBs-E aqueous solution was negatively charged and the absolute values of the zeta potential increased with increasing pH. A storage experiment showed that TBs-E were more stable at pH 7.0 and in low-temperature environments around 25°C. HT-29 human colon cancer cells were used to evaluate the biological activity of TBs-E through 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT), H&E staining, propidium iodide immunofluorescent staining, flow cytometry, and real-time PCR assays. The TBs-E significantly inhibited cell growth and caused late apoptosis, particularly at the dose of 500 μg/ml. The TBs-E markedly reduced the expression of antioxidant enzyme genes and increased the generation of reactive oxygen species to break the redox balance, which may have led to cell damage and death. These results will promote research and industrialization of TBs.

Kaempferol suppresses androgen-dependent and androgen-independent prostate cancer by regulating Ki67 expression.

Kaempferol is a natural flavonoid that has been reported to be active against many cancers, including prostate cancer, breast cancer and colon cancer. In our previous study, we found kaempferol could act as a selective androgen receptor modulator, thereby suppress development of benign prostatic hyperplasia. This finding inspired us to further explore the effect and the mechanism of action of kaempferol on prostate cancer. Plate clone formation assay was performed to detect the effect of kaempferol on cell proliferation. Flow cytometry was used to detect the impact of kaempferol on cell apoptosis and cell cycle distribution. qPCR, immunofluorescence staining, and enzyme-linked immunosorbent assay were used to detect the expression of gene and protein of Ki67 which is a biomarker of cell proliferation. In the present study, we found kaempferol could dramatically suppress androgen-dependent and androgen-independent prostate cancer cells proliferation and induce their apoptosis. Furthermore, we found that kaempferol induced cell cycle to be arrested at G1 phase in 22Rv1 cells but at S and G2 phase in PC-3 cells. In addition, we detected the mRNA and protein of Ki67 which is corresponding to the cell proliferation and found that kaempferol could significantly inhibit Ki67 expression at mRNA level but increase its expression at protein levels in both androgen-dependent and androgen-independent prostate cancer cells. Taken together, kaempferol inhibited the proliferation of androgen-dependent and androgen-independent prostate cancer cells by regulating the expression of Ki67. These findings further shed light on the mechanism of action of kaempferol on anti-prostate cancer.

Extracellular vesicle-mediated delivery of miR-127-3p inhibits the proliferation and invasion of choriocarcinoma cells by targeting ITGA6

BackgroundChoriocarcinoma (CC) is a highly aggressive malignant tumor that mostly occurs in women of childbearing age. Chemotherapy is the main treatment for CC, but it has side effects and causes drug resistance, which can lead to treatment failure. Extracellular vesicles (EVs) that deliver microRNAs (miRNAs) have emerged as a novel and promising therapeutic tool for inhibiting tumor progression and metastasis. This research aimed to study the effects of miR-127-3p-enriched EVs (EV-miR-127-3p) on CC and underlying mechanisms. MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were performed to determine the miR-127-3p and integrin subunit alpha-6 (ITGA6) expression levels. The interaction between miR-127-3p and ITGA6 was confirmed by a dual-luciferase reporter assay. Human umbilical cord mesenchymal stem cells (hUCMSCs) were identified using flow cytometry and multilineage differentiation. Uptake of labeled EVs was demonstrated using immunofluorescence staining and flow cytometry assays. EV-miR-127-3p were isolated from the culture medium of hUCMSCs and co-cultured with JEG-3 or JAR cells to evaluate their effects on cell proliferation, invasion, migration, and apoptosis, using the cell counting kit-8, Transwell, and flow cytometry assays. Epithelial-mesenchymal transition (EMT) and the transforming growth factor (TGF)-β1/Smad pathway were investigated using qRT-PCR and western blotting. ResultsThe expression of miR-127-3p was downregulated, while that of ITGA6 was upregulated in CC cell lines. ITGA6 was identified as a target gene of miR-127-3p. EV-miR-127-3p could inhibit the proliferation, invasion, migration, and promote the apoptosis of CC cells. We observed that EV-miR-127-3p suppressed EMT of CC cells by targeting ITGA6. In addition, the knockdown of ITGA6 inhibited the TGF-β1/Smad pathway and reversed the EMT-promoting effect. ConclusionThese results indicate that EV-miR-127-3p from hUCMSCs exhibits anti-tumor effects by targeting ITGA6, which may be used as a novel therapeutic strategy for CC treatment.

The anti-cancer role of microRNA-143 in papillary thyroid carcinoma by targeting high mobility group AT-hook 2

ABSTRACT Papillary thyroid carcinoma (PTC), a common thyroid cancer (TC) subtype, rapidly increases in occurrence. MicroRNAs (miRNAs), which are non-coding small RNAs, have been demonstrated to play a role in cancer pathogenic mechanisms. Although miR-143 is involved in suppressing certain malignant tumor progression, its biological role is unknown in PTC. The present study found that miR-143 levels were strongly lower in PTC patient samples and cell lines, implying that miR-143 may play a biological role in PTC. Down-regulation of miR-143 resulted in the increased expression of HMGA2. Furthermore, HMGA2 was found to be a direct target of miR-143. A dual-luciferase assay confirmed a direct binding site for miR-143 was confirmed on HMGA2 using a dual-luciferase assay. Next, over-expression of miR-143 suppressed PTC cell growth as analyzed by MTT, clone formation, and Ki-67 immunofluorescence staining assays. miR-143 mimics transfection downregulated the expression of PCNA, CDK4, CDK1, and Cyclin E1. In addition, wound healing and trans-well assays revealed that miR-143 up-regulation inhibited PTC cells invasion and migration. Co-transfection of HMGA2 expression vector restored HMGA2 expression and rescued PTC cells proliferation capability in miR-143 mimics transfected PTC cells, indicating that miR-143 inhibited PTC cells proliferation via HMGA2. These observations were also obtained in xenografts experiments in nude mice. Altogether, our study shed light on miR-143ʹs anti-cancer biological functions in PTC progression through targeting HMGA2, suggesting that restoration of miR-143 could be a potential therapeutic approach for PTC treatment.

Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development.

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow-associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; Rosa26fs-tdTomato and Col10a1Cre; Rosa26fs-tdTomato) in combination with a PDGFRaH2B-GFP transgenic line, single-cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow-associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte-derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

Disruption of LTBP4 Inhibition-Induced TGFβ1 Activation Promoted Cell Proliferation and Metastasis in Skin Melanoma by Inhibiting the Activation of the Hippo-YAP1 Signaling Pathway.

Melanoma is a malignant tumor derived from melanocytes, which is the most fatal skin cancer. The present study aimed to explore and elucidate the candidate genes in melanoma and its underlying molecular mechanism. A total of 1,156 differentially expressed genes were obtained from the GSE46517 dataset of Gene Expression Omnibus database using the package “limma” in R. Based on two algorithms (LASSO and SVM-RFE), we obtained three candidate DEGs (LTBP4, CDHR1, and MARCKSL1). Among them, LTBP4 was identified as a diagnostic marker of melanoma (AUC = 0.985). Down-regulation of LTBP4 expression was identified in melanoma tissues and cells, which predicted poor prognosis of patients with melanoma. Cox analysis results discovered that LTBP4 with low expression was an independent prognostic factor for overall survival in patients with melanoma. LTBP4 inhibition reduced cell apoptosis and promoted cell proliferation and metastasis. These changes were correlated with the expression levels of caspase-3, Ki67 and E-cadherin. Further, as indicated by tumor formation study of nude mice, LTBP4 silencing improved the tumorigenic ability of melanoma cells. Knockdown of LTBP4 increased the percentage of active TGFβ1 secreted by melanoma cells. CTGF, Gyr61, and Birc5 expression levels were reduced, YAP1 phosphorylation was inhibited, and YAP1 was translocated from the cytoplasm to the nucleus in melanoma cells treated with TGF-β1. These effects were reversed by LTBP4 overexpression. As evidenced by immunofluorescent staining, Western blotting and luciferase reporter assay, LTBP4 overexpression activated the Hippo signaling pathway, which was characterized by the increased nuclear-cytoplasmic translocation of YAP1 and the enhanced phosphorylation of YAP1, MST1, and MOB1. In addition, the effects of LTBP4 overexpression on inhibiting CTGF, Cyr61 and Birc5 expression, promoting the apoptosis, and inhibiting the metastasis and proliferation of melanoma cells were reversed by the overexpression of YAP1 or MST1. In conclusion, the LTBP4-TGFβ1-Hippo-YAP1 axis is a critical pathway for the progression of skin melanoma.

Abstract P5-12-11: NSDHL knockdown decreases tightly cohesive tumorsphere formation and breast cancer stem cell population

Abstract Background: Cholesterol biosynthesis pathway is commonly elevated or dysregulated in cancer cells and high cholesterol levels are associated with cancer progression. NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an enzyme involved in cholesterol biosynthesis, has been implicated in maintaining cancer stem cell, but its role in regulating breast cancer stem cells (BCSC) remains unclear. This study aimed to uncover the molecular mechanisms by which NSDHL regulates BCSC properties.Methods: The knockdown of NSDHL was induced by an introduction of siRNA/shRNA into MCF-7 cells. A non-adherent 3D culture method based on polymer-X coating was applied for growing tumorspheres under serum-free condition. 3’ quant mRNA-sequencing, qRT-PCR, western blot, immunofluorescent staining, ELISA, flow cytometry and ALDEFLUOR assay were carried out. Immunodeficient NSG mice were used for orthotropic xenograft tumor models by injecting tumorspheres.Result: NSDHL knockdown reduced a tightly cohesive sphere formation. RNA-seq data revealed a significant upregulation of 253 DEGs and downregulation of 364 DEGs with >2-fold change in NSDHL knockdown tumorsphere. The KEGG pathway analysis showed that DEGs were mainly enriched in TGF-β signaling pathway and cell cycle. NSDHL knockdown reduced the population of BCSCs with CD44+/CD24- phenotype and high ALDH activity as well as luminal progenitors with CD49f+/EpCAM+ phenotype along with decrease in TGF-β 1 and 3 and GLI1 in tumorsphere. In orthotropic xenograft tumor models, NSDHL knockdown strongly suppressed the tumor initiation and growth.Conclusions: Our findings reveal that NSDHL has an important role in maintaining BCSC population and tumor initiating capacity, suggesting NSDHL as an attractive therapeutic target to eliminate BCSCs, and thus preventing breast cancer initiation and progression. Citation Format: So-Hyun Yoon, Hoe Suk Kim, Junhyuk Song, Sunkyu Kwon, Seungyeon Ryu, Moonjou Baek, Sangeun Lee, Han-Byoel Lee, Hyeong-Gon Moon, Dong-Young Noh, Sangyong Jon, Wonshik Han. NSDHL knockdown decreases tightly cohesive tumorsphere formation and breast cancer stem cell population [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-11.

Long non-coding RNA FAM83A antisense RNA 1 (lncRNA FAM83A-AS1) targets microRNA-141-3p to regulate lung adenocarcinoma cell proliferation, migration, invasion, and epithelial-mesenchymal transition progression

ABSTRACT The current paper investigates how long non-coding RNA (lncRNA) FAM83A antisense RNA 1 (lncRNA FAM83A-AS1) affected the epithelial-mesenchymal transformation (EMT), growth, invasion and migration of lung adenocarcinoma (LUAD) via targeting miRNA-141-3p. The GEPIA and ENCORI databases were used to analyze differences in lncRNA FAM83A-AS1 levels within LUAD samples. FAM83A-AS1 and miR-141-3p levels were assessed using qRT-PCR among 30 LUAD samples and surrounding normal tissues. In addition, we analyzed how FAM83A-AS1 affected proliferation, invasion, migration, and EMT processes of LUAD cells by targeting miR-141-3p through EdU, CCK-8 assay, scratch assay, transwell migration and invasion assay, immunofluorescence (IF) staining and WB assay. MicroRNAs targeting FAM83A-AS1 were screened using AnnoLnc2 and identified by RT-qPCR. Dual-luciferase assays were utilized to evaluate the connection between FAM83A-AS1 and miR-141-3p. FAM83A-AS1 expression was remarkably raised in lung cancer cells and tissue samples; however, miR-141-3p level markedly reduced relative to healthy samples. FAM83A-AS1 silencing suppressed EMT, growth, invasion and migration of LUAD cells. MiR-141-3p was the possible FAM83A-AS1 binding target negatively associated with FAM83A-AS1. The miR-141-3p inhibitor partly abolished the FAM83A-AS1 knockdown-induced inhibition on EMT, cell growth, invasion and migration in LUAD cells. In addition, miR-141-3p down-regulation abolished the inhibition of E-box-bound zinc finger protein 1 and 2 protein production following FAM83A-AS1 knockdown. According to our results, FAM83A-AS1/miR-141-3p axis plays an important role in LUAD occurrence and development. FAM83A-AS1 sponged miR-141-3p to down-regulate the level of the latter within LUAD and thereby encouraging LUAD development and suggesting a possible novel therapeutic approach for LUAD.

Qufeng Xuanbi Formula Ameliorates Airway Remodeling in Asthmatic Mice by Suppressing Airway Smooth Muscle Cell Proliferation through MEK/ERK Signaling Pathway.

Asthma is a common chronic respiratory disease. The Qufeng Xuanbi formula (QFXBF), a Chinese herbal decoction, has shown efficacy in the management of asthma. The purpose of this study was to investigate the potential therapeutic effects of QFXBF in the treatment of asthma both in vitro and in vivo. Platelet-derived growth factor (PDGF)-induced airway smooth muscle cell (ASMC) proliferation and MTT assays were used to explore the effects of QFXBF on the proliferation of ASMCs. Moreover, 40 female BALB/c mice were randomly divided into five groups: control group, ovalbumin (OVA) group, high QFXBF group, low QFXBF group, and dexamethasone (DEX) group (n = 8 per group). A mouse allergic asthma model was established using the intranasally administered OVA sensitization method. Morphological changes in the lung tissue were examined by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. Finally, the protein expression of alpha-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), phospho-mitogen-activated protein kinase (p-MEK1/2), mitogen-activated protein kinase (MEK1/2), phospho-extracellular signal-regulated kinases (p-ERK1/2), and extracellular signal-regulated kinases (ERK1/2) in ASMCs and lung tissue were determined by western blotting and immunofluorescent staining assays. PDGF significantly increased the viability of ASMCs. Compared with mice in the control group, the airway walls and airway smooth muscle of mice in the OVA group were thickened, and the number of inflammatory cells around the bronchus significantly increased. Moreover, the administration of QFXBF markedly inhibited the proliferation of ASMCs and alleviated the pathological changes induced by OVA. Furthermore, the protein expressions of p-ERK1/2, p-MEK1/2, PCNA, and α-SMA were significantly increased in OVA-treated mice and PDGF-treated ASMCs. Finally, treatment with QFXBF also significantly decreased the protein expression of p-ERK1/2, p-MEK1/2, α-SMA, and PCNA. QFXBF inhibited the proliferation of ASMCs by suppressing MEK/ERK signaling in PDGF-induced ASMCs and OVA-induced mice.

Anatomical Transcriptome Atlas of the Male Mouse Reproductive System During Aging.

The elderly males undergo degenerative fertility and testicular endocrine function that jeopardize the reproductive health and well-being. However, the mechanisms underlying reproductive aging are unclear. Here, we tried to address this by investigating the phenotypes and transcriptomes of seven regions of the male mouse reproductive tract: the testis, efferent ductules, initial segment, caput, corpus and cauda epididymidis, and vas deferens, in adult (3 months) and aged (21 months) mice. Quantitative PCR, immunohistochemistry, immunofluorescent staining, and enzyme-linked immunosorbent assay were performed for the analysis of gene expression in mice, human tissues, and semen samples. Aged male mice showed both systematic and reproductive changes, and remarkable histological changes were detected in the testis and proximal epididymis. Transcriptomes of the male reproductive tract were mapped, and a series of region-specific genes were identified and validated in mouse and/or human tissues, including Protamine 1 (Prm2), ADAM metallopeptidase domain 28 (Adam28), Ribonuclease A family member 13 (Rnase13), WAP four-disulfide core domain 13 (Wfdc13), and Wfdc9. Meanwhile, age-related transcriptome changes of different regions of the male reproductive tract were characterized. Notably, increased immune response was functionally related to the male reproductive aging, especially the T cell activation. An immune response-associated factor, phospholipase A2 group IID (Pla2g2d), was identified as a potential biomarker for reproductive aging in mice. And the PLA2G2D level in human seminal plasma surged at approximately 35 years of age. Furthermore, we highlighted Protein tyrosine phosphatase receptor type C (Ptprc), Lymphocyte protein tyrosine kinase (Lck), Microtubule associated protein tau (Mapt), and Interferon induced protein with tetratricopeptide repeats 3 (Ifit3) as critical molecules in the aging of initial segment, caput, caput, and cauda epididymidis, respectively. This study provides an RNA-seq resource for the male reproductive system during aging in mice, and is expected to improve our understanding of male reproductive aging and infertility.

TLR13 contributes to skeletal muscle atrophy by increasing insulin resistance in chronic kidney disease.

ObjectivesInsulin resistance in chronic kidney disease (CKD) stimulates muscle wasting, but the molecular processes behind the resistance are undetermined. However, inflammation in skeletal muscle is implicated in the pathogenesis of insulin resistance and cachexia. Toll‐like receptors (TLRs) are known to regulate local innate immune responses, and microarray data have shown that Tlr13 is upregulated in the muscles of mice with CKD, but the relevance is unknown.Materials and MethodsWe performed in vitro experiments in C2C12 myotubes and constructed a CKD murine model using subtotal nephrectomy to conduct experiments in vivo.Results Tlr13 expression was stimulated in C2C12 myotubes treated with uremic serum. The expression of Tlr13 was also upregulated in the tibialis anterior muscles of mice with CKD. Tlr13 knockdown with siRNAs in skeletal muscle cells decreased insulin resistance despite the inclusion of uremic serum. This led to increased levels of p‐AKT and suppression of protein degradation. Using immunofluorescence staining and coimmunoprecipitation assay, we found that TLR13 recruits IRF3, which activates Irf3 expression, resulting in decreased AKT activity. Moreover, insulin resistance and proteolysis are re‐induced by Irf3 overexpression under Tlr13 deletion.ConclusionsOur results indicate that TLR13 is involved in CKD‐mediated insulin resistance in muscle. In catabolic conditions where insulin signaling is impaired, targeting TLR13 may improve insulin sensitivity and prevent muscle atrophy.

Human placental mesenchymal stromal cells are ciliated and their ciliation is compromised in preeclampsia

BackgroundThe development of the human placenta is tightly coordinated by a multitude of placental cell types, including human chorionic villi mesenchymal stromal cells (hCV-MSCs). Defective hCV-MSCs have been reported in preeclampsia (PE), a gestational hypertensive disease characterized by maternal endothelial dysfunction and systemic inflammation. Our goal was to determine whether hCV-MSCs are ciliated and whether altered ciliation is responsible for defective hCV-MSCs in preeclamptic placentas, as the primary cilium is a hub for signal transduction, which is important for various cellular activities.MethodsIn the present work, we collected placental tissues from different gestational stages and we isolated hCV-MSCs from 1st trimester, term control, and preeclamptic placentas. We studied their ciliation, functionality, and impact on trophoblastic cell lines and organoids formed from human trophoblast stem cells (hTSCs) and from the trophoblastic cell line JEG-3 with various cellular and molecular methods, including immunofluorescence staining, gene analysis, spheroid/organoid formation, motility, and cellular network formation assay. The statistical evaluation was performed using a Student’s t test (two-tailed and paired or homoscedastic) or an unpaired Mann–Whitney U test (two-tailed).ResultsThe results show that primary cilia appeared abundantly in normal hCV-MSCs, especially in the early development of the placenta. Compared to control hCV-MSCs, the primary cilia were truncated, and there were fewer ciliated hCV-MSCs derived from preeclamptic placentas with impaired hedgehog signaling. Primary cilia are necessary for hCV-MSCs’ proper signal transduction, motility, homing, and differentiation, which are impaired in preeclamptic hCV-MSCs. Moreover, hCV-MSCs derived from preeclamptic placentas are significantly less capable of promoting growth and differentiation of placental organoids, as well as cellular network formation.ConclusionsThese data suggest that the primary cilium is required for the functionality of hCV-MSCs and primary cilia are impaired in hCV-MSCs from preeclamptic placentas.

Brain-Type Glycogen Phosphorylase Is Crucial for Astrocytic Glycogen Accumulation in Chronic Social Defeat Stress-Induced Depression in Mice.

Astrocytic glycogen plays an important role in brain energy metabolism. However, the contribution of glycogen metabolism to stress-induced depression remains unclear. Chronic social defeat stress was used to induce depression-like behaviors in mice, assessed with behavioral tests. Glycogen concentration in the medial prefrontal cortex (mPFC) and the expression of key enzymes of the glycogen metabolism were investigated using Western blots, immunofluorescent staining, electron microscopy, and biochemical assays. Stereotaxic surgery and viral-mediated gene transfer were applied to knockdown or overexpress brain-type glycogen phosphorylase (PYGB) in the mPFC. The glycogen content increased in the mPFC after stress. Glycogenolytic dysfunction due to inactivation of PYGB was responsible for glycogen accumulation. Behavioral tests on astrocyte-specific PYGB overexpression mice showed that augmenting astrocytic PYGB reduces susceptibility to depression when compared with stress-susceptible mice. Conversely, PYGB genetic down-regulation in the mPFC was sufficient to induce glycogen accumulation and depression-like behaviors. Furthermore, PYGB overexpression in the mPFC decreases susceptibility to depression, at least partially by rescuing glycogen phosphorylase activity to maintain glycogen metabolism homeostasis during stress. These findings indicate that (1) glycogen accumulation occurs in mice following stress and (2) glycogenolysis reprogramming leads to glycogen accumulation in astrocytes and PYGB contributes to stress-induced depression-like behaviors. Pharmacological tools acting on glycogenolysis might constitute a promising therapy for depression.