Β-catenin Gene Research Articles

MiR-194 Up-Regulates Cholesterol 7 Alpha-Hydroxylase Expression via β-Catenin Signaling and Aggravates Cholestatic Liver Diseases

miR-194 is abundantly expressed in hepatocytes, and its depletion increases hepatic resistance to acetaminophen-induced acute injuries. In this study, the biological role of miR-194 in cholestatic liver injury was investigated by using miR-194/miR-192 cluster liver-specific knockout (LKO) mice, in which no liver injuries or metabolic disorders were predisposed. Bile duct ligation (BDL) and 1-naphthyl isothiocyanate (ANIT) were applied to LKO and matched control wild-type (WT) mice to induce hepatic cholestasis. Periportal liver damage, mortality rate, and liver injury biomarkers in LKO mice were significantly less than in WT mice after BDL and ANIT injection. Intrahepatic bile acid level was significantly lower in the LKO liver within 48 hours of BDL- and ANIT-induced cholestasis compared with WT. Western blot analysis showed that β-catenin (CTNNB1) signaling and genes involved in cellular proliferation were activated in BDL- and ANIT-treated mice. The expression levels of cholesterol 7 alpha-hydroxylase (CYP7A1), pivotal in bile synthesis, and its upstream regulator hepatocyte nuclear factor 4α were reduced in primary LKO hepatocytes and liver tissues compared with WT. The knockdown of miR-194 using miRNA inhibitors reduced CYP7A1 expression in WT hepatocytes. In contrast, the knockdown of CTNNB1 and overexpression of miR-194, but not miR-192, in LKO hepatocytes and AML12 cells increased CYP7A1 expression. In conclusion, the results suggest that the loss of miR-194 ameliorates cholestatic liver injury and may suppress CYP7A1 expression via activation of CTNNB1 signaling.

MicroRNA-122 mimic/microRNA-221 inhibitor combination as a novel therapeutic tool against hepatocellular carcinoma.

Therapeutic microRNAs (miRNAs) delivery holds a lot of promise for treating human malignancies. So, this study was carried out to examine the potential of miR-122 mimic and/or miR-221 inhibitor as an innovative therapeutic strategy for HCC in an animal model. Mice were categorized into five groups comprising: (1) a normal control group, (2) an HCC group subjected to diethylnitrosamine (DEN) injection for 12 weeks, (3) a miR-122 mimic-treated HCC group, (4) a miR-221 inhibitor-treated HCC group, and (5) a miR-122 mimic/miR-221 inhibitor-treated HCC group. After 16 weeks, all animals were sacrificed and underwent biochemical, miRNAs and genes expression, histopathological, and immunohistochemical examinations. The miR-122 mimic/miR-221 inhibitor combination dramatically reduced the levels of pro-inflammatory, liver cancer, angiogenesis, and cell proliferation markers when compared to either treatment alone. It also down-regulated the expression of cyclin D1, TGF-β, and β-catenin genes, which are involved in promoting cell cycle progression and cancer cell proliferation. Furthermore, it caused the resolution of nearly all the histological malignant features as well as the reduction of malignant cellular markers, including α-smooth muscle actin, arginase-1, and tropomyosin-1. The co-treatment with miR-122 mimic and miR-221 inhibitor amplifies the benefits of either treatment on HCC through targeting the SENP1 and ARF4 genes, respectively. This combination can inhibit cancer cell proliferation and angiogenesis while inducing tumor apoptosis and necrosis. This study demonstrates the therapeutic potential of reversing a dysregulated miRNAs expression pattern in HCC. As a result, future research should concentrate on turning miRNA understanding into therapeutic applications.

The Efficiency of Neurospheres Derived from Human Wharton's Jelly Mesenchymal Stem Cells for Spinal Cord Injury Regeneration in Rats.

Spinal cord injury (SCI) causes inflammation and neuronal degeneration, resulting in functional movement loss. Since the availability of SCI treatments is still limited, stem cell therapy is an alternative clinical treatment for SCI and neurodegenerative disorders. Human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) are an excellent option for cell therapy. This study aimed to induce hWJ-MSCs into neural stem/progenitor cells in sphere formation (neurospheres) by using neurogenesis-enhancing small molecules (P7C3 and Isx9) and transplant to recover an SCI in a rat model. Inducted neurospheres were characterized by immunocytochemistry (ICC) and gene expression analysis. The best condition group was selected for transplantation. The results showed that the neurospheres induced by 10 µM Isx9 for 7 days produced neural stem/progenitor cell markers such as Nestin and β-tubulin 3 through the Wnt3A signaling pathway regulation markers (β-catenin and NeuroD1 gene expression). The neurospheres from the 7-day Isx9 group were selected to be transplanted into 9-day-old SCI rats. Eight weeks after transplantation, rats transplanted with the neurospheres could move normally, as shown by behavioral tests. MSCs and neurosphere cells were detected in the injured spinal cord tissue and produced neurotransmitter activity. Neurosphere-transplanted rats showed the lowest cavity size of the SCI tissue resulting from the injury recovery mechanism. In conclusion, hWJ-MSCs could differentiate into neurospheres using 10 µM Isx9 media through the Wnt3A signaling pathway. The locomotion and tissue recovery of the SCI rats with neurosphere transplantation were better than those without transplantation.

The molecular basis of odontogenic cysts and tumours.

The advances in molecular technologies have allowed a better understanding of the molecular basis of odontogenic cysts and tumours. PTCH1 mutations have been reported in a high proportion of odontogenic keratocyst. BRAF p.V600E are recurrent in ameloblastoma and KRAS p.G12V/R in adenomatoid odontogenic tumour, dysregulating the MAPK/ERK pathway. Notably, BRAF p.V600E is also detected in ameloblastic carcinoma, but at a lower frequency than in its benign counterpart ameloblastoma. Recently, adenoid ameloblastoma has been shown to be BRAF wild-type and to harbour CTNNB1 (β-catenin gene) mutations, further suggesting that it is not an ameloblastoma subtype. CTNNB1 mutations also occur in other ghost-cell-containing tumours, including calcifying odontogenic cysts, dentinogenic ghost cell tumours and odontogenic carcinoma with dentinoid, but the link between CTNNB1 mutations and ghost cell formation in these lesions remains unclear. Regarding mixed tumours, BRAF p.V600E has been reported in a subset of ameloblastic fibromas, ameloblastic-fibrodentinomas and fibro-odontomas, in addition to ameloblastic fibrosarcoma. Such mutation-positivity in a subset of samples can be helpful in differentiating some of these lesions from odontoma, which is BRAF-wild-type. Recently, FOS rearrangements have been reported in cementoblastoma, supporting its relationship with osteoblastoma. Collectively, the identification of recurrent mutations in these aforementioned lesions has helped to clarify their molecular basis and to better understand the interrelationships between some tumours, but none of these genetic abnormalities is diagnostic. Since the functional effect of pathogenic mutations is context and tissue-dependent, a clear role for the reported mutations in odontogenic cysts and tumours in their pathogenesis remains to be elucidated.

Electrical Stimulation Therapy and HA/TCP Composite Scaffolds Modulate the Wnt Pathways in Bone Regeneration of Critical-Sized Defects.

Critical bone defects are the most difficult challenges in the area of tissue repair. Polycaprolactone (PCL) scaffolds, associated with hydroxyapatite (HA) and tricalcium phosphate (TCP), are reported to have an enhanced bioactivity. Moreover, the use of electrical stimulation (ES) has overcome the lack of bioelectricity at the bone defect site and compensated the endogenous electrical signals. Such treatments could modulate cells and tissue signaling pathways. However, there is no study investigating the effects of ES and bioceramic composite scaffolds on bone tissue formation, particularly in the view of cell signaling pathway. This study aims to investigate the application of HA/TCP composite scaffolds and ES and their effects on the Wingless-related integration site (Wnt) pathway in critical bone repair. Critical bone defects (25 mm2) were performed in rats, which were divided into four groups: PCL, PCL + ES, HA/TCP and HA/TCP + ES. The scaffolds were grafted at the defect site and applied with the ES application twice a week using 10 µA of current for 5 min. Bone samples were collected for histomorphometry, immunohistochemistry and molecular analysis. At the Wnt canonical pathway, HA/TCP and HA/TCP + ES groups showed higher Wnt1 and β-catenin gene expression levels, especially HA/TCP. Moreover, HA/TCP + ES presented higher Runx2, Osterix and Bmp-2 levels. At the Wnt non-canonical pathway, HA/TCP group showed higher voltage-gated calcium channel (Vgcc), calmodulin-dependent protein kinase II, and Wnt5a genes expression, while HA/TCP + ES presented higher protein expression of VGCC and calmodulin (CaM) at the same period. The decrease in sclerostin and osteopontin genes expressions and the lower bone sialoprotein II in the HA/TCP + ES group may be related to the early bone remodeling. This study shows that the use of ES modulated the Wnt pathways and accelerated the osteogenesis with improved tissue maturation.

Chronic GPER activation prompted the proliferation of ileal stem cell in ovariectomized mice depending on Paneth cell-derived Wnt3.

Menopausal women often face long-term estrogen treatment. G protein-coupled estrogen receptor (GPER) expressed in intestinal crypt was activated by estrogen therapy, but it was unclear whether chronic GPER activation during menopause had an effect on intestinal stem cells (ISCs). We tested the effect of chronic GPER activation on ISCs of ovariectomized (OVX) mice by injection of the selective GPER agonist G-1 for 28 days, or G-1 stimulation of organoids derived from crypts of OVX mice. G-1 up-regulated crypt depth, the number of Ki67+, bromodeoxyuridine+ cells and Olfm4+ ISCs, and the expression of ISCs marker genes (Lgr5, Olfm4 and Axin2). G-1 administration promoted organoid growth, increased the number of EdU+ cells per organoid and protein expression of Cyclin D1 and cyclin B1 in organoids. After G-1 treatment in vivo or in vitro, Paneth cell-derived Wnt3, Wnt3 effector β-catenin and Wnt target genes c-Myc and Cyclin D1 increased in ileum or organoids. Once blocking the secretion of Wnt3 from Paneth cells, the effects of G-1 on organoids growth, ISCs marker genes and Wnt/β-catenin signaling were abolished. G-1 did not affect the number of Paneth cells in ex vivo organoids, while activated Mmp7/cryptdin program in Paneth cells, promoted their maturation, and increased the expression of lysozyme protein. G-1 pretreatment in OVX mice inhibited radiation-induced ISCs proliferation injury and enhanced the resistance of mice to intestinal injury. In conclusion, chronic GPER activation prompted the Wnt3 synthesis in Paneth cells, thus increased the proliferation of ISCs via activation of Wnt3/β-catenin signaling in OVX mice.

Skin-specific transgenic overexpression of ovine β-catenin in mice.

β-catenin is a conserved molecule that plays an important role in hair follicle development. In this study, we generated skin-specific overexpression of ovine β-catenin in transgenic mice by pronuclear microinjection. Results of polymerase chain reaction (PCR) testing and Southern blot showed that the ovine β-catenin gene was successfully transferred into mice, and the exogenous β-catenin gene was passed down from the first to sixth generations. Furthermore, real-time fluorescent quantitative PCR (qRT-PCR) and western blot analysis showed that β-catenin mRNA was specifically expressed in the skin of transgenic mice. The analysis of F6 phenotypes showed that overexpression of β-catenin could increase hair follicle density by prematurely promoting the catagen-to-anagen transition. The results showed that ovine β-catenin could also promote hair follicle development in mice. We, therefore, demonstrate domestication traits in animals.

AMPK activator decelerates osteoarthritis development by inhibition of β-catenin signaling in chondrocytes.

Osteoarthritis (OA) is a common degenerative joint disease with significant negative impact on the quality of life. It has been reported that abnormal upregulation of β-catenin signaling could lead to OA development; however, the upstream regulatory mechanisms of β-catenin signaling have not been determined. Primary rat chondrocytes and ATDC5 chondrocyte cell line were stimulated with AKT2 and treated with or without metformin, an adenosine 5'-monophosphate-activated protein kinase (AMPK) activator. Westerrn blot analysis, luciferase reporter assay and immunofluorescent (IF) staining were performed to examine changes in β-cateninS552 phosphorylation and β-catenin nuclear translocation in ATDC5 cells and in primary chondrocytes. We found that metformin inhibited β-cateninS552 phosphorylation in ATDC5 cells and in primary chondrocytes in a time-dependent manner. Metformin inhibited β-catenin nuclear translocation and β-catenin reporter activity. In addition, metformin also attenuated the expression of β-catenin downstream target genes. We also demonstrated that metformin inhibited β-cateninS552 phosphorylation in articular cartilage in mice. These findings suggest that metformin may exert its chondro-protective effect at least in part through the inhibition of β-catenin signaling in chondrocytes. This study demonstrated the interaction between AMPK and β-catenin signaling in chondrocytes and defined novel molecular targets for the treatment of OA disease.

Diallyl trisulfide ameliorates bone loss and alters specific gut microbiota and serum metabolites in natural aging mice.

Aging is a major cause of bone loss and osteoporosis. Diallyl trisulfide (DATS), one of the main organic sulfides in garlic oil, has been shown to alleviate arthritis in mice. However, further research is still needed to determine how DATS affects bone formation and bone loss in aging mice. Here, we established a mouse model of natural aging for dietary DATS intervention. DATS treatment improved the bone microstructure, including the disorganized arrangement of bone trabeculae and promoted collagen synthesis, as confirmed by micro-CT and histological analyses. The abundance of beneficial bacteria for bone formation, such as Clostridiaceae and Erysipelotrichaceae, and the microbial diversity and community richness were all altered by DATS, according to 16S rRNA sequencing data. 14 potential biomarkers and 9 important metabolic pathways were examined using serum metabolomics analysis. Additionally, there has been a significant reduction in sphingosine, which is directly associated with bone metabolism. The level of sphingosine and relative abundance of Clostridium were found to be negatively correlated by correlation analysis, indicating that bacteria may regulate bone reconstruction via influencing metabolites. Furthermore, Runx2 and β-catenin gene expression levels increased in bones, which may be related to the ameliorative mechanism of DATS. Our results suggested that DATS may prevent age-related bone loss by upregulating osteogenic gene expression through altering gut microbes and serum metabolism.

Multi-Regulatory Effects of Agmatine on Akt/GSK-3β/β-Catenin Signaling Pathway in SH-SY5Y cells: A Potential Explanation for its Conflicting Roles in Schizophrenia

Objectives: Schizophrenia is a common psychiatric disease with its complex neurobiology. The current knowledge about the neurobiology of schizophrenia is based on dopaminergic and glutamatergic dysregulation of the central nervous system. The facts that all current antipsychotic drugs act on dopaminergic D2 receptor antagonism and glutamatergic N-Methyl D-Aspartate (NMDA) receptor antagonists reveal schizophrenia-like behavioral and structural alteration in rodents and humans are the major evidences of these hypotheses. However, they are not sufficient to explain the disease neurobiology and lead novel therapeutics. In recent years, it has been shown that the functions of certain intracellular pathways are disrupted in schizophrenia, especially Akt/glycogen synthase kinase 3beta (GSK-3β)/β-catenin signaling pathway. Certain studies showed that GSK-3β inhibition accompanied to the effects of antipsychotic drugs and specific inhibition of the GSK-3β might be a valuable approach for the novel treatments for schizophrenia. Agmatine is an endogenous amine that interacts with many receptors and enzymes including adrenergic, glutamatergic, cholinergic, serotonergic and imidazoline receptors and nitric oxide synthase enzyme. The limited number of studies showed conflicting results about the role of agmatine on schizophrenia and there was no study to evaluate the intracellular molecular mechanisms of agmatine`s schizophrenia-related effects. Herein, we aimed to investigate the effects of agmatine on MK-801 induced neurotoxicity and the disruption of the Akt/GSK-3β/β-catenin signaling pathway in SH-SY5Y human neuroblastoma cells. Methods: SH-SY5Y cells were grouped as the medium, agmatine (50μM), agmatine (100μM), olanzapine (10μM), MK-801 (100μM), MK-801+Agmatine (50μM), MK-801+Agmatine (100μM) and MK-801+Olanzapine (10μM). Real-time cell analysis (RTCA), real-time polymerase chain reaction (Rt-PCR) and western blot (WB) were used for monitoring the cell viabilities, quantifying gene and protein expressions, respectively. Cell viabilities monitored for the 24 hours and comparative viabilities (%) were calculated at the 6th, 12th and 24th hours of treatments. Akt, GSK-3β and β-Catenin gene expressions phosphorylated (p) and non-phosphorylated protein expressions were investigated at the 24th hour of treatments. Results: In RTCA, MK-801 administration decreased cell viabilities at the 6th (p<0.01) and 24th (p<0.001) hours compared to medium in cells. Agmatine (50μM) and agmatine (100μM) pre-treatments increased the cell viabilities at the 6th (p<0.05), 12th (p<0.01), and 24th (p<0.05) hours while olanzapine at 24th (p<0.05) compared to MK-801. In Rt-PCR, MK-801 administration increased GSK-3B decreased CTNNB1 gene expressions (p<0.001) compared to the medium. Agmatine (50μM) and olanzapine reversed (p<0.001) the effects of MK-801 for both gene expressions while agmatine (100μM) reversed (p<0.001) only GSK-3B. In WB, MK-801 administration decreased the p-GSK-3β/GSK-3β ratio compared to medium while only agmatine (50μM) increased the ratio compared to MK-801 administration. Conclusion: Herein, it has been shown that agmatine had a neuroprotective effect on MK-801 induced cell toxicity in SH-SY5Y cells. Our results have suggested a low dose (50μM) agmatine shows antipsychotic like effects on Akt/GSK-3β/β-catenin signaling pathway in SH-SY5Y cells bot, not high dose (100μM). We have suggested that these doses dependent regulatory effects of agmatine might be a possible explanation for its conflicting role in schizophrenia.

The expression of NOTUM in replantation of severed fingers may be an important treatment factor.

After years of development, digital replantation has become a mature treatment. Although the NOTUM gene has been shown to be involved in the formation of vertebrate nerves, whether it contributes to the osteogenic mechanism of severed finger replantation remains unknown. In response to this, this study investigates the specific details of NOTUM involvement in replantation of severed fingers. The experimental subjects are patientswithreplantation of severed fingers from Shulan International Medical College of Shulan (Hangzhou) Hospital affiliated to Zhejiang Shuren University. In addition to using bone marrow mesenchymal stem cells (BMSCs) as an in vitro system, this experiment also involves quantitative polymerase chain reaction, microarray analysis, cell counting Kit-8, ethynyl deoxyuridine staining and Western blot analysis. The expression level of NOTUM in the severed finger replantation group is lower than that in the normal group. NOTUM inhibits cell growth and cell transfer, osteogenic differentiation and β-catenin gene expression in BMSCs. Luciferase reporter assay illustrated that β-catenin wild type closely correlated with NOTUM. The inhibition of β-catenin increases the effects of NOTUM on cell growth, cell transfer and osteogenic differentiation of BMSCs. Considering that NOTUM can inhibit cell growth, cell transfer, osteogenic differentiation of BMSCs, as well as the gene expression of β-catenin, it may be a biomarker of osteogenic differentiation and a potential therapeutic target for replantation of severed fingers.

Evaluation of in vivo lithium chloride effects as a GSK3-β inhibitor on human adipose derived stem cells differentiation into oligodendrocytes and re-myelination in an animal model of multiple sclerosis

The application of neuroprotective agents in combination with stem cells is considered a potential effective treatment for multiple sclerosis (MS). Therefore, the effects of lithium chloride as a neuroprotective agent and a GSK3-β inhibitor were evaluated in combination with human adipose derived stem cells on re-myelination, oligodendrocyte differentiation, and functional recovery. After inducing a mouse model of MS and proving it by the hanging wire test, the mice were randomly assigned to five experimental groups: Cup, Sham, Li, hADSC, and Li + hADSC. Additionally, a control group with normal feeding was considered. Finally, toluidine blue staining was carried out to estimate the level of myelination. Furthermore, immunofluorescent staining was used to evaluate the mean of OLIG2 and MOG positive cells. The mRNA levels of β-Catenin, myelin and oligodendrocyte specific genes were determined via the Real-Time PCR. The results of the hanging wire test and toluidine blue staining showed a significant increase in myelin density and improvements in motor function in groups, which received lithium and stem cells, particularly in the Li + hADSC group compared with the untreated groups (P < 0.01). Moreover, immunostaining results indicated that the mean percentages of MOG and OLIG2 positive cells were significantly higher in the Li + hADSC group than in the other groups (P < 0.01). Finally, gene expression studies indicated that the use of lithium could increase the expression of β-Catenin, myelin and oligodendrocyte specific genes. The use of Lithium Chloride can increase stem cells differentiation into oligodendrocytes and improve re-myelination in MS.

Effect of Co-culturing both placenta-derived mesenchymal stem cells and their condition medium in the cancer cell (HepG2) migration, damage through apoptosis and cell cycle arrest

Human placental-derived mesenchymal stem cells (hPMSCs) are a promising candidate to inhibit the proliferation of hepatocellular carcinoma (HCC) cell lines such as HepG2. The effects of hPMSCs and their conditioned media on HepG2 are, however, still a mystery. As a result, the goal of this study was to look into the effects of hPMSCs and their conditioned media on HepG2 and figure out what was going on. Fluorescence-activated cell sorting and the MTT test were used to determine the percentage of cells that died (early apoptosis, late apoptosis). The DIO and DID colors were used to detect cell fusion and cell death in both cells. HepG2 cells were co-treated with hPMSCs or hPMSCs-conditioned medium (hPMSCs-CM) to reduce growth and promote apoptosis. Morphological changes were also seen in the 30 percent, 50 percent, and 60 percent cases. The secretion of cytokine was determined by the ELISA. Flow cytometry, caspase 9 immunofluorescence, qPCR (detection of Bax, Bcl-2, and β-catenin genes), western blot, and immunophenotyping revealed that treatment with hPMSCs or hPMSCs-CM caused HepG2 cell death through apoptosis (detection of caspase 9, caspase 3 protein). HepG2 cell cycle arrest could be induced by hPMSCs and hPMSCs-CM. Following treatment with hPMSCs or hPMSCs-CM, HepG2 cell development was stopped in the G0/G1 phase. These treatments also inhibited HepG2 cells from migrating, with the greatest effect when the highest ratio/concentration of hPMSCs (70%) and hPMSCs-CM were used (90%). Our findings indicated that hPMSCs and hPMSCs-CM could be promising treatment options for liver cancer. To elucidate the proper effect, more research on liver cancer-induced rat/mice is needed.

Identification of odontogenic ameloblast associated as a novel target gene of the Wnt/β-catenin signaling pathway.

The Wnt/β-catenin signaling pathway plays a key role in development and carcinogenesis. Although some target genes of this signaling have been identified in various tissues and neoplasms, the comprehensive understanding of the target genes and their roles in the development of human cancer, including hepatoma and colorectal cancer remain to be fully elucidated. In this study, we searched for genes regulated by the Wnt signaling in liver cancer using HuH-7 hepatoma cells. A comparison of the expression profiles between cells expressing an active form of mutant β-catenin and cells expressing enhanced green fluorescent protein (EGFP) identified seven genes upregulated by the mutant β-catenin gene (CTNNB1). Among the seven genes, we focused in this study on ODAM, odontogenic,ameloblast associated, as a novel target gene. Interestingly, its expression was frequently upregulated in hepatocellular carcinoma, colorectal adenocarcinoma, and hepatoblastoma. We additionally identified a distant enhancer region that was associated with the β-catenin/TCF7L2 complex. Further analyses revealed that ODAM plays an important role in the regulation of the cell cycle, DNA synthesis, and cell proliferation. These data may be useful for clarification of the main molecular mechanism(s) underlying these cancers.

478 MICAL2, upregulated by β-catenin, induces invasion in melanoma

The Wnt/β-catenin signaling pathway plays a key role during embryonic development, homeostasis and cancer. The role of β-catenin in melanocyte lineage establishment and homeostasis has been elucidated, but its role in melanoma remains poorly defined. In particular, its function in cell invasion is controversial. Especially, we generated a signature of β-catenin activation to emerge proteins of interest including known β-catenin target genes (AXIN2, APCDD1, NKD1, SP5, NOTUM, ZNRF3, CCND1 and PPARD) and new genes (SLC1A5, SLC7A11, SLC24A4 and MICAL2).

TMEM196 inhibits lung cancer metastasis by regulating the Wnt/β-catenin signaling pathway.

The TMEM196 protein, which comprises four membrane-spanning domains, belongs to the TMEM protein family. TMEM196 was identified as a candidate tumor suppressor gene in lung cancer. However, its role and mechanism in lungcancer metastasis remain unclear. Here, we study the role of TMEM196 in tumor metastasis to further verify the function in lung cancer. In this study, we used qRT-PCR, western blot analysis and immunohistochemistry to examine the expression levels of TMEM196 and related proteins in lung cancer tissues and tumor cells. We utilized Transwell assays, xenograft nude mouse models, and TMEM196-/- mouse models to evaluate the effects of TMEM196 on tumor invasion and metastasis. Finally, we used bioinformatics analysis and dual-luciferase reporter gene assays to explore the molecular mechanism of TMEM196 as a tumor suppressor. We found that TMEM196 mRNA and protein expression levels were significantly decreased in lung cancer tissues and cells. Low expression of TMEM196 in clinical patients was associated with poor prognosis. TMEM196 strongly inhibited tumor metastasis and progression in vitro and in vivo. The primary lung tumors induced by tail vein-inoculated B16 cells in TMEM196-/- mice were significantly larger than those in TMEM196+/+ mice. Mechanistically, TMEM196 inhibited the Wnt signaling pathway and repressed β-catenin promoter transcription. TMEM196 silencing in lung cancer cells and mice resulted in significant upregulation of the expression of β-catenin and Wnt signaling pathway downstream target genes (MMP2 and MMP7). Decreasing β-catenin expression in TMEM196-silenced cancer cells attenuated the antimetastatic effect of TMEM196. Our results revealed that TMEM196 acts as a novel lung cancer metastasis suppressor via the Wnt/β-catenin signaling pathway.

Pygo1 Regulates the Behavior of Human Non-Small-Cell Lung Cancer via the Wnt/β-Catenin Pathway.

Abnormal activation of the classical Wnt pathway has been reported in non-small-cell lung cancer (NSCLC) previously. Pygo family genes, the core regulators of Wnt/β-catenin signaling, were also reported to be involved in tumorigenesis. However, the role of the homolog Pygo1 in human lung cancer remains unclear. In the current study, we demonstrated an association of increased Pygo1 expression with consistent high nuclear β-catenin signals across pathological tissue samples of early-stage human NSCLC. Overexpression of Pygo1 in lung cancer cells resulted in enhanced G1/S cell phase transformation, reduced apoptosis, and increased cell proliferation. These changes were accompanied by the downregulation of cell cycle-related proteins, such as RB, p16, p53, and p27Kip1, and increased expression of CyclinE1. Migration, wound healing, and colony formation assays revealed that Pygo1 overexpression enhanced the invasion and migration of lung cancer cells, increased the formation of clones, and suppressed E-cadherin expression. In addition, overexpression of Pygo1 in lung cancer cells led to an increase of β-catenin/TCF4 complex, as well as upregulated expression of target genes of β-catenin. In vivo experiments also revealed that Pygo1 overexpression promoted the tumorigenicity of a xenograft tumor model, while Wnt inhibition partially blocked the effect of Pygo1 overexpression. In conclusion, Pygo1 affects human NSCLC via the canonical Wnt/β-catenin pathway, which provides new clues for lung cancer pathology.

RF33 | PSAT79 Chronic Activation of Gq-Signaling in the Mouse Adrenal Cortex Disrupts Zonation and Causes Aldosterone Production in the Zona Fasciculata

Abstract Background The mammalian adrenal cortex is composed of the zona glomerulosa (ZG), zona fasciculata (ZF), and in some species the zona reticularis (ZR). Each layer is characterized by a specific steroidogenic enzyme expression pattern that leads to selective production of mineralocorticoids, glucocorticoids or adrenal androgens. ZG-specific expression of cytochrome P450 aldosterone synthase (Cyp11b2) and aldosterone biosynthesis are renin-angiotensin-dependent processes in which the Gq-signaling pathway is activated by angiotensin II (AngII). Gq-signaling can be exogenously activated using Gq-coupled designer receptors exclusively activated by designer drug (GqDREADD) technology. Using transgenic mice with targeted expression of GqDREADD to the adrenal cortex, we previously demonstrated that short-term receptor activation increased adrenal Cyp11b2 and aldosterone production. Herein, we hypothesized that prolonged activation of mouse adrenal cortex Gq-signaling would exacerbate the aldosterone levels and promote the transdifferentiation of ZF cells to ZG cells. Methods Transgenic AS+/Cre hM3Dq (GqDREADD) female mice were engineered through crossing GqDREADD mice with Cyp11b2-regulated Cre recombinase mice. Mice were placed on a high sodium diet (to suppress physiologic renin) and orally provided the GqDREADD ligand, clozapine N-oxide (CNO; n=10) or control (n=8) for 6 weeks followed by collection of blood and adrenals. Mineralocorticoid levels were quantified using LC-MS/MS. Adrenal RNA was isolated and assessed by RNAseq for transcriptomic changes, and RT-qPCR analysis for ZG- and ZF-specific transcript markers, Cyp11b2 and Cyp11b1 (marker of ZF), respectively. Transcripts for β-catenin (Ctnnb1) and axin-2 (Axin2) (markers of the WNT signaling pathway) were also analyzed. Immunofluorescence (IF) studies examined adrenal expression of Cyp11b1 and Cyp11b2 in mice treated for 1 month. Results CNO treatment caused plasma aldosterone to increase 613-fold from 7.9 ±2.5 pg/mL to 4810.7 ±1110.7 pg/mL (p&amp;lt;0.01). Levels of 18-hydroxycorticosterone increased 70-fold from 144.9 ±22.0 to 10143.2 ±1479.6 pg/mL (p&amp;lt;0.0001). Corticosterone levels were unaffected. RT-qPCR analysis showed adrenal Cyp11b2 transcripts to be 224-fold higher (p&amp;lt;0.0001) in the CNO-treated group while a 30% decrease in Cyp11b1 levels was observed (p&amp;lt;0.01) compared to the controls. IF demonstrated hybrid ZF cells aberrantly expressing both Cyp11b2 and Cyp11b1. Within the normal mouse adrenal, the outer ZG exhibits high WNT/β-catenin (Ctnnb1) activation. To determine if chronic CNO treatment impacted overall adrenal WNT signaling, Ctnnb1 and Axin2 (a downstream β-catenin gene target) were examined. Analysis revealed no statistical difference in Ctnnb1 and Axin2 transcript levels in treated mice. RNAseq revealed that 242 transcripts were increased in adrenals from treated mice, while 109 decreased (&amp;gt;3-fold change; p&amp;lt;0.01). Induced transcripts include Cyp11b2 and its transcriptional regulators, Nr4a1 and Nr4a2. Conclusion Overall, these findings demonstrate that chronic Gq-signaling is sufficient to cause ZF cells to produce aldosterone and express Cyp11b2. The findings also suggest that the adrenocortical WNT signaling pathway is not increased by the activation of Gq-signaling. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Monday, June 13, 2022 12:36 p.m. - 12:41 p.m.

Anti-proliferative action, molecular investigation and computational studies of novel fused heterocyclic cellulosic compounds on human cancer cells

Reactivity of MCEC (3) with diazonium chloride, DMF-DMA and NH2NH2 to afford fused heterocyclic cellulosic derivative which confirmed through spectral analysis. Also, the cellulosic compounds were evaluated their growth inhibitory activity contra cancer cells A549 and Caco2 using neutral red uptake assay. Compounds, MCEN(6), MCPT(5a), MCPT(5b) showed better growth inhibitory activity on A549 and Caco2 growth compared to control values. While compound MCPY(7) exerted the best cytotoxic activity against A549 cells after 48 h. The expression levels of HIF-1α, β-Catenin, MYC, Cyclin D1 and MMP7 genes in A549 cells were examined using QRT-PCR. The compounds MCEN(6) and MCPY(7) down regulated levels of β-Catenin, MYC, Cyclin D1 and MMP7 genes and up-regulated levels of HIF-1α when treated with A549 cells compared to control values. Also, these biological studies confirmed through docking stimulation with different proteins and showed least binding energy with amino acids and attached with NH2 and OH of cellulose with hydrogen bond interaction. Moreover, optimization of compounds using DFT/6-311(G) showed the stability of them and identifies their physical descriptors which showed excellent correlation with experimental results. Noteworthy, compounds, MCEN(6) and MCPY(7) were most promising anticancer agents against lung cancer through the regulation of apoptosis, cell cycle, proliferation, progression, chemo-resistance, and angiogenesis.

Secondary bile acids mediate high-fat diet-induced upregulation of R-spondin 3 and intestinal epithelial proliferation.

A high-fat diet (HFD) contributes to the increased incidence of colorectal cancer, but the mechanisms are unclear. We found that R-spondin 3 (Rspo3), a ligand for leucine-rich, repeat-containing GPCR 4 and 5 (LGR4 and LGR5), was the main subtype of R-spondins and was produced by myofibroblasts beneath the crypts in the intestine. HFD upregulated colonic Rspo3, LGR4, LGR5, and β-catenin gene expression in specific pathogen-free rodents, but not in germ-free mice, and the upregulations were prevented by the bile acid (BA) binder cholestyramine or antibiotic treatment, indicating mediation by both BA and gut microbiota. Cholestyramine or antibiotic treatments prevented HFD-induced enrichment of members of the Lachnospiraceae and Rumincoccaceae, which can transform primary BA into secondary BA. Oral administration of deoxycholic acid (DCA), or inoculation of a combination of the BA deconjugator Lactobacillus plantarum and 7α-dehydroxylase-containing Clostridium scindens with an HFD to germ-free mice increased serum DCA and colonic Rspo3 mRNA levels, indicating that formation of secondary BA by gut microbiota is responsible for HFD-induced upregulation of Rspo3. In primary myofibroblasts, DCA increased Rspo3 mRNA via TGR5. Finally, we showed that cholestyramine or conditional deletion of Rspo3 prevented HFD- or DCA-induced intestinal proliferation. We conclude that secondary BA is responsible for HFD-induced upregulation of Rspo3, which, in turn, mediates HFD-induced intestinal epithelial proliferation.