Serum long-noncoding RNA H19 and beta-catenin as biomarkers for early diagnosis of colorectal cancer in egyptian patients: a case control study

Colorectal cancer (CRC) is the third most prevalent cancer and the second most common cause of cancer death; however, its early detection can improve the survival. Colonic polyps are considered one of the CRC's major risk factors. Throughout many biological processes and malignancies, the non-coding RNAs have essential functions. Certain long noncoding RNAs (lncRNAs), including H19, were supposed to be CRC possible biomarkers. Also, H19 has been reported to play a role in regulating the activity of beta-catenin, a protein that regulates cell-to-cell adhesion, as well as gene transcription. The current work aimed to investigate the potential significance of LncRNA H19 relative serum expression level by quantitative polymerase chain reaction (q-PCR) and beta-catenin by enzyme-linked immunosorbent assay (ELISA) as noninvasive biomarkers to discriminate between colorectal cancer and colonic polyps. The statistical analysis of the studied factors revealed that the serum expression of H19 and beta-catenin in cancer cases were substantially greater than colonic polyp cases and normal control. Conclusion: The relative expressions of H19 and beta-catenin in the serum can significantly discriminate patients with CRC from those with polyp and normal controls, which could help when screening for CRC. (AU)

Experimental study of proteasome inhibitor MG132 up-regulates Wnt/β-catenin signaling pathway to improve osteoporosis / 中国骨伤

OBJECTIVE@#To explore the mechanism of proteasome inhibitor MG132 in improving osteoporosis.@*METHODS@#Total of 32 female SD rats, weighing 220 to 250 g and 8 weeks old, were selected. They were randomly divided into 4 groups(n=8). Rats of group A and group B were cut off ovaris on both sides to make model of osteoporosis, and then they were given proteasome inhibitors MG132 and dimethyl sufoxide (DMSO) respectively. Group C was a sham group and rats were given MG132. Group D was a normal group and rats were given MG132 too. The rats were killed in batches at 6 and 12 weeks after administration, and the femoral neck tissues were obtained. Relevant data were analyzed, such as pathomorphological observation, micro-CT analysis, detection of 20S proteasome activity in tissues, and expression of Wnt and β-catenin.@*RESULTS@#Morphological observation showed that the trabecular were slightly thinner, reticulated, and occasionally interrupted in group A, while the trabecular were obviously thinner and discontinuous in group B. And the trabecular were intact and arranged reticulated in group C and D. The analysis results of bone mineral density(BMD), bone surface(BS), bone volume/total volume(BV/TV) and trabecular thickness(Tb.Th) showed that group B was worse than other groups in all parameters at different time points(P<0.05), and group A was worse than group C and group D in BS(P<0.05), there was no significant difference in all parameters between group C and group D. RFU value of 20S proteasome in group B was significantly higher than that in other groups(P<0.05). According to the results of Western blot, the gray values of Wnt protein and β-catenin protein in group A were significantly higher than those in other groups (P<0.05).@*CONCLUSION@#MG-132, a ubiquitin proteasome inhibitor, can regulate Wnt/β-catenin signaling pathway by inhibiting the degradation of β-catenin protein, and delaying the occurrence and development of osteoporosis.

Naringin attenuates acute myocardial ischemia-reperfusion injury via miR126/GSK-3ß/ß-catenin signaling pathway

Introduction: Myocardial ischemia-reperfusion (I/R) injury is one of the mechanisms contributing to the high mortality rate of acute myocardial infarction. Purpose: This study intended to study the role of naringin in cardiac I/R injury. Methods: AC16 cells (human cardiomyocyte cell line) were subjected to oxygen-glucose deprivation/recovery (OGD/R) treatment and/or naringin pretreatment. Then, the apoptosis was examined by flow cytometry and Western blotting. The concentration of IL-6, IL-8 and TNF-α was measured by enzyme-linked immunosorbent assay (ELISA) kits. How naringin influenced microRNA expression was examined by microarrays and quantitative real-time polymerase chain reaction (qRT-PCR). Dual luciferase reporter assay was employed to evaluate the interaction between miR-126 and GSK-3ß. The GSK-3ß/ß-catenin signaling pathway was examined by Western blotting. Finally, rat myocardial I/R model was created to examine the effects of naringin in vivo. Results: Naringin pretreatment significantly decreased the cytokine release and apoptosis of cardiomyocytes exposed to OGD/R. Bioinformatical analysis revealed that naringin upregulated miR-126 expression considerably. Also, it was found that miR-126 can bind GSK-3ß and downregulate its expression, suggesting that naringin could decrease GSK-3ß activity. Next, we discovered that naringin increased ß-catenin activity in cardiomyocytes treated with OGD/R by inhibiting GSK-3ß expression. Our animal experiments showed that naringin pre-treatment or miR-126 agomir alleviated myocardial I/R. Conclusions: Naringin preconditioning can reduce myocardial I/R injury via regulating miR-126/GSK-3ß/ß-catenin signaling pathway, and this chemical can be used to treat acute myocardial infarction.

β-Catenin Deletion in Regional Neural Progenitors Leads to Congenital Hydrocephalus in Mice / 神经科学通报·英文版

Congenital hydrocephalus is a major neurological disorder with high rates of morbidity and mortality; however, the underlying cellular and molecular mechanisms remain largely unknown. Reproducible animal models mirroring both embryonic and postnatal hydrocephalus are also limited. Here, we describe a new mouse model of congenital hydrocephalus through knockout of β-catenin in Nkx2.1-expressing regional neural progenitors. Progressive ventriculomegaly and an enlarged brain were consistently observed in knockout mice from embryonic day 12.5 through to adulthood. Transcriptome profiling revealed severe dysfunctions in progenitor maintenance in the ventricular zone and therefore in cilium biogenesis after β-catenin knockout. Histological analyses also revealed an aberrant neuronal layout in both the ventral and dorsal telencephalon in hydrocephalic mice at both embryonic and postnatal stages. Thus, knockout of β-catenin in regional neural progenitors leads to congenital hydrocephalus and provides a reproducible animal model for studying pathological changes and developing therapeutic interventions for this devastating disease.

Effect of lathyrol derivatives on non-small cell lung cancer and the possible mechanism / 中南大学学报(医学版)

OBJECTIVES@#Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer, with highmorbidity and mortality rate. Nove drug development for NSCLC is urgently needed.This study aims to investigate the activity of lathyrol derivatives and the mechanism for its inhibitory effect on the growth of NSCLC cells.@*METHODS@#Three lathyrol derivatives were synthesized from lathyrol and their structures were verified by nuclear magnetic resonance. MTT assay was used to detect the effects of the lathyrol derivatives on the proliferation activity of NSCLC cells (A549 and H1299 cells), and the compound with the best activity was selected for subsequent experiments. Colony forming assay, wound-healing assay, and transwell assay were applied to detect in vitro cell proliferation, migration and invasion ability in A549 and H1299 cells, respectively. Quantitative real-time RT-PCR and Western blotting were performed to detect mRNA and protein levels of E-cadherin, N-cadherin, β-catenin, and MMP2 in A549 cells, respectively.@*RESULTS@#Three lathyrol derivatives inhibited the growth of A549 and H1299 cells in a dose-dependent manner, and they showed a weak inhibitory effect on normal cells Beas-2B and 16HBE, indicating that they possessed certain selective toxic effects. Therefore, C-5 benzoylated lathyrol with the best activity was selected as the ideal drug for the subsequent experiments. Compared with the control group, the number and size of cell clusters in the treatment group of A549 and H1299 cells were significantly decreased, the relative mobility were significantly decreased, and the number of invaded cells were significantly decreased (all P<0.05), indicating that the in vitro cell proliferation, migration and invasion ability were decreased. The mRNA levels of integrin α2, integrin β1, MMP2, MMP9, β-catenin, and N-cadherin were decreased, while the expression of E-cadherin was increased (all P<0.05). The protein levels of N-cadherin, β-catenin, MMP2, and integrin αV were decreased, while the expression of E-cadherin was increased (all P<0.05).@*CONCLUSIONS@#The lathyrol derivatives synthesized in this study possess good inhibitory activity against NSCLC. Among them, C-5 benzoylated lathyrol significantly inhibits the proliferation, migration, and invasion ability of NSCLC cells in vitro through regulating the process of epithelial-mesenchymal transition.

Total Ginsenoside Extract from Panax ginseng Enhances Neural Stem Cell Proliferation and Neuronal Differentiation by Inactivating GSK-3β / 中国结合医学杂志

OBJECTIVE@#To study the effects of total ginsenosides (TG) extract from Panax ginseng on neural stem cell (NSC) proliferation and differentiation and their underlying mechanisms.@*METHODS@#The migration of NSCs after treatment with various concentrations of TG extract (50, 100, or 200 µ g/mL) were monitored. The proliferation of NSCs was examined by a combination of cell counting kit-8 and neurosphere assays. NSC differentiation mediated by TG extract was evaluated by Western blotting and immunofluorescence staining to monitor the expression of nestin and microtubule associated protein 2 (MAP2). The GSK-3β/β-catenin pathway in TG-treated NSCs was examined by Western blot assay. The NSCs with constitutively active GSK-3β mutant were made by adenovirus-mediated gene transfection, then the proliferation and differentiation of NSCs mediated by TG were further verified.@*RESULTS@#TG treatment significantly enhanced NSC migration (P<0.01 or P<0.05) and increased the proliferation of NSCs (P<0.01 or P<0.05). TG mediation also significantly upregulated MAP2 expression but downregulated nestin expression (P<0.01 or P<0.05). TG extract also significantly induced GSK-3β phosphorylation at Ser9, leading to GSK-3β inactivation and, consequently, the activation of the GSK-3β/β-catenin pathway (P<0.01 or P<0.05). In addition, constitutive activation of GSK-3β in NSCs by the transfection of GSK-3β S9A mutant was found to significantly suppress TG-mediated NSC proliferation and differentiation (P<0.01 or P<0.05).@*CONCLUSION@#TG promoted NSC proliferation and neuronal differentiation by inactivating GSK-3β.

Mechanism of miR-155 Promoting Drug Resistance in Childhood Acute Lymphoblastic Leukemia by Regulating Wnt/β-Catenin Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanism of miR-155 promoting drug resistance of children B-ALL to Ara-C by regulating Wnt/β-Catenin signaling pathway.@*METHODS@#The expression of miR-155 in bone marrow tissue and cell line of B-ALL was detected by PCR. The chemotherapy resistant strain REH/ Ara-C was constructed by using REH cells. REH/ Ara-C cells were transfected with miR-155 inhibitor. The proliferation of REH/Ara-C cells was detected by EdU. The apoptosis of REH/ Ara-C cells was detected by flow cytometry. The drug resistance of REH/Ara-C cells were analyzed by CCK-8 method and colony formation assay. The expression of Wnt/β-Catenin signaling pathway related proteins were determined by Western blot. MiR-155 inhibitor and Wnt activator agonist were used to transfect REH/Ara-C cells, and their effects on cell proliferation, apoptosis and drug resistance were determined.@*RESULTS@#Compared with normal tissues and cells, the expression level of miR-155 in B-ALL bone marrow tissue/cell line was increased (P<0.05); Compared with drug sensitive B-ALL tissues/cell lines, the expression level of miR-155 in drug resistant B-ALL tissues and cell lines was increased (P<0.05); Inhibition of miR-155 expression decreased the proliferation of REH/Ara-C cells (P<0.05), promoted apoptosis (P<0.05), enhanced the cytotoxicity of Ara-C (P<0.05), and inhibited Wnt/β-Catenin signaling pathway related protein and MDR1 gene expression (P<0.05), which could be reversed by activating Wnt expression (P<0.05).@*CONCLUSION@#The expression of miR-155 is up-regulated in bone marrow of children with B-ALL, which may be related to the activation of Wnt/β-Catenin signaling pathway promotes the proliferation of B-ALL cells and inhibits apoptosis, which leads to chemotherapy resistance.

Aspirin inhibits the growth of hypertrophic scar in rabbit ears via regulating Wnt/β-catenin signal pathway / 中南大学学报(医学版)

OBJECTIVES@#Steroidal anti-inflammatory drugs have certain side effects in the treatment of hypertrophic scar, and the scar recurrence is easy after withdrawal of steroid anti-inflammatory drugs. Finding reliable alternative drugs is an effective means to improve this defect. Aspirin, a traditional non-steroidal anti-inflammatory drug, is safe for topical use and has anti-inflammatory effects similar to those of steroidal anti-inflammatory drugs, which may have similar effects on the treatment of hypertrophic scar. This study aims to investigate the inhibitory effect of aspirin on the proliferation of hypertrophic scar in rabbit ears and the underlying mechanism.@*METHODS@#The rabbit ear hypertrophic scar models were prepared. The rabbits were randomly divided into a normal skin group (group A), a blank control group (group B), a 0.9% NaCl group (group C), a 0.2% aspirin group (group D), a 0.5% aspirin group (group E), a 2% aspirin group (group F), and a triamcinolone acetonide group (group G). Macroscopic observation of hyperplasia was performed 8 weeks after local injection of the scar, followed by collecting the scar tissue samples for HE staining, Masson staining, and immunohistochemistry, respectively to assess the proliferation of fibroblasts and collagen fibers, and calculate the hypertrophic index, microvessel density, and immunohistochemical score.@*RESULTS@#All rabbit ear hypertrophic scar models were successfully constructed. In groups B and C, the hypertrophic scar edge was irregular, with reddish protruding epidermis, significant contracture and hard touch. In group D, E, and F, with the increase of aspirin administration concentration, the scar became thinner and gradually flat, the proliferation of fibrocytes and collagen fibers was weakened, and the hypertrophic index was gradually decreased (P<0.05). Immunohistochemistry showed that the expression of β-catenin was decreased in the group D, E and F in turn, and the immunohistochemical score was gradually decreased (P<0.05). There was no significant difference in hypertrophic index, microvessel density, and immunohistochemical score (all P>0.05).@*CONCLUSIONS@#Local injection of aspirin can reduce the generation of hypertrophic scar in a dose-dependent manner within a certain concentration range; aspirin inhibits the growth of hypertrophic scar in rabbit ears by inhibiting Wnt/β-catenin signal pathway; 2% aspirin and 40 mg/mL triamcinolone acetonide have similar curative efficacy on hypertrophic scar.

20-Hydroxyecdysone Improves Neuronal Differentiation of Adult Hippocampal Neural Stem Cells in High Power Microwave Radiation-Exposed Rats / 生物医学与环境科学（英文）

Objective@#The hippocampus is thought to be a vulnerable target of microwave exposure. The aim of the present study was to investigate whether 20-hydroxyecdysone (20E) acted as a fate regulator of adult rat hippocampal neural stem cells (NSCs). Furthermore, we investigated if 20E attenuated high power microwave (HMP) radiation-induced learning and memory deficits.@*Methods@#Sixty male Sprague-Dawley rats were randomly divided into three groups: normal controls, radiation treated, and radiation+20E treated. Rats in the radiation and radiation+20E treatment groups were exposed to HPM radiation from a microwave emission system. The learning and memory abilities of the rats were assessed using the Morris water maze test. Primary adult rat hippocampal NSCs were isolated in vitro and cultured to evaluate their proliferation and differentiation. In addition, hematoxylin & eosin staining, western blotting, and immunofluorescence were used to detect changes in the rat brain and the proliferation and differentiation of the adult rat hippocampal NSCs after HPM radiation exposure.@*Results@#The results showed that 20E induced neuronal differentiation of adult hippocampal NSCs from HPM radiation-exposed rats via the Wnt3a/β-catenin signaling pathway in vitro. Furthermore, 20E facilitated neurogenesis in the subgranular zone of the rat brain following HPM radiation exposure. Administration of 20E attenuated learning and memory deficits in HPM radiation-exposed rats and frizzled-related protein (FRZB) reduced the 20E-induced nuclear translocation of β-catenin, while FRZB treatment also reversed 20E-induced neuronal differentiation of NSCs in vitro.@*Conclusion@#These results suggested that 20E was a fate regulator of adult rat hippocampal NSCs, where it played a role in attenuating HPM radiation-induced learning and memory deficits.

TRIM21 suppresses invasion of hepatocellular carcinoma cells by promoting β-catenin ubiquitylation and degradation / 南方医科大学学报

OBJECTIVE@#To explore the role of TRIM21 in modulating the invasive phenotype of hepatocellular carcinoma (HCC) cells and its mechanism of action.@*METHODS@#RNA interference technique was used to knock down the expression of TRIM21 and β-catenin, alone or in combination, in HCC cell lines 97H and LM3, and the interfering efficiency and the activity of closely related pathways were determined using Western blotting. The two cells with TRIM21 knockdown (siTRIM21 97H and siTRIM21 LM3 cells) were assessed for their invasion ability in vitro using Transwell invasion assay, and the lung metastasis capacity of siTRIM21 LM3 cells following tail vein injection was evaluated in nude mice. The binding of TRIM21 with β-catenin and the ubiquitylation level of β-catenin in TRIM21-overexpressing HEK293 cells were determined with Western blotting and co-immunoprecipitation assay. We also compared the overall survival of patients with CTNNB1highTRIM21high and CTNNB1highTRIM21low HCC subtypes using Kaplan-Meier method based on filtrated and grouped HCC clinical data from TCGA database.@*RESULTS@#TRIM21 knockdown significantly enhanced the invasion ability of 97H and LM3 cells in vitro (P < 0.01 or 0.05) and the lung metastasis ability of LM3 cells in nude mice (P < 0.01), and simultaneous knockdown of β -catenin obviously suppressed the in vitro invasiveness of the cells (P < 0.0001 or 0.05). Co-immunoprecipitation assay showed that TRIM21 was capable of directly binding with β-catenin protein to accelerate the ubiquitination and degradation of the latter, leading to inhibition of nuclear translocation of β-catenin and hence reduced invasiveness of HCC cells. Bioinformatic analysis showed that compared patients with CTNNB1highTRIM21low HCC subtype where Wnt pathway was activated, the patients with CTNNB1highTRIM21high HCC subtype had a significantly better survival outcomes (P < 0.05).@*CONCLUSION@#A high expression of TRIM21 suppresses the invasion of HCC cells by promoting β-catenin ubiquitylation and degradation, which possibly explains the poor prognosis of CTNNB1highTRIM21low HCC patients.

Wnt/β-catenin signaling in kidney repair and fibrosis after injury / 生理学报

Wnt/β-catenin is an evolutionarily conserved, complex developmental signal pathway that regulates embryogenesis, cell fate, tissue homeostasis, injury repair, and the pathogenesis of human diseases. Mounting evidence demonstrates that Wnt/β-catenin signaling plays a key role in early nephrogenesis. It is relatively silent in normal adult kidneys but reactivated in a wide variety of animal models of nephropathies and in human kidney diseases. Activation of Wnt/β-catenin after acute kidney injury contributes to proper repair and regeneration of damaged renal tubules. However, sustained activation of this signal cascade is closely related to the development and progression of fibrotic chronic kidney disease. In this paper, we systematically review the components and mechanisms of Wnt/β-catenin signaling and its role in kidney repair and fibrosis after injury. A better delineation of the mechanisms of this pathway will provide novel targets and new strategies for designing effective treatment of various kidney diseases.

Hypoxia promotes differentiation of human induced pluripotent stem cells into embryoid bodies in vitro / 南方医科大学学报

OBJECTIVE@#To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms.@*METHODS@#EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.@*RESULTS@#The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05).@*CONCLUSION@#Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.

Papillary thyroid carcinoma with nodular fasciitis-like stroma: a rare variant

Papillary thyroid carcinoma with desmoid-type fibromatosis (PTC-DTF) or nodular fasciitis-like stroma (PTC-NFS) is a rare morphological variant of PTC with a favorable prognosis. There is a paucity of molecular data regarding this entity. We present the case of a 20-year-old female who presented with a palpable mass over the anterior aspect of the neck for the past 3-4 months, which was diagnosed as PTC-NFS. Ultrasonogram of the neck revealed a bulky left lobe of thyroid that contained a well-defined heterogenous lesion measuring around 24 × 26 × 36 mm with involvement of the adjacent isthmus. She underwent a total thyroidectomy with central compartment lymph node dissection. Histological examination revealed a biphasic tumor with epithelial and stromal components resembling nodular fasciitis. Two dissected lymph nodes showed metastasis of the epithelial component only. On immunohistochemistry, BRAF mutant protein expression was evident in the epithelial component only, while β-catenin was negative in both the components. The histopathological diagnosis of papillary thyroid carcinoma with nodular fasciitis-like stroma was offered. Sanger sequencing revealed a BRAFV600E (c.1799T>A, Val600Glu) mutation. Post-operatively, no residual tumor was detected on ultrasound and radioiodine scans. The patient was doing well at follow-up of 9 months. PTC-NFS/DTF is a histological variant of PTC with a favorable prognosis. Our index case was associated with the BRAF mutation, which was restricted to the epithelial component. Thorough sampling of the excised specimen is essential in order not to miss the epithelial component, which, in most reported cases (including ours) appears to be small.

Tobramycin promotes fracture healing by upregulating expressions of ALP and RUNX2 proteins through activating Wnt/β-catenin pathway / 中国骨伤

OBJECTIVE@#To explore effect of tobramycin (TOB) on healing of femoral fractures in rats.@*METHODS@#Totally 32 male sprague-dawley (SD) rats were selected and randomly divided into sham group (group A), fracture group (group B), fracture with TOB group (group C) and fracture + TOB + IWR-1 group (group D), 8 rats in each group. Close femoral fracture model in rats were established in group B, C and D, group A was sham operation without otherwise process. Group D was intraperitoneal injected 100 μl (8 μM) of Wnt pathway inhibitor IWR-1-endo (IWR-1) before molding at 1 day. At 1 day after molding, 100 μl (100 μM) of TOB was intraperitoneally injected into group C and D at once a day for 7 days. At 7 weeks after modling, fracture healing of group B, C and D were observed by X-ray, Western blotting was appilied to detect alkaline phosphatase(ALP) and Runt related transcription factor 2 (RUNX2) and β-catenin of Wnt passway.@*RESULTS@#X-ray results showed fracture line disappeared, callus formation and fracture healing well in group C compared with begning of molding; while a little fracture line, callus formation and fracture malunion in group B and d could be seen. Western blotting results showed ALP, RUNX2 and expression of β-catenin in group B, C and D were higher than that of group A (@*CONCLUSION@#Tobramycin could promote osteoblast differentiation and fracture healing by stimulating Wnt / β-catenin signaling pathway, up regulating expression of ALP and RUNX2.

Optimizations of an ELISA-like high-throughput screening assay for the discovery of β-catenin/TCF4 interaction antagonists / 生物工程学报

In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.

Mechanism of electroacupuncture for regulation of lipid production and improvement in obesity by mediating Wnt/ β-catenin pathway through activating SIRT1 / 中国针灸

OBJECTIVE@#To explore the mechanism of electroacupuncture (EA) for the regulation of lipid production and improvement in obesity by mediating Wnt/β-catenin pathway through activating silent information regulator 1 (SIRT1).@*METHODS@#Of 75 Wistar male rats, 10 rats were selected randomly as the normal group and fed with standard diet. The rest rats were fed with high-fat diet for 8 weeks to establish the obesity model. Forty rats of successful modeling were randomized into a model group, an EA group, an EA plus inhibitor group (EA+I group) and an agonist group, 10 rats in each one. In the EA group, EA was applied at "Guanyuan" (CV 4), "Zhongwan" (CV 12), "Zusanli" (ST 36) and "Fenglong" (ST 40), with continuous wave, 2 Hz in frequency and around 1 mA in intensity. The needles were retained for 20 min. In the EA+I group, sirtinol solution was injected from caudal vein and EA was exerted simultaneously. In the agonist group, resveratrol solution was given by intragastric administration. The intervention of the above three groups was given once every two days, 3 times a week, consecutively for 8 weeks. Before and after intervention, body mass and Lee's index were recorded in the rats of each group. After intervention, the levels of serum total cholesterol (TC), triglyceride (TG) and free fatty acid (FFA) were detected in the rats of each group. After intervention, the mass of white adipose tissue (WAT) and the area of adipocytes were compared in the rats among the 5 groups. Using Western blot method, the protein expressions of SIRT1, glycogen synthase kinase-3β (GSK3β), β-catenin, cyclin D1 and peroxisome proliferators-activated receptor γ (PPARγ) were detected in WAT in the rats of each group.@*RESULTS@#After intervention, compared with the model group, the body mass and Lee's index were reduced in the rats of the EA group and the agonist group (@*CONCLUSION@#Electroacupuncture remarkably improves the body mass, Lee's index and blood lipid metabolism and reduces WAT mass and adipocyte size in obesity model rats, which is probably related to up-regulating the protein expression of SIRT1 in WAT, activating Wnt/β-catenin pathway and inhibiting the expression of PPARγ of downstream lipogenic gene so as to affect lipid production.

Dexmedetomidine-mediated Wnt Pathway Inhibits Sevoflurane-induced Cognitive Impairment in Neonatal Rats / 中国医学科学院学报

Objective To explore the effect of dexmedetomidine(Dex)on sevoflurane-induced cognitive impairment in neonatal rats through Wnt signaling pathway. Methods Sixty 7-day-old SD rats were assigned into five groups:control group(without any intervention),Dex group(intraperitoneal injection of 25 μg/kg Dex),sevoflurane group(3% sevoflurane treatment for 4 hours),sevoflurane+Dex group(inhalation of 3% sevoflurane after injection of 25 μg/kg Dex for 4 hours),and sevoflurane+Dex+Wnt inhibitor group(Wnt inhibitor XAV393 and 25 μg/kg Dex were injected and 3% sevoflurane was inhaled for 4 hours).Three weeks later,Morris water maze was used to detect the cognitive function;TdT-mediated dUTP nick end labeling(TUNEL)staining was performed to detect the apoptosis of hippocampal neurons;neuronal nuclei (NeuN) staining was conducted to detect the survival of hippocampal neurons;Western blot was carried out to detect the expression of apoptosis-related proteins.The expression of the factors involved in Wnt/GSK-3β/β-catenin signaling pathway was detected by fluorescence quantitative polymerase chain reaction,and Western blot. Results Compared with the control group,there was no significant difference in the escape latency of Dex group(t=0.304,P=0.768);the escape latency in sevoflurane group(t=5.823,P=0.002),sevoflurane+Dex group(t=3.188,P=0.010),and sevoflurane+Dex+Wnt inhibitor group(t=5.784,P=0.002)was significantly prolonged.Compared with that in the sevoflurane group,the escape latency in sevoflurane+Dex group(t=3.646,P=0.005)was significantly shortened.Compared with that in sevoflurane+Dex group,the escape latency in sevoflurane+Dex+Wnt inhibitor group(t=3.296,P=0.008)was prolonged.Compared with that in the control group,the times of crossing platform in sevoflurane group(t=5.179, P=0.004),sevoflurane+Dex group(t=2.309,P=0.043),and sevoflurane+Dex+Wnt inhibitor group(t=3.871, P=0.003)decreased.Compared with that in sevoflurane group,the times of crossing platform in sevoflurane+Dex group(t=3.296,P=0.008)significantly increased.Compared with that in sevoflurane+Dex group,the times of crossing platform in sevoflurane+Dex+Wnt inhibitor group(t=2.361, P=0.041)reduced.Compared with the control group,there was no significant difference in the number of apoptotic cells in Dex group(t=1.920,P=0.127),and the number of apoptotic cells in sevoflurane group,sevoflurane+Dex group,and sevoflurane+Dex+Wnt inhibitor group increased by 16%(t=13.436,P=0.002),5%(t=7.752, P=0.001),and 11.5%(t=12.612,P=0.002),respectively.Compared with that in the sevoflurane group,the number of apoptotic cells in sevoflurane+Dex group and sevoflurane+Dex+Wnt inhibitor group decreased by 11%(t=8.521,P=0.002)and 5.5%(t=3.123,P=0.036),respectively.Compared with that in the sevoflurane+Dex group,the number of apoptotic cells in sevoflurane+Dex+Wnt inhibitor group increased by 6.5%(t=6.250,P=0.003).Compared with that in the control group,the number of positive cells in 0.15 mm

Sex determining region Y-box 9 induced microtubule formation and epithelial⁃mesenchymal transition in human oral squamous cell carcinoma CAL27 cells / 华西口腔医学杂志

OBJECTIVES@#This study aimed to explore the effect of sex determining region Y-box 9 (SOX9) on the microtubule formation and epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (OSCC) CAL27 and the underlying mechanism.@*METHODS@#SOX9-shRNA1 and SOX9-shRNA2 were designed and synthesized and then transfected into CAL27 cells. The expression of SOX9 was detected by quantitative real-time polymerase chain reaction. Microtubule formation assay was used to detect the change in the number of microtubule nodules after interfering with SOX9. Immunofluorescence was used to detect the Vimentin content. Western blot was used to detect the protein expression of EMT marker molecules and Wnt/β-catenin pathway-related proteins, such as E-cadherin, N-cadherin, Fibronectin, Wnt, β-catenin, T-cell factor-4 (TCF-4).@*RESULTS@#The expression level of SOX9 significantly decreased after transfection with SOX9-shRNA1 and SOX9-shRNA2 in CAL27 cells (@*CONCLUSIONS@#Interference with SOX9 decreased Vimentin content and inhibited the microtubule formation and protein expression of EMT marker molecules, as well as the expression of proteins related to the Wnt/β-catenin pathway. Thus, SOX9 can induce microtubule formation and EMT in CAL27, which was related to the inhibition of the Wnt/β-catenin pathway activation.

LINC00665 promotes HeLa cell proliferation, migration, invasion and epithelial-mesenchymal transition by activating the WNT-CTNNB1/β‑catenin signaling pathway / 生理学报

There is increasing evidence that long non-coding RNA (lncRNA) plays critical roles in cancer progression. However, the role of long non-coding RNA 00665 (LINC00665) in most cancers is poorly understood. The purpose of the present study was to reveal the functional role of LINC00665 in cervical cancer cells. HeLa cells were subjected to LINC00665 short hairpin RNA (shRNA) or control shRNA treatment to investigate the metastasis and proliferation phenotype of cervical cancer cells in vitro and in vivo. Transcriptome sequencing experiments of HeLa cells in LINC00665 silencing or control group were conducted, and the differentially expressed genes (DEGs) were screened. The DEGs were subjected to Metascape database functional analysis and gene set enrichment analysis. Epithelial-mesenchymal transition (EMT) related markers and a key element of WNT/β‑catenin pathway, CTNNB1 (catenin beta 1), were detected by Western blot and immunofluorescence assay. The results showed that silencing LINC00665 reduced cell viability of Hela cells, up-regulated protein expression level of E-cadherin, down-regulated protein expression levels of N-cadherin, Vimentin and CTNNB1, and inhibited cell migration and invasion of HeLa cells. Bioinformatics analysis results showed that LINC00665 might promote EMT by activating WNT-CTNNB1/β‑catenin signaling pathway. These results indicate that LINC00665 has functions in transcriptional EMT regulation via WNT-CTNNB1/β‑catenin signaling pathway and therefore can be developed as a therapeutic target for cervical cancer.

Effects of HDAC4 on IL-1β-induced matrix metalloproteinase expression regulated partially through the WNT3A/β-catenin pathway / 中华医学杂志(英文版)

BACKGROUND@#Histone deacetylase 4 (HDAC4) regulates chondrocyte hypertrophy and bone formation. The aim of the present study was to explore the effects of HDAC4 on Interleukin 1 beta (IL-1β)-induced chondrocyte extracellular matrix degradation and whether it is regulated through the WNT family member 3A (WNT3A)/β-catenin signaling pathway.@*METHODS@#Primary chondrocytes (CC) and human chondrosarcoma cells (SW1353 cells) were treated with IL-1β and the level of HDAC4 was assayed using Western blotting. Then, HDAC4 expression in the SW1353 cells was silenced using small interfering RNA to detect the effect of HDAC4 knockdown on the levels of matrix metalloproteinase 3 (MMP3) and MMP13 induced by IL-1β. After transfection with HDAC4 plasmids, the overexpression efficiency was examined using Real-time quantitative polymerase chain reaction (qRT-PCR) and the levels of MMP3 and MMP13 were assayed using Western blotting. After incubation with IL-1β, the translocation of β-catenin into the nucleus was observed using immunofluorescence staining in SW1353 cells to investigate the activation of the WNT3A/β-catenin signaling pathway. Finally, treatment with WNT3A and transfection with glycogen synthase kinase 3 beta (GSK3β) plasmids were assessed for their effects on HDAC4 levels using Western blotting.@*RESULTS@#IL-1β downregulated HDAC4 levels in chondrocytes and SW1353 cells. Furthermore, HDAC4 knockdown increased the levels of MMP3 and MMP13, which contributed to the degradation of the extracellular matrix. Overexpression of HDAC4 inhibited IL-1β-induced increases in MMP3 and MMP13. IL-1β upregulated the levels of WNT3A, and WNT3A reduced HDAC4 levels in SW1353 cells. GSK-3β rescued IL-1β-induced downregulation of HDAC4 in SW1353 cells.@*CONCLUSION@#HDAC4 exerted an inhibitory effect on IL-1β-induced extracellular matrix degradation and was regulated partially by the WNT3A/β-catenin signaling pathway.