Human bone marrow mesenchymal stem cells (HBMSCs) can promote new bone formation. Previous studies have proven the ability of long non-coding RNAs (lncRNAs) to modulate the osteogenic differentiation of mesenchymal stem cells. However, the molecular mechanism modulated by lncRNAs in affecting the osteogenic differentiation of HBMSCs remains largely unknown. Thus, this study aims to reveal the role of lncRNA ubiquitin-specific peptidase 2 antisense RNA 1 (USP2-AS1) in regulating the osteogenic differentiation of HBMSCs and investigate its regulatory mechanism. Through bioinformatics analysis and RT-qPCR, we confirmed that USP2-AS1 expression was increased in HBMSCs after culturing in osteogenic differentiation medium (OM-HBMSCs). Moreover, we uncovered that knockdown of USP2-AS1 inhibited the osteogenic differentiation of HBMSCs. Further exploration indicated that USP2-AS1 positively regulated the expression of its nearby gene USP2. Mechanistically, USP2-AS1 recruited lysine demethylase 3A (KDM3A) to stabilize ETS proto-oncogene 1 (ETS1), transcription factor that transcriptionally activated USP2. Additionally, USP2-induced Wnt/β-catenin signalling pathway activation via deubiquitination of β-catenin protein. In summary, our study proved that lncRNA USP2-AS1 facilitates the osteogenic differentiation of HBMSCs by targeting KDM3A/ETS1/USP2 axis to activate the Wnt/β-catenin signalling pathway.

Graphene quantum dots (GQDs), a type of carbon-based nanomaterial, have remarkable biological, physical, and chemical properties. This study investigated the biological mechanisms of the proliferation and osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) induced by GQDs in an inflammatory microenvironment.
PDLSCs were cultured in osteogenic-induced medium with various concentrations of GQDs in standard medium or medium mimicking a proinflammatory environment. The effects of GQDs on the proliferation and osteogenic differentiation activity of PDLSCs were tested by CCK-8 assay, Alizarin Red S staining, and qRT‒PCR. In addition, Wnt/β-catenin signalling pathway-related gene expression was measured by qRT‒PCR.
Compared with the control group, the mRNA expression levels of ALP, RUNX2, and OCN and the number of mineralized nodules were all increased in PDLSCs after treatment with GQDs. Moreover, during the osteogenic differentiation of PDLSCs, the expression levels of LRP6 and β-catenin, which are Wnt/β-catenin signalling pathway-related genes, were upregulated.
In the inflammatory microenvironment, GQDs might promote the osteogenic differentiation ability of PDLSCs by activating the Wnt/β-catenin signalling pathway.

Chronic kidney disease (CKD) is a major worldwide public health problem. The increase in the number of patients with CKD and end-stage kidney disease requesting renal dialysis or transplantation will progress to epidemic proportions in the next several decades. Although blocking the renin-angiotensin system (RAS) has been used as a first-line standard therapy in patients with hypertension and CKD, patients still progress towards end-stage kidney disease, which might be closely associated with compensatory renin expression subsequent to RAS blockade through a homeostatic mechanism. The Wnt/β-catenin signalling pathway is the master upstream regulator that controls multiple intrarenal RAS genes. As Wnt/β-catenin regulates multiple RAS genes, we inferred that this pathway might also be implicated in blood pressure control. Therefore, discovering new medications to synchronously target multiple RAS genes is necessary and essential for the effective treatment of patients with CKD. We hypothesized that Shenkang injection (SKI), which is widely used to treat CKD patients, might ameliorate CKD by inhibiting the activation of multiple RAS genes via the Wnt/β-catenin signalling pathway. To test this hypothesis, we used adenine-induced CKD rats and angiotensin II (AngII)-induced HK-2 and NRK-49F cells. Treatment with SKI inhibited renal function decline, hypertension and renal fibrosis. Mechanistically, SKI abrogated the increased protein expression of multiple RAS elements, including angiotensin-converting enzyme and angiotensin II type 1 receptor, as well as Wnt1, β-catenin and downstream target genes, including Snail1, Twist, matrix metalloproteinase-7, plasminogen activator inhibitor-1 and fibroblast-specific protein 1, in adenine-induced rats, which was verified in AngII-induced HK-2 and NRK-49F cells. Similarly, our results further indicated that treatment with rhein isolated from SKI attenuated renal function decline and epithelial-to-mesenchymal transition and repressed RAS activation and the hyperactive Wnt/β-catenin signalling pathway in both adenine-induced rats and AngII-induced HK-2 and NRK-49F cells. This study first revealed that SKI repressed epithelial-to-mesenchymal transition by synchronously targeting multiple RAS elements by blocking the hyperactive Wnt/β-catenin signalling pathway.

UNASSIGNED: Bone defects remain an unsolved clinical problem due to the lack of effective osteogenic induction protocols. Nanomaterials play an important role in bone defect repair by stimulating osteogenesis. However, constructing an effective bioactive nanomaterial remains a substantial challenge.
UNASSIGNED: In this study, mesoporous silica nanoparticles (MSNs) were prepared and used as nanocarriers for basic fibroblast growth factor (bFGF). The characteristics and biological properties of the synthetic bFGF@MSNs were tested. The osteogenic effects of the particles on the behavior of MC3T3-E1 cells were investigated in vitro. In addition, the differentially expressed genes during induction of osteogenesis were analyzed by transcriptomic sequencing. Radiological and histological observations were carried out to determine bone regeneration capability in a distal femur defect model.
UNASSIGNED: Achieving bFGF sustained release, bFGF@MSNs had uniform spherical morphology and good biocompatibility. In vitro osteogenesis induction experiments showed that bFGF@MSNs exhibited excellent osteogenesis performance, with upregulation of osteogenesis-related genes (RUNX2, OCN, Osterix, ALP). Transcriptomic sequencing revealed that the Wnt/β-catenin signalling pathway could be activated in regulation of biological processes. In vivo, bone defect repair experiments showed enhanced bone regeneration, as indicated by radiological and histological analysis, after the application of bFGF@MSNs.
UNASSIGNED: bFGF@MSNs can promote bone regeneration by activating the Wnt/β-catenin signalling pathway. These particles are expected to become a potential therapeutic bioactive material for clinical application in repairing bone defects in the future.

OBJECTIVE: The epithelial-mesenchymal transition (EMT) pathway can mediate tumour migration, and the occurrence of EMT is closely related to the Wnt/β-catenin signalling pathway. The purpose of this paper was to study the effect of Lactobacillus fermentum ZS09 (L. fermentum ZS09) on the EMT pathway in mouse with azoxymethane/dextran sulfate sodium salt (AOM/DSS) induced colon cancer and the potential underlying mechanism.
METHODS: In this study, a mouse colon cancer model was established through intraperitoneal injection of 10 mg/kg azoxymethane (AOM) and three cycles of 2.5% dextran sulfate sodium salt (DSS) in the drinking water. H&E staining, enzyme-linked immunosorbent assay (ELISA), real-time fluorescent quantitative PCR (RT-qPCR) and Western blotting (WB) were used to study the antitumour mechanisms of L. fermentum ZS09 through the EMT pathway.
RESULTS: The results of this study showed that compared with the model group, the high-dose L. fermentum ZS09 intervention group exhibited decreased serum levels of MMP-9, TNF-α, IL-6R, Ang-2 and VEGFR-2 and increased contents of DKK1 (P<0.05). The expression of Wnt/β-catenin signalling pathway-related genes (Dv1, GSK-3β, β-catenin, c-myc, cyclinD1, Vim, and MMP-9) was significantly reduced, and the gene expression levels of APC, CDH1, and Axin were increased. The levels of related proteins (β-catenin, N-cadherin, and VEGF) were downregulated, and the levels of p-β-catenin and E-cadherin were upregulated.
CONCLUSIONS: The results indicate that L. fermentum ZS09 could inhibit EMT and angiogenesis pathways by inhibiting the Wnt/β-catenin signalling pathway, which could inhibit tumour metastasis.

We examined the mechanism by which lithium chloride (LiCl) attenuates the impaired learning capability and memory function of dual-transgenic APP/PS1 mice. Six- or 12-month-old APP/PS1 and wild-type (WT) mice were randomized into four groups, namely WT, WT+Li (100 mg LiCl/kg body weight, gavage once daily), APP/PS1 and APP/PS1+Li. Primary rat hippocampal neurons were exposed to β-amyloid peptide oligomers (AβOs), LiCl and/or XAV939 (inhibitor of Wnt/β-catenin) or transfected with small interfering RNA against the β-catenin gene. In the cerebral zone of APP/PS1 mice, the level of Aβ was increased and those of α7 nicotinic acetylcholine receptors (nAChR), phosphor-GSK3β (ser9), β-catenin and cyclin D1 (protein and/or mRNA levels) reduced. Two-month treatment with LiCl at ages of 4 or 10 months weakened all of these effects. Similar expression variations were observed for these proteins in primary neurons exposed to AβOs, and these effects were attenuated by LiCl and aggravated by XAV939. Inhibition of β-catenin expression lowered the level of α7 nAChR protein in these cells. LiCl attenuates the impaired learning capability and memory function of APP/PS1 mice via a mechanism that might involve elevation of the level of α7 nAChR as a result of altered Wnt/β-catenin signalling.

BACKGROUND: As a prevalent type of cryptogenic fibrotic disease with high mortality, idiopathic pulmonary fibrosis (IPF) still lacks effective therapeutic drugs. The compounds extracted from buds and flowers of Chrysanthemum indicum Linné with supercritical-carbon dioxide fluid (CISCFE) has been confirmed to have antioxidant, anti-inflammatory, and lung-protective effects. This paper aimed to clarify whether CISCFE could treat IPF induced by bleomycin (BLM) and elucidate the related mechanisms.
METHODS: Rats (Sprague-Dawley, male) were separated into the following groups: normal, model, pirfenidone (50 mg/kg), CISCFE-L, -M, and -H (240, 360, and 480 mg/kg/d, i.g., respectively, for 4 weeks). Rats were given BLM (5 mg/kg) via intratracheal installation to establish the IPF model. A549 and MRC-5 cells were stimulated by Wnt-1 to establish a cell model and then treated with CISCFE. Haematoxylin-eosin (H&E) and Masson staining were employed to observe lesions in the lung tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) were performed to observe changes in genes and proteins connected with the Wnt/β-catenin pathway.
RESULTS: CISCFE inhibited the proliferation of MRC-5 cells (IC50: 2.723 ± 0.488 μg/mL) and A549 cells (IC50: 2.235 ± 0.229 μg/mL). In rats, A549 cells, and MRC-5 cells, BLM and Wnt-1 obviously induced the protein expression of α-smooth muscle actin (α-SMA), vimentin, type I collagen (collagen-I), and Nu-β-catenin. The mRNA levels of matrix metalloproteinase-3 (MMP-3) and - 9 (MMP-9), two enzymes that degrade and reshape the extracellular matrix (ECM) were also increased while those of tissue inhibitor of metalloproteinase 1 (TIMP-1) were decreased. However, CISCFE reversed the effects of BLM and Wnt-1 on the expression pattern of these proteins and genes.
CONCLUSIONS: These findings showed that CISCFE could inhibit IPF development by activating the Wnt/β-catenin pathway and may serve as a treatment for IPF after further investigation.

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/β-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of β-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/β-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the β-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/β-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.

As a secretory cell transcription factor, muscle intestine stomach expression 1 (Mist1) is associated with serous secretory cell development and gastric chief cell maturation. Here, we focus on the function of Mist1 in gastric adenocarcinoma carcinogenesis. Based on clinical data and a mouse model of gastric cancer, we found that Mist1 expression was reduced in gastric cancer. Then, we overexpressed Mist1 using a lentivirus system and found that overexpression of Mist1 could inhibit gastric cancer cell proliferation, migration and invasion in vitro. Additionally, in vivo, we assessed the function of Mist1 in a gastric cancer xenograft model and distant pulmonary metastasis model. Overexpression of Mist1 decreased tumour growth and distant metastasis in vivo, suggesting that Mist1 acts as a tumour suppressor in gastric carcinogenesis. Furthermore, Mist1 overexpression inhibited epithelial-mesenchymal transition (EMT) in gastric cancer by suppressing β-catenin transcription activity and then the Wingless and INT-1 (Wnt)/β-catenin signalling pathway, which could be reversed by a Wnt/β-catenin-specific agonist. In conclusion, this study indicated that overexpression of Mist1 could reverse EMT in gastric carcinogenesis by inhibiting the Wnt/β-catenin signalling pathway and that Mist1 might be a novel marker for early gastric cancer screening.

Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β-catenin signalling and assess its potential to activate lung epithelial cells and repair.
We screened 1216 human-approved compounds for Wnt/β-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model.
The primary screen identified 16 compounds that significantly induced Wnt/β-catenin-dependent luciferase activity. Selected compounds activated Wnt/β-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β-catenin inhibition, confirming the Wnt/β-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signalling in emphysema.
Using a drug screen based on Wnt/β-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.