Peripheral nerve-derived Sema3A promotes osteogenic differentiation of mesenchymal stem cells through the Wnt/ß-catenin/Nrp1 positive feedback loop.

Sensory nerves play a crucial role in maintaining bone homeostasis by releasing Semaphorin 3A (Sema3A). However, the specific mechanism of Sema3A in regulation of bone marrow mesenchymal stem cells (BMMSCs) during bone remodelling remains unclear. The tibial denervation model was used and the denervated tibia exhibited significantly lower mass as compared to sham operated bones. In vitro, BMMSCs cocultured with dorsal root ganglion cells (DRGs) or stimulated by Sema3A could promote osteogenic differentiation through the Wnt/ß-catenin/Nrp1 positive feedback loop, and the enhancement of osteogenic activity could be inhibited by SM345431 (Sema3A-specific inhibitor). In addition, Sema3A-stimulated BMMSCs or intravenous injection of Sema3A could promote new bone formation in vivo. To sum up, the coregulation of bone remodelling is due to the ageing of BMMSCs and increased osteoclast activity. Furthermore, the sensory neurotransmitter Sema3A promotes osteogenic differentiation of BMMSCs via Wnt/ß-catenin/Nrp1 positive feedback loop, thus promoting osteogenesis in vivo and in vitro.

The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation.

Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/ß-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/ß-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.

Wnt, glucocorticoid and cellular prion protein cooperate to drive a mesenchymal phenotype with poor prognosis in colon cancer.

BACKGROUND: The mesenchymal subtype of colorectal cancer (CRC), associated with poor prognosis, is characterized by abundant expression of the cellular prion protein PrPC, which represents a candidate therapeutic target. How PrPC is induced in CRC remains elusive. This study aims to elucidate the signaling pathways governing PrPC expression and to shed light on the gene regulatory networks linked to PrPC. METHODS: We performed in silico analyses on diverse datasets of in vitro, ex vivo and in vivo models of mouse CRC and patient cohorts. We mined ChIPseq studies and performed promoter analysis. CRC cell lines were manipulated through genetic and pharmacological approaches. We created mice combining conditional inactivation of Apc in intestinal epithelial cells and overexpression of the human prion protein gene PRNP. Bio-informatic analyses were carried out in two randomized control trials totalizing over 3000 CRC patients. RESULTS: In silico analyses combined with cell-based assays identified the Wnt-ß-catenin and glucocorticoid pathways as upstream regulators of PRNP expression, with subtle differences between mouse and human. We uncover multiple feedback loops between PrPC and these two pathways, which translate into an aggravation of CRC pathogenesis in mouse. In stage III CRC patients, the signature defined by PRNP-CTNNB1-NR3C1, encoding PrPC, ß-catenin and the glucocorticoid receptor respectively, is overrepresented in the poor-prognosis, mesenchymal subtype and associates with reduced time to recurrence. CONCLUSIONS: An unleashed PrPC-dependent vicious circle is pathognomonic of poor prognosis, mesenchymal CRC. Patients from this aggressive subtype of CRC may benefit from therapies targeting the PRNP-CTNNB1-NR3C1 axis.

A microfluidic model to study the effects of arrhythmic flows on endothelial cells.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and an important contributor to morbidity and mortality. Endothelial dysfunction has been postulated to be an important contributing factor in cardiovascular events in patients with AF. However, how vascular endothelial cells respond to arrhythmic flow is not fully understood, mainly due to the limitation of current in vitro systems to mimic arrhythmic flow conditions. To address this limitation, we developed a microfluidic system to study the effect of arrhythmic flow on the mechanobiology of human aortic endothelial cells (HAECs). The system utilises a computer-controlled piezoelectric pump for generating arrhythmic flow with a unique ability to control the variability in both the frequency and amplitude of pulse waves. The flow rate is modulated to reflect physiological or pathophysiological shear stress levels on endothelial cells. This enabled us to systematically dissect the importance of variability in the frequency and amplitude of pulses and shear stress level on endothelial cell mechanobiology. Our results indicated that arrhythmic flow at physiological shear stress level promotes endothelial cell spreading and reduces the plasma membrane-to-cytoplasmic distribution of ß-catenin. In contrast, arrhythmic flow at low and atherogenic shear stress levels does not promote endothelial cell spreading or redistribution of ß-catenin. Interestingly, under both shear stress levels, arrhythmic flow induces inflammation by promoting monocyte adhesion via an increase in ICAM-1 expression. Collectively, our microfluidic system provides opportunities to study the effect of arrhythmic flows on vascular endothelial mechanobiology in a systematic and reproducible manner.

Endothelial DR6 in blood-brain barrier malfunction in Alzheimer's disease.

The impairment of the blood-brain barrier (BBB) has been increasingly recognised as a critical element in the early pathogenesis of Alzheimer's disease (AD), prompting a focus on brain endothelial cells (BECs), which serve as the primary constituents of the BBB. Death receptor 6 (DR6) is highly expressed in brain vasculature and acts downstream of the Wnt/ß-catenin pathway to promote BBB formation during development. Here, we found that brain endothelial DR6 levels were significantly reduced in a murine model of AD (APPswe/PS1dE9 mice) at the onset of amyloid-ß (Aß) accumulation. Toxic Aß25-35 oligomer treatment recapitulated the reduced DR6 in cultured BECs. We further showed that suppressing DR6 resulted in BBB malfunction in the presence of Aß25-35 oligomers. In contrast, overexpressing DR6 increased the level of BBB functional proteins through the activation of the Wnt/ß-catenin and JNK pathways. More importantly, DR6 overexpression in BECs was sufficient to rescue BBB dysfunction in vitro. In conclusion, our findings provide new insight into the role of endothelial DR6 in AD pathogenesis, highlighting its potential as a therapeutic target to tackle BBB dysfunction in early-stage AD progression.

Colorectal cancer cells with stably expressed SIRT3 demonstrate proliferating retardation by Wnt/ß-catenin cascade inactivation.

Colorectal cancer (CRC) is a typical and lethal digestive system malignancy. In this study, we investigated the effect of sirtuin 3 (SIRT3) expression, a fidelity mitochondrial protein, on the proliferation of CRC cells and the mechanisms involved. Using the University of Alabama at Birmingham Cancer Data Analysis Portal database and the Clinical Proteomic Tumour Analysis Consortium database, we discovered that low expression of SIRT3 in CRC was a negative factor for survival prognosis (P < .05). Meanwhile, SIRT3 expression was correlated with distant metastasis and tumour, node, metastasis stage of CRC patients (P < .05). Subsequently, we observed that CRC cells with stable SIRT3 expression exhibited a significant decrease in proliferative capacities both in vitro and in vivo, compared to their counterparts (P < .05). Further investigation using western blot, immunoprecipitation and TOPflash/FOPflash assay showed the mechanism of growth retardation of these cells was highly associated with the degradation of ß-catenin in cytosol, and the localization of ß-catenin/α-catenin complex in the nucleus. In conclusion, our findings suggest that the inhibition of CRC cell proliferation by SIRT3 is closely associated with the inactivation of the Wnt/ß-catenin signalling pathway.

[Banxia Xiexin Decoction inhibiting colitis-associated colorectal cancer infected with Fusobacterium nucleatum by regulating Wnt/ß-catenin pathway].

This paper investigates the intervention effect and mechanism of Banxia Xiexin Decoction(BXD) on colitis-associated colorectal cancer(CAC) infected with Fusobacterium nucleatum(Fn). C57BL/6 mice were randomly divided into a control group, Fn group, CAC group [azoxymethane(AOM)/dextran sulfate sodium salt(DSS)](AOM/DSS), model group, and BXD group. Except for the control and AOM/DSS groups, the mice in the other groups were orally administered with Fn suspension twice a week. The AOM/DSS group, model group, and BXD group were also injected with a single dose of 10 mg·kg~(-1) AOM combined with three cycles of 2.5% DSS taken intragastrically. The BXD group received oral administration of BXD starting from the second cycle until the end of the experiment. The general condition and weight changes of the mice were monitored during the experiment, and the disease activity index(DAI) was calculated. At the end of the experiment, the colon length and weight of the mice in each group were compared. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-2, IL-4, and IL-6 inflammatory factors in the serum. Immunohistochemistry(IHC) was used to detect the expression of Ki67, E-cadherin, and ß-catenin in the colon tissue. Western blot was used to detect the protein content of Wnt3a, ß-catenin, E-cadherin, annexin A1, cyclin D1, and glycogen synthase kinase-3ß(GSK-3ß) in the colon tissue. The results showed that compared with the control group, the Fn group had no significant lesions. The mice in the AOM/DSS group and model group had decreased body weight, increased DAI scores, significantly increased colon weight, and significantly shortened colon length, with more significant lesions in the model group. At the same time, the colon histology of the model group showed more severe adenomas, inflammatory infiltration, and cellular dysplasia. The levels of IL-4 and IL-6 in the serum were significantly increased, while the IL-2 content was significantly decreased. The IHC results showed low expression of E-cadherin and high expression of Ki67 and ß-catenin in the model group, with a decreased protein content of E-cadherin and GSK-3ß and an increased protein content of Wnt3a, ß-catenin, annexin A1, and cyclin D1. After intervention with BXD, the body weight of the mice increased; the DAI score decreased; the colon length increased, and the tumor decreased. The histopathology showed reduced tumor proliferation and reduced inflammatory infiltration. The levels of IL-6 and IL-4 in the serum were significantly decreased, while the IL-2 content was increased. Meanwhile, the expression of E-cadherin was upregulated, and that of Ki67 and ß-catenin was downregulated. The protein content of E-cadherin and GSK-3ß increased, while that of Wnt3a, ß-catenin, annexin A1, and cyclin D1 decreased. In conclusion, BXD can inhibit CAC infected with Fn, and its potential mechanism may be related to the inhibition of Fn binding to E-cadherin, the decrease in annexin A1 protein level, and the regulation of the Wnt/ß-catenin pathway.

The multifaceted role of Wnt canonical signalling in neurogenesis, neuroinflammation, and hyperexcitability in mesial temporal lobe epilepsy.

Epilepsy is a neurological disorder characterised by unprovoked, repetitive seizures caused by abnormal neuronal firing. The Wnt/ß-Catenin signalling pathway is involved in seizure-induced neurogenesis, aberrant neurogenesis, neuroinflammation, and hyperexcitability associated with epileptic disorder. Wnt/ß-Catenin signalling is crucial for early brain development processes including neuronal patterning, synapse formation, and N-methyl-d-aspartate receptor (NMDAR) regulation. Disruption of molecular networks such as Wnt/ß-catenin signalling in epilepsy could offer encouraging anti-epileptogenic targets. So, with a better understanding of the canonical Wnt/-Catenin pathway, we highlight in this review the important elements of Wnt/-Catenin signalling specifically in Mesial Temporal Lobe Epilepsy (MTLE) for potential therapeutic targets.

Exploring the effect of 6-BIO and sulindac in modulation of Wnt/ß-catenin signaling pathway in chronic phase of temporal lobe epilepsy.

The prospective involvement of the Wnt/ß-catenin signaling pathway in epilepsy, with the proposed therapeutic uses of its modulators, has been suggested; however, comprehensive knowledge in this regard is currently limited. Despite postulations about the pathway's significance and treatment potential, a systematic investigation is required to better understand its implications in chronic epilepsy. We investigated the role of key proteins like ß-catenin, GSK-3ß, and their modulators sulindac and 6-BIO, in Wnt/ß-catenin pathway during chronic phase of temporal lobe epilepsy. We also evaluated the role of modulators in seizure score, seizure frequency and neurobehavioral parameters in temporal lobe epilepsy. We developed status epilepticus model using lithium-pilocarpine. The assessment of neurobehavioral parameters was done followed by histopathological examination and immunohistochemistry staining of hippocampus as well as RT-qPCR and western blotting to analyse gene and protein expression. In SE rats, seizure score and frequency were significantly high compared to control rats, with notable changes in neurobehavioral parameters and neuronal damage observed in hippocampus. Our study also revealed a substantial upregulation of the Wnt/ß-catenin pathway in chronic epilepsy, as evidenced by gene and protein expression studies. Sulindac emerged as a potent modulator, reducing seizure score, frequency, neuronal damage, apoptosis, and downregulating the Wnt/ß-catenin pathway when compared to 6-BIO. Our findings emphasize the potential of GSK-3ß and ß-catenin as promising drug targets for chronic temporal lobe epilepsy, offering valuable treatment options for chronic epilepsy. The promising outcomes with sulindac encourages further exploration in clinical trials to assess its therapeutic potential.

ZNF554 Inhibits Endometrial Cancer Progression via Regulating RBM5 and Inactivating WNT/ß-Catenin Signaling Pathway.

OBJECTIVE: Uterine corpus endometrial carcinoma (UCEC), a kind of gynecologic malignancy, poses a significant risk to women's health. The precise mechanism underlying the development of UCEC remains elusive. Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein superfamily, was reported to be dysregulated in various illnesses, including malignant tumors. This study aimed to examine the involvement of ZNF554 in the development of UCEC. METHODS: The expression of ZNF554 in UCEC tissues and cell lines were examined by qRT-PCR and Western blot assay. Cells with stably overexpressed or knocked-down ZNF554 were established through lentivirus infection. CCK-8, wound healing, and Transwell invasion assays were employed to assess cell proliferation, migration, and invasion. Propidium iodide (PI) staining combined with fluorescence-activated cell sorting (FACS) flow cytometer was utilized to detect cell cycle distribution. qRT-PCR and Western blotting were conducted to examine relative mRNA and protein levels. Chromatin immunoprecipitation assay and luciferase reporter assay were used to explore the regulatory role of ZNF554 in RNA binding motif 5 (RBM5). RESULTS: The expression of ZNF554 was found to be reduced in both UCEC samples and cell lines. Decreased expression of ZNF554 was associated with higher tumor stage, decreased overall survival, and reduced disease-free survival in UCEC. ZNF554 overexpression suppressed cell proliferation, migration, and invasion, while also inducing cell cycle arrest. In contrast, a decrease in ZNF554 expression resulted in the opposite effect. Mechanistically, ZNF554 transcriptionally regulated RBM5, leading to the deactivation of the Wingless (WNT)/ß-catenin signaling pathway. Moreover, the findings from rescue studies demonstrated that the inhibition of RBM5 negated the impact of ZNF554 overexpression on ß-catenin and p-glycogen synthase kinase-3ß (p-GSK-3ß). Similarly, the deliberate activation of RBM5 reduced the increase in ß-catenin and p-GSK-3ß caused by the suppression of ZNF554. In vitro experiments showed that ZNF554 overexpression-induced decreases in cell proliferation and migration were counteracted by RBM5 knockdown. Additionally, when RBM5 was overexpressed, it hindered the improvements in cell proliferation and migration caused by reducing the ZNF554 levels. CONCLUSION: ZNF554 functions as a tumor suppressor in UCEC. Furthermore, ZNF554 regulates UCEC progression through the RBM5/WNT/ß-catenin signaling pathway. ZNF554 shows a promise as both a prognostic biomarker and a therapeutic target for UCEC.

Dysregulation of Wnt/ß-catenin signaling contributes to intestinal inflammation through regulation of group 3 innate lymphoid cells.

RORγt+ group 3 innate lymphoid cells (ILC3s) are essential for intestinal homeostasis. Dysregulation of ILC3s has been found in the gut of patients with inflammatory bowel disease and colorectal cancer, yet the specific mechanisms still require more investigation. Here we observe increased ß-catenin in intestinal ILC3s from inflammatory bowel disease and colon cancer patients compared with healthy donors. In contrast to promoting RORγt expression in T cells, activation of Wnt/ß-catenin signaling in ILC3s suppresses RORγt expression, inhibits its proliferation and function, and leads to a deficiency of ILC3s and subsequent intestinal inflammation in mice. Activated ß-catenin and its interacting transcription factor, TCF-1, cannot directly suppress RORγt expression, but rather alters global chromatin accessibility and inhibits JunB expression, which is essential for RORγt expression in ILC3s. Together, our findings suggest that dysregulated Wnt/ß-catenin signaling impairs intestinal ILC3s through TCF-1/JunB/RORγt regulation, further disrupting intestinal homeostasis, and promoting inflammation and cancer.

STUB1 is acetylated by KAT5 and alleviates myocardial ischemia-reperfusion injury through LATS2-YAP-ß-catenin axis.

Myocardial ischemia-reperfusion injury (MIRI) is involved in the pathogenesis of multiple cardiovascular diseases. This study elucidated the biological function of lysine acetyltransferase 5 (KAT5) in cardiomyocyte pyroptosis during MIRI. Oxygen-glucose deprivation/reoxygenation and left anterior descending coronary artery ligation were used to establish MIRI models. Here we show, KAT5 and STIP1 homology and U-box-containing protein 1 (STUB1) were downregulated, while large tumor suppressor kinase 2 (LATS2) was upregulated in MIRI models. KAT5/STUB1 overexpression or LATS2 silencing repressed cardiomyocyte pyroptosis. Mechanistically, KAT5 promoted STUB1 transcription via acetylation modulation, and subsequently caused ubiquitination and degradation of LATS2, which activated YAP/ß-catenin pathway. Notably, the inhibitory effect of STUB1 overexpression on cardiomyocyte pyroptosis was abolished by LATS2 overexpression or KAT5 depletion. Our findings suggest that KAT5 overexpression inhibits NLRP3-mediated cardiomyocyte pyroptosis to relieve MIRI through modulation of STUB1/LATS2/YAP/ß-catenin axis, providing a potential therapeutic target for MIRI.

A WNT mimetic with broad spectrum FZD-specificity decreases fibrosis and improves function in a pulmonary damage model.

BACKGROUND: Wnt/ß-catenin signaling is critical for lung development and AT2 stem cell maintenance in adults, but excessive pathway activation has been associated with pulmonary fibrosis, both in animal models and human diseases such as idiopathic pulmonary fibrosis (IPF). IPF is a detrimental interstitial lung disease, and although two approved drugs limit functional decline, transplantation is the only treatment that extends survival, highlighting the need for regenerative therapies. METHODS: Using our antibody-based platform of Wnt/ß-catenin modulators, we investigated the ability of a pathway antagonist and pathway activators to reduce pulmonary fibrosis in the acute bleomycin model, and we tested the ability of a WNT mimetic to affect alveolar organoid cultures. RESULTS: A WNT mimetic agonist with broad FZD-binding specificity (FZD1,2,5,7,8) potently expanded alveolar organoids. Upon therapeutic dosing, a broad FZD-binding specific Wnt mimetic decreased pulmonary inflammation and fibrosis and increased lung function in the bleomycin model, and it impacted multiple lung cell types in vivo. CONCLUSIONS: Our results highlight the unexpected capacity of a WNT mimetic to effect tissue repair after lung damage and support the continued development of Wnt/ß-catenin pathway modulation for the treatment of pulmonary fibrosis.

Recurrent mutations in tumor suppressor FBXW7 bypass Wnt/ß-catenin addiction in cancer.

Pathologic Wnt/ß-catenin signaling drives various cancers, leading to multiple approaches to drug this pathway. Appropriate patient selection can maximize success of these interventions. Wnt ligand addiction is a druggable vulnerability in RNF43-mutant/RSPO-fusion cancers. However, pharmacologically targeting the biogenesis of Wnt ligands, e.g., with PORCN inhibitors, has shown mixed therapeutic responses, possibly due to tumor heterogeneity. Here, we show that the tumor suppressor FBXW7 is frequently mutated in RNF43-mutant/RSPO-fusion tumors, and FBXW7 mutations cause intrinsic resistance to anti-Wnt therapies. Mechanistically, FBXW7 inactivation stabilizes multiple oncoproteins including Cyclin E and MYC and antagonizes the cytostatic effect of Wnt inhibitors. Moreover, although FBXW7 mutations do not mitigate ß-catenin degradation upon Wnt inhibition, FBXW7-mutant RNF43-mutant/RSPO-fusion cancers instead lose dependence on ß-catenin signaling, accompanied by dedifferentiation and loss of lineage specificity. These FBXW7-mutant Wnt/ß-catenin-independent tumors are susceptible to multi-cyclin-dependent kinase inhibition. An in-depth understanding of primary resistance to anti-Wnt/ß-catenin therapies allows for more appropriate patient selection and use of alternative mechanism-based therapies.

Identification of Novel FZD4 Mutations in Familial Exudative Vitreoretinopathy and Investigating the Pathogenic Mechanisms of FZD4 Mutations.

Purpose: The purpose of this study is to report five novel FZD4 mutations identified in familial exudative vitreoretinopathy (FEVR) and to analyze and summarize the pathogenic mechanisms of 34 of 96 reported missense mutations in FZD4. Methods: Five probands diagnosed with FEVR and their family members were enrolled in the study. Ocular examinations and targeted gene panel sequencing were conducted on all participants. Plasmids, each carrying 29 previously reported FZD4 missense mutations and five novel mutations, were constructed based on the selection of mutations from each domain of FZD4. These plasmids were used to investigate the effects of mutations on protein expression levels, Norrin/ß-catenin activation capacity, membrane localization, norrin binding ability, and DVL2 recruitment ability in HEK293T, HEK293STF, and HeLa cells. Results: All five novel mutations (S91F, V103E, C145S, E160K, C377F) responsible for FEVR were found to compromise Norrin/ß-catenin activation of FZD4 protein. After reviewing a total of 34 reported missense mutations, we categorized all mutations based on their functional changes: signal peptide mutations, cysteine mutations affecting disulfide bonds, extracellular domain mutations influencing norrin binding, transmembrane domain (TM) 1 and TM7 mutations impacting membrane localization, and intracellular domain mutations affecting DVL2 recruitment. Conclusions: We expanded the spectrum of FZD4 mutations relevant to FEVR and experimentally demonstrated that missense mutations in FZD4 can be classified into five categories based on different functional changes.

RBM10 C761Y mutation induced oncogenic ASPM isoforms and regulated ß-catenin signaling in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) comprises a heterogeneous group of biliary tract cancer. Our previous CCA mutation pattern study focused on genes in the post-transcription modification process, among which the alternative splicing factor RBM10 captured our attention. However, the roles of RBM10 wild type and mutations in CCA remain unclear. METHODS: RBM10 mutation spectrum in CCA was clarified using our initial data and other CCA genomic datasets from domestic and international sources. Real-time PCR and tissue microarray were used to detect RBM10 clinical association. Function assays were conducted to investigate the effects of RBM10 wild type and mutations on CCA. RNA sequencing was to investigate the changes in alternative splicing events in the mutation group compared to the wild-type group. Minigene splicing reporter and interaction assays were performed to elucidate the mechanism of mutation influence on alternative splicing events. RESULTS: RBM10 mutations were more common in Chinese CCA populations and exhibited more protein truncation variants. RBM10 exerted a tumor suppressive effect in CCA and correlated with favorable prognosis of CCA patients. The overexpression of wild-type RBM10 enhanced the ASPM exon18 exon skipping event interacting with SRSF2. The C761Y mutation in the C2H2-type zinc finger domain impaired its interaction with SRSF2, resulting in a loss-of-function mutation. Elevated ASPM203 stabilized DVL2 and enhanced ß-catenin signaling, which promoted CCA progression. CONCLUSIONS: Our results showed that RBM10C761Y-modulated ASPM203 promoted CCA progression in a Wnt/ß-catenin signaling-dependent manner. This study may enhance the understanding of the regulatory mechanisms that link mutation-altering splicing variants to CCA.

RSPO3 induced by Helicobacter pylori extracts promotes gastric cancer stem cell properties through the GNG7/ß-catenin signaling pathway.

BACKGROUND: Helicobacter pylori (H. pylori) accounts for the majority of gastric cancer (GC) cases globally. The present study found that H. pylori promoted GC stem cell (CSC)-like properties, therefore, the regulatory mechanism of how H. pylori promotes GC stemness was explored. METHODS: Spheroid-formation experiments were performed to explore the self-renewal capacity of GC cells. The expression of R-spondin 3 (RSPO3), Nanog homeobox, organic cation/carnitine transporter-4 (OCT-4), SRY-box transcription factor 2 (SOX-2), CD44, Akt, glycogen synthase kinase-3ß (GSK-3ß), p-Akt, p-GSK-3ß, ß-catenin, and G protein subunit gamma 7 (GNG7) were detected by RT-qPCR, western blotting, immunohistochemistry (IHC), and immunofluorescence. Co-immunoprecipitation (CoIP) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were performed to identify proteins interacting with RSPO3. Lentivirus-based RNA interference constructed short hairpin (sh)-RSPO3 GC cells. Small interfering RNA transfection was performed to inhibit GNG7. The in vivo mechanism was verified using a tumor peritoneal seeding model in nude mice. RESULTS: H. pylori extracts promoted a CSC-like phenotype in GC cells and elevated the expression of RSPO3. RSPO3 knockdown significantly reduced the CSC-like properties induced by H. pylori. Previous studies have demonstrated that RSPO3 potentiates the Wnt/ß-catenin signaling pathway, but the inhibitor of Wnt cannot diminish the RSPO3-induced activation of ß-catenin. CoIP and LC-MS/MS revealed that GNG7 is one of the transmembrane proteins interacting with RSPO3, and it was confirmed that RSPO3 directly interacted with GNG7. Recombinant RSPO3 protein increased the phosphorylation level of Akt and GSK-3ß, and the expression of ß-catenin in GC cells, but this regulatory effect of RSPO3 could be blocked by GNG7 knockdown. Of note, GNG7 suppression could diminish the promoting effect of RSPO3 to CSC-like properties. In addition, RSPO3 suppression inhibited MKN45 tumor peritoneal seeding in vivo. IHC staining also showed that RSPO3, CD44, OCT-4, and SOX-2 were elevated in H. pylori GC tissues. CONCLUSION: RSPO3 enhanced the stemness of H. pylori extracts-infected GC cells through the GNG7/ß-catenin signaling pathway.

[Downregulation of SETDB1 suppresses epithelial mesenchymal transition and invasion and metastasis in oral cancer via SOX7 methylation].

PURPOSE: To explore the mechanism of SETDB1 inhibiting epithelial mesenchymal transition (EMT),migration and invasion in oral cancer via SOX 7 methylation. METHODS: SETDB1 and SOX7 mRNA and protein expression levels in KB cells of oral cancer and oral mucosal epithelial ATCC cells were determined by qRT-PCR and Western blot (WB). SETDB1 si-RNA was structured, then transfect into KB cells of oral cancer by liposome-mediated method. siRNA-SETDB1 was the experimental group (si-S), siRNA empty vector was the negative control group (si-N), and untransfected KB cells were the blank control group(NC). SETDB1 mRNA and protein expression levels were detected by qRT-PCR and Western blot(WB), to verify the transfection effect. The methylation levels of SOX7 were determined by pyrosequencing. The expression of N-cadherin, Vimentin, ß-catenin, and Slug proteins was detected by WB. Cell viability was measured by MTT assay, migration ability was tested by scratch healing assay, and invasion ability was tested by Transwell chamber assay. Statistical analysis was performed with SPSS 21.0 software package. RESULTS: The results of Rt-qPCR and WB showed that the SETDB1 mRNA and protein expression decreased significantly in si-S group(P＜0.05). Pyrosequencing test results showed that the regulation of SETDB1 could significantly reduce the SOX7 methylation rate and increased the SOX7 protein expression. WB results showed that knockdown of SETDB1 significantly inhibited the expression of EMT-related proteins N-cadherin, Vimentin, ß-catenin and Slug in oral cancer KB cells (P＜0.05). The results of cell functology experiments showed that knockdown of SETDB1 could significantly inhibit survival, migration and invasion of KB cells. CONCLUSIONS: Downregulation of SETDB1 could suppress EMT, migration and invasion of oral cancer cells by regulating SOX7 methylation level, providing new ideas and targets for the diagnosis and treatment of oral cancer.

The Role of Heat-Induced Stress Granules in the Blood-Testis Barrier of Mice.

Stress granules (SGs) are membraneless ribonucleoprotein (RNP)-based cellular foci formed in response to stress, facilitating cell survival by protecting against damage. Mammalian spermatogenesis should be maintained below body temperature for proper development, indicating its vulnerability to heat stress (HS). In this study, biotin tracer permeability assays showed that the inhibition of heat-induced SG assembly in the testis by 4-8 mg/kg cycloheximide significantly increased the percentage of seminiferous tubules with a damaged blood-testis barrier (BTB). Western blot results additionally revealed that the suppression of heat-induced SG assembly in Sertoli cell line, TM4 cells, by RNA inference of G3bp1/2 aggravated the decline in the BTB-related proteins ZO-1, ß-Catenin and Claudin-11, indicating that SGs could protect the BTB against damage caused by HS. The protein components that associate with SGs in Sertoli cells were isolated by sequential centrifugation and immunoprecipitation, and were identified by liquid chromatography with tandem mass spectrometry. Gene Ontology and KEGG pathway enrichment analysis revealed that their corresponding genes were mainly involved in pathways related to proteasomes, nucleotide excision repair, mismatch repair, and DNA replication. Furthermore, a new SG component, the ubiquitin associated protein 2 (UBAP2), was found to translocate to SGs upon HS in TM4 cells by immunofluorescence. Moreover, SG assembly was significantly diminished after UBAP2 knockdown by RNA inference during HS, suggesting the important role of UBAP2 in SG assembly. In addition, UBAP2 knockdown reduced the expression of ZO-1, ß-Catenin and Claudin-11, which implied its potential role in the function of the BTB. Overall, our study demonstrated the role of SGs in maintaining BTB functions during HS and identified a new component implicated in SG formation in Sertoli cells. These findings not only offer novel insights into the biological functions of SGs and the molecular mechanism of low fertility in males in summer, but also potentially provide an experimental basis for male fertility therapies.

Gynostemma pentaphyllum Hydrodistillate and Its Major Component Damulin B Promote Hair Growth-Inducing Properties In Vivo and In Vitro via the Wnt/ß-Catenin Pathway in Dermal Papilla Cells.

Alopecia, a prevalent yet challenging condition with limited FDA-approved treatments which is accompanied by notable side effects, necessitates the exploration of natural alternatives. This study elucidated the hair growth properties of Gynostemma pentaphyllum leaf hydrodistillate (GPHD) both in vitro and in vivo. Furthermore, damulin B, a major component of GPHD, demonstrated hair growth-promoting properties in vitro. Beyond its established anti-diabetic, anti-obesity, and anti-inflammatory attributes, GPHD exhibited hair growth induction in mice parallel to minoxidil. Moreover, it upregulated the expression of autocrine factors associated with hair growth, including VEGF, IGF-1, KGF, and HGF. Biochemical assays revealed that minoxidil, GPHD, and damulin B induced hair growth via the Wnt/ß-catenin pathway through AKT signaling, aligning with in vivo experiments demonstrating improved expression of growth factors. These findings suggest that GPHD and damulin B contribute to the hair growth-inducing properties of dermal papilla cells through the AKT/ß-catenin signaling pathway.