Protein Expression Levels Of Β-catenin Research Articles

Mitotic Arrest Deficient 2 Like 1 Contributes to Colorectal Cancer Cell Migration, Invasion, and Oxaliplatin Resistance Through the Wnt/β-Catenin Pathway.

Colorectal cancer (CRC) is a highly prevalent malignancy, requiring chemotherapy for advanced stages of the disease. Previously, we found that mitotic arrest deficient 2 like 1 (MAD2L1) was upregulated and facilitated malignant proliferation in CRC. However, the association between MAD2L1 expression and tumor progression, as well as chemotherapy resistance in CRC, remains unclear. The progression capacities of CRC cells were assessed using transwell and wound healing assays, and the resistance to cisplatin in oxaliplatin-resistant CRC cells was assessed using CCK-8 assay and flow cytometry. Relevant protein levels of epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin pathway were analyzed using western blotting. Revealing the impact of MAD2L1 on metastasis and drug resistance in CRC through inhibition of the Wnt/β-catenin pathway. Knockdown of MAD2L1 attenuated the malignant progression of CRC cells, inhibited EMT, and blocked the Wnt/β-catenin pathway. MAD2L1 was significantly upregulated in oxaliplatin-resistant CRC cells, accompanied by the activation of the Wnt/β-catenin pathway. Knockdown of MAD2L1 effectively reversed oxaliplatin resistance, leading to apoptosis and downregulation of the protein expression levels of β-catenin, P-glycoprotein (P-gp), and ABCG2. After the knockdown of MAD2L1, the inhibition of the Wnt/β-catenin pathway exhibited a synergistic effect, effectively suppressing malignant progression and reversing oxaliplatin resistance in CRC cells. So, knockdown of MAD2L1 suppressed cell malignant progression, equally sensitized resistant CRC cells to oxaliplatin, potentially by blocking the activation of the Wnt/β-catenin pathway.

Abstract C141: Inactivating the mitotic kinases TTK and NEK2 as a promising approach for the reduction of metastasis in triple-negative breast cancer in non-Hispanic Blacks and Hispanic/Latinas

Abstract Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer in women worldwide. It is associated with uncontrolled cell proliferation, increased rates of centrosome amplification, genetic instability, and invasive capacity. Advanced stages of TNBC with distant metastasis are virtually incurable, with chemotherapy as the primary treatment option. Non- Hispanic Black (NHB) and Hispanic/Latino (H/L) women have lower 5-year survival rates when compared to non-Hispanic White (NHW) women with breast cancer, in part because their breast tumors are detected at higher stages, and due to elevated rates of TNBC. Women with a higher percentage of African ancestry have been shown to have a higher expression of multiple mitotic kinases. Mitotic kinases, such as TTK, maintain genomic integrity, while NEK2 modulates centrosome separation and microtubule organization. However, their overexpression in breast cancer is associated with cell proliferation, chromosome instability, aneuploidy, and early stages of metastasis (the epithelial-to-mesenchymal transition, or EMT, cell migration and invasion). A tissue microarray confirmed associations between TTK overexpression, proliferation, and EMT markers in breast tumors and the TNBC subtype. Thus, we aim to measure protein and mRNA expression levels of EMT markers upon downregulation of TTK and NEK2. To address this, mesenchymal TNBC cell lines, MDA-MB-231 (NHW) or MDA-MB-157 (NHB), were cultured in DMEM and 10% FBS, followed by TTK siRNA, NEK2 siRNA, or double transfection. TRIzol was then used for protein and RNA extraction. Western blot and qPCR analyses measured EMT markers' protein and mRNA expression levels, respectively. Preliminary results from these experiments showed that silencing of TTK, NEK2, or TTK/NEK2 resulted in a reduction of the mRNA expression levels of Vimentin, N-cadherin, and β-Catenin in MDA-MB- 231 cell lines. Moreover, data showed a decrease in Vimentin in all treatment groups at the protein level. TTK/NEK2 knockdown also reduced N-cadherin and β-Catenin protein expression levels. These preliminary data suggest that silencing of mitotic kinases TTK and NEK2 may be a promising approach for targeting early stages of metastasis in TNBCs. Because of their increased expression in women of African heritage, anti-mitotic kinase inactivation can be a novel approach that can highly benefit women of the West African diaspora with breast cancer, including African American and Caribbean Hispanics, specifically by diminishing cancer cell survival and metastasis. Citation Format: Alexandra N Aquino-Acevedo, Ángel D. Colón-Burgos, Gretchen M. Albarrán-Acosta, Marileana Rodríguez-Ruiz, Joel A. Orengo-Orengo, Melanie E. Cruz- Robles, Harold I. Saavedra. Inactivating the mitotic kinases TTK and NEK2 as a promising approach for the reduction of metastasis in triple-negative breast cancer in non-Hispanic Blacks and Hispanic/Latinas [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C141.

Impact of heterogeneous nuclear ribonucleoprotein A/B subtype overexpression on the expression of cancer stem cell markers CD133 and CD44 and cellular proliferation capacity.

The occurrence and progression of intestinal cancer are complex, multifactorial processes in which tumor stem cells are believed to play a crucial role. An in-depth understanding of their molecular mechanisms holds imperative clinical significance for improving intestinal cancer treatment. HT-29 and HCT116intestinal cancer cell lines were utilized as research models. The experimental group (group E) and control group (group C) were established by transfecting the hnRNPA1 subtype and empty vector, respectively. The expression (EP) levels of hnRNPA1 and changes in Wnt/β-catenin signaling-related markers were evaluated using techniques such as RNA extraction, reverse transcription reactions, real-time quantitative PCR (RT qPCR), and protein extraction. The EP of tumor stem cell markers CD133 and CD44 was assessed using immunohistochemistry. Additionally, cell invasion assays, scratch assays, and cell counting kit-8 (CCK-8) proliferation assays were conducted. Furthermore, a mouse tumor model was established to observe the growth of tumors in both groups. overexpression (OP) of hnRNPA1 in group E greatly increased the protein EP levels of β-catenin, Axin2, and Cyclin D1 and exhibited a higher positivity rate for CD133 and CD44. The invasive, migratory, and proliferative abilities of cells in group E were notably enhanced, and tumor growth was observably faster versus group C. OP of hnRNPA1 was closely associated with the activation of Wnt/β-catenin signaling and promoted the proliferation and invasive capacity of tumor cells. hnRNPA1 played an imperative role in intestinal cancer stem cells.

BMSCs alleviate liver cirrhosis by regulating Fstl1/Wnt/β-Catenin signaling pathway

Researchers have shown that bone mesenchymal stem cells (BMSCs) can alleviate the progression of liver cirrhosis; however, it is unclear how exactly BMSCs function to cure liver disease. In this study, we used bioinformatics methods to assess differentially expressed genes (DEGs) in liver cirrhosis and found a significantly upregulated gene, Fstl1, in liver cirrhosis. In vivo and in vitro experiments showed that compared with those in the disease model group, the mRNA, and protein expression levels of Fstl1 were significantly reduced after BMSCs treatment, and the β-Catenin protein level was also significantly reduced after BMSCs treatment. Subsequently, we downregulated Fstl1 in activated hepatic stellate cells (HSCs) and found that Wnt and β-Catenin protein expression levels also decreased. Finally, we found that in BMSCs-treated activated HSCs, overexpression of Fstl1 reversed the inhibitory effect of BMSCs on the Wnt/β-Catenin signaling pathway to a certain extent. In summary, our results show that BMSCs can inhibit Wnt/β-Catenin signaling pathway activation by downregulating the protein expression level of Fstl1, thus alleviating cirrhosis. Therefore, targeted regulation of Fstl1 may provide a new therapeutic strategy for the progression of liver cirrhosis.

Low Levels of Amyloid Precursor Protein (APP) Promote Neurogenesis and Decrease Gliogenesis in Human Neural Stem Cells.

Amyloid precursor protein (APP) has been widely studied due to its association with Alzheimer's disease (AD). However, the physiological functions of APP are still largely unexplored. APP is a transmembrane glycoprotein whose expression in humans is abundant in the central nervous system. Specifically, several studies have revealed the high expression of APP during brain development. Previous studies in our laboratory revealed that a transient increase in APP expression induces early cell cycle exit of human neural stem cells (hNSCs) and directs their differentiation towards glial cells (gliogenesis) while decreasing their differentiation towards neurons (neurogenesis). In the present study, we have evaluated the intrinsic cellular effects of APP down-expression (using siRNA) on cell death, cell proliferation, and cell fate specification of hNSCs. Our data indicate that APP silencing causes cellular effects opposite to those obtained in previous APP overexpression assays, inducing cell proliferation in hNS1 cells (a model line of hNSCs) and favoring neurogenesis instead of gliogenesis in these cells. In addition, we have analyzed the gene and protein expression levels of β-Catenin as a possible molecule involved in these cellular effects. These data could help to understand the biological role of APP, which is necessary to deepen the knowledge of AD.

Differential histological features and myogenic protein levels in distinct muscles of d-sarcoglycan null muscular dystrophy mouse model.

Skeletal muscle (SkM) comprises slow and fast-twitch fibers, which differ in molecular composition, function, and systemic energy consumption. In addition, muscular dystrophies (DM), a group of diverse hereditary diseases, present different patterns of muscle involvement, progression, and severity, suggesting that the regeneration-degeneration process may differ depending on the muscle type. Therefore, the study aimed to explore the expression of proteins involved in the repair process in different muscles at an early stage of muscular dystrophy in the δ-sarcoglycan null mice (Sgcd-null), a limb-girdle muscular dystrophy 2F model. Hematoxylin & Eosin (H&E) Staining showed a high number of central nuclei in soleus (Sol), tibialis (Ta), gastrocnemius (Gas), and extensor digitorum longus (Edl) from four months Sgcd-null mice. However, fibrosis, determined by trichrome of Gomori modified staining, was only observed in Sgcd-null Sol. In addition, the number of Type I and II fibers variated differentially in the Sgcd-null muscles vs. wild-type muscles. Besides, the protein expression level of β-catenin, myomaker, MyoD, and myogenin also presented different expression levels in all the Sgcd-null muscles studied. In summary, our study reveals that muscles with different metabolic characteristics showed distinct expression patterns of proteins involved in the muscle regeneration process. These results could be relevant in designing therapies for genetic and acquired myopathy.

Differential expression levels of β-catenin are associated with invasive behavior of both functional and non-functional pituitary neuroendocrine tumor (PitNET).

Although research continues to elucidate the molecular mechanism underlying pituitary tumor pathogenesis, limited information is available on the potential role and expression profile of β-catenin in functional and non-functional pituitary neuroendocrine tumors (PitNETs). In the current study, 104 pituitary samples (tumors and cadaveric healthy pituitary tissues) were included and the gene and protein expression levels of β-catenin were assessed by Real-Time PCR and immunohistochemistry, respectively. The correlation between expression level of β-catenin and tumor invasive feature and size as well as patient age, gender, and hormonal level was measured. The data showed that PitNET samples expressed higher levels of the β-catenin gene and protein compared to healthy pituitary tissues. Although there was no difference in β-catenin expression level between non-functioning (NF-PitNETs) and growth hormone-producing tumors (GH-PitNETs), both tumor types showed significantly elevated β-catenin levels compared to healthy pituitary tissues. The high level of β-catenin in the invasive functional and non-functional tumors is indicative of the association of β-catenin with PitNETs invasion. The expression pattern of the β-catenin gene and protein was consistently and significantly associated with these tumor types. The correlation between β-catenin and insulin-like growth factor 1 (IGF-1) in GH-PitNETs indicates the potential relevance of β-catenin and IGF-1 for GH-PitNETs. The simultaneous increase in the expression of β-catenin gene and protein level in PitNET tissues and their relationship to the tumor severity indicates the possible contributing role of β-catenin and its underlying signaling mediators in PitNET pathogenesis.

Isoquercitrin restrains the proliferation and promotes apoptosis of human osteosarcoma cells by inhibiting the Wnt/β-catenin pathway

Currently, chemotherapeutic drugs are widely used for the treatment of osteosarcoma. However, many of these drugs exhibit shortcomings such as poor efficacy, high toxicity, and tolerance. Isoquercitrin (ISO) is a traditional Chinese medicine that has been proved to exert good therapeutic effects on various tumors; however, its role in osteosarcoma has not been reported. Here, we observed that ISO exerted a marked inhibitory effect on the occurrence and development of osteosarcoma in a time- and dose-dependent manner. First, we determined that ISO significantly inhibited proliferation, induced EMT-related migration and invasion and induced apoptosis of osteosarcoma cells in vitro. Concurrently, we also observed that both β-catenin and its downstream genes (c-Myc, CyclinD1, and Survivin) were significantly down-regulated. To verify if the anti-tumor effect of ISO was related to the Wnt/β-catenin signaling pathway, we altered the protein expression level of β-catenin using recombinant lentivirus, then we observed that the effects of ISO on the proliferation, metastasis, and apoptosis of osteosarcoma cells were significantly reversed. Additionally, we used a nude mouse xenograft model and observed that ISO significantly inhibited the growth of osteosarcoma and improved the survival rate of the animal models. In conclusion, this study demonstrates that ISO can exert anti-tumor effects in part by inhibiting the Wnt/β-catenin signaling pathway, thus providing a new potential therapeutic strategy for the treatment of osteosarcoma.

Effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/PPARγ pathway in arrhythmic rats

This paper aimed to investigate the effect of total flavonoids of buckwheat flower and leaf on myocardial cell apoptosis and Wnt/β-catenin/peroxisome proliferator-activated receptor γ(PPARγ) pathway in arrhythmic rats. SD rats were randomly divided into a control group, a model group, a low-dose(20 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a medium-dose(40 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a high-dose(80 mg·kg~(-1)) group of total flavonoids of buckwheat flower and leaf, a propranolol hydrochloride(2 mg·kg~(-1)) group, with 12 rats in each group. Except the control group, rats in other groups were prepared as models of arrhythmia by sublingual injection of 1 mL·kg~(-1) of 0.002% aconitine. After grouping and intervention with drugs, the arrhythmia, myocardial cells apoptosis, myocardial tissue glutathione peroxidase(GSH-Px), catalase(CAT), malondialdehyde(MDA), serum interleukin-6(IL-6), prostaglandin E2(PGE2) levels, myocardial tissue apoptosis, and Wnt/β-catenin/PPARγ pathway-related protein expression of rats in each group were measured. As compared with the control group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA levels in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels increased significantly in the model group, whereas the GSH-Px and CAT levels, and Bcl-2 and PPARγ protein expression levels in myocardial tissues reduced significantly. As compared with the model group, the arrhythmia score, the number of ventricular premature beats, ventricular fibrillation duration, myocardial cell apoptosis rate, MDA leve in myocardial tissues, serum IL-6 and PGE2 levels, Bax in myocardial tissues, and Wnt1 and β-catenin protein expression levels reduced in the drug intervention groups, whereas the GSH-Px and CAT levels and Bcl-2 and PPARγ protein expression levels in myocardial tissues increased. The groups of total flavonoids of buckwheat flower and leaf were in a dose-dependent manner. There was no significant difference in the levels of each index in rats between the propranolol hydrochloride group and the high-dose group of total flavonoids of buckwheat flower and leaf. The total flavonoids of buckwheat flower and leaf inhibit the activation of Wnt/β-catenin pathway, up-regulate the expression of PPARγ, reduce oxidative stress and inflammatory damage in myocardial tissues of arrhythmic rats, reduce myocardial cell apoptosis, and improve the symptoms of arrhythmia in rats.

Melittin Inhibits Growth of Human Osteosarcoma 143B Cells through Induction of Apoptosis via Suppressing the Wnt/β-catenin Signaling Pathway.

Osteosarcoma is the most commonly seen type of primary malignant bone tumors in children and adolescents. Partial patients with osteosarcoma cannot tolerate the side effects of chemotherapy drugs. Hence, it is urgent to find anti-osteosarcoma drugs with low side effects. Melittin is an anti-tumor Traditional Chinese Medicine with low side effects. The purpose of this study was to explore the anti-osteosarcoma effect of melittin and its possible molecular mechanisms. The effects of melittin on cell growth were detected by CCK-8, clonal formation, and flow cytometry. The related molecules were also investigated by Real-time PCR and Western blot. A xenograft model in nude mice was established to observe the effects of melittin on tumor growth and the related molecular expression was detected by immunohistochemistry. Melittin can inhibit the proliferation of osteosarcoma 143B cells, reduce colony formation, and induce apoptosis while significantly up-regulating the expression of Bax and Caspase-3 and down-regulating the expression of Bcl-2 proteins. Moreover, treatment with melittin significantly reduced the mRNA and protein levels of β-catenin and Wnt/β- catenin related genes (LRP5, c-Myc, and Survivin) in osteosarcoma 143B cells in vitro. The xenograft model found that melittin significantly inhibited tumor growth and decreased the protein expression levels of β-catenin and Wnt/β- catenin related genes in vivo. These findings show that melittin could inhibit the growth of osteosarcoma 143B cells, which may be related to the inhibition of Wnt/β-catenin signaling pathway activity and induce apoptosis by up-regulating the ratio of Bax/Bcl-2 in osteosarcoma 143B cells. Therefore, melittin is a promising anti-tumor drug for the treatment of osteosarcoma.

Overexpression of PYGO1 promotes early cardiac lineage development in human umbilical cord mesenchymal stromal/stem cells by activating the Wnt/β-catenin pathway.

Cardiovascular disease still has the highest mortality. Gene-modified mesenchymal stromal/stem cells could be a promising therapy. Pygo plays an important role in embryonic development and regulates life activities with a variety of regulatory mechanisms. Therefore, this study aimed to investigate whether the overexpression of the PYGO1 gene can promote the differentiation of human umbilical cord-derived mesenchymal stromal/stem cells (HUC-MSCs) into early cardiac lineage cells and to preliminary explore the relevant mechanisms. In this study, HUC-MSCs were isolated by the explant method and were identified by flow cytometry and differentiation assay, followed by transfected with lentivirus carrying the PYGO1 plasmid. In PYGO1 group (cells were incubated with lentiviral-PYGO1), the mRNA expressions of cardiac differentiation-specific markers (MESP1, NKX2.5, GATA4, MEF2C, ISL1, TBX5, TNNT2, ACTC1, and MYH6 genes) and the protein expressions of NKX2.5 and cTnT were significantly up-regulated compared with the NC group (cells were incubated with lentiviral-empty vector). In addition, the proportion of NKX2.5, GATA4, and cTnT immunofluorescence-positive cells increased with the inducement time. Overexpression of PYGO1 statistically significantly increased the relative luciferase expression level of Topflash plasmid, the protein expression level of β-catenin and the mRNA expression level of CYCLIND1. Compared with the control group, decreased protein levels of NKX2.5 and cTnT were detected in PYGO1 group after application of XAV-939, the specific inhibitor of the canonical Wnt/β-catenin pathway. Our study suggests that overexpression of PYGO1 significantly promotes the differentiation of HUC-MSCs into early cardiac lineage cells, which is regulated by the canonical Wnt/β-catenin signaling.

PaThe regulation mechanism of MUC5AC secretion in airway of obese asthma

The purpose of this study was to establish a rat asthma model and extract MUC5AC to explore the mechanism of mucin 5AC (MUC5AC) signaling pathway regulating the function of asthmatic airway smooth muscle cells (ASMC) and participating in asthmatic airway remodeling. Western blot was used to detect β-catenin (β-catenin), glycogen synthase kinase-3β (GSK-3β), proto-oncogene MUC5AC and cyclin D1 (cyclin D1) in MUC5AC of asthmatic and normal groups. After inhibiting the interaction between β-catenin and transcription cofactor p300 / CBP in ASMC of the asthma group and control group, the cell viability and cycle changes of ASMC were detected by the CCK-8 method and flow cytometry. After inhibiting the activity of P38 mitogen-activated protein kinase (MAPK), the protein expression changes of c-Myc and cyclin D1 were detected by Western blot. Results showed that comprehensive HE staining results of lung tissue sections indicate that the experimental rat model of asthma airway remodeling was successfully established. Compared with the control group, 100 fxmol and L1 Efaroxan promoted insulin secretion (P <0.01), and administration of the MUC5AC antagonist KU14R significantly inhibited the effect of MUC5AC.Western blot showed that the protein expression levels of β-catenin, c-Myc and cyclin D1 in ASMC of the obese asthma group were significantly higher than those of the control group (P <0.05), while the protein expression level of GSK-3β was lower than Control group (P <0.05). After inhibiting the interaction between β-catenin and p300 / CBP, the decrease in cell viability and the degree of cell cycle change of ASMC in the asthma group were more obvious than those in the control group (P <0.05). After inhibiting the activity of P38 MAPK, the expressions of the target proteins c-Myc and cyclin D1 in the MUC5AC signaling pathway in ASMC model rats and control rats were down-regulated, and the difference was statistically significant (P <0.05). The conclusion was that the Wnt/β-catenin signaling pathway can regulate the proliferation and differentiation of ASMC by up-regulating the expression level of cMyc. Cyclin D1 interacts with the MAPK signaling pathway, thereby affecting the function of ASMC and participating in asthma airway remodeling.

RRM2 regulates osteogenesis of mouse embryo fibroblasts via the Wnt/β‑catenin signaling pathway.

Osteoporosis is a widespread bone metabolic disease characterized by reduced bone mass and bone microstructure deterioration. Ribonucleotide reductase M2 (RRM2) is a key enzyme in DNA synthesis and repair. The present study investigated the effect of RRM2 on osteogenesis of mouse embryo fibroblasts (MEFs) and its molecular mechanism. Bioinformatics analysis revealed that RRM2 expression was increased during osteogenesis of MEFs triggered by bone morphogenetic protein 9. Subsequently, MEFs were used as a mesenchymal stem cell model and osteogenic inducing medium was used to induce osteogenic differentiation. RRM2 protein expression was measured by western blotting during osteogenic differentiation induction of MEFs. RRM2 levels in MEFs were upregulated and downregulated by RRM2-overexpressing recombinant adenovirus and small interfering RNA-RRM2, respectively. Bone formation markers (RUNX family transcription factor 2, osterix, distal-less homeobox 5, collagen type I α1 chain, osteopontin and osteocalcin) were detected by reverse transcription-quantitative (RT-q) PCR and alkaline phosphatase (ALP) and Alizarin Red S staining were examined. The protein expression levels of β-catenin and the ratio of phosphorylated (p-)GSK-3β to GSK-3β were detected by western blotting and the RNA expression of downstream related target genes (β-catenin, axis inhibition protein 2 (AXIN2), transcription factor 7 like 2, lymphoid enhancer binding factor 1, c-MYC and Cyclin D1) in the Wnt/β-catenin signaling pathway was measured by RT-qPCR. RRM2 protein expression increased as the osteogenic differentiation induction period was extended. RRM2 overexpression increased osteogenic marker RNA expression, ALP activity, bone mineralization, the protein expression levels of β-catenin, the ratio of p-GSK-3β to GSK-3β and the RNA expression of downstream related target genes in the Wnt/β-catenin signaling pathway, whereas RRM2 knockdown had the opposite effect. The findings of the present study revealed that RRM2 overexpression enhanced osteogenic differentiation, while RRM2 knockdown reduced osteogenic differentiation. RRM2 may regulate osteogenic differentiation of MEFs via the canonical Wnt/β-catenin signaling pathway, providing a possible therapeutic target for osteoporosis.

Exosome Component 4 Promotes Epithelial Ovarian Cancer Cell Proliferation, Migration, and Invasion via the Wnt Pathway.

BackgroundOf gynecologic malignancies, ovarian cancer is the leading cause of death, mainly due to the lack of sensitive tumor markers, which means it almost always presents at an advanced stage. Exosome Component 4 (EXOSC4) is involved in RNA degradation, but its role in epithelial ovarian cancer (EOC) is unclear.MethodsThe expression levels of EXOSC4 in EOC and normal ovarian tissue specimens were determined by immunohistochemical staining. The overall survival (OS) and progression-free survival (PFS) of patients with EOC were evaluated after patients were classified into high and low EXOSC4 expression groups, and the Cox regression model was established to identify independent predictors of patient prognosis. The effects of EXOSC4 on proliferation, colony formation, migration, and invasion were examined in the SKOV-3 and HO8910 cell lines by lentivirus-mediated shRNA knockdown. Flow cytometry was used to detect cell cycle changes. The mRNA levels of cyclin D1, CDK4, and c-myc were detected by RT-PCR. The protein expression levels of β-catenin, cyclin D1, CDK4, c-myc, vimentin, N-cadherin, and E-cadherin were assessed by western blot. Wnt/β-catenin activation was measured by TCF/LEF reporter assay.ResultsEXOSC4 was significantly elevated in EOC tissues and cell lines. High EXOSC4 expression was correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage and pathological grade, and identified as an independent predictor of shorter OS and PFS. EXOSC4 knockdown suppressed proliferation, migration, and invasion in EOC cell lines. Cells were arrested at G0/G1 phase after EXOSC4 knockdown. The mRNA levels of cyclin D1, CDK4, and c-myc were decreased. β-catenin, cyclin D1, CDK4, c-myc, vimentin, and N-cadherin protein expression levels were reduced, while those of E-cadherin was increased. Wnt/β-catenin activity was suppressed after the EXOSC4 knockdown.ConclusionsEXOSC4 is involved in EOC. Knockdown of EXOSC4 can inhibit the proliferation, migration, and invasion ability of EOC by suppressing the Wnt pathway. EXOSC4 is expected to be a novel biomarker and molecular target in EOC.

Sanguisorba officinalis L. suppresses 5-fluorouracil-sensitive and-resistant colorectal cancer growth and metastasis via inhibition of the Wnt/β-catenin pathway

BackgroundColorectal cancer (CRC) is a widespread cancer with high morbidity and mortality. Chemoresistance and metastasis are the current challenges for CRC treatment. Sanguisorba officinalis Linn. (called DiYu in Chinese, DY) is a traditional Chinese medicine (TCM) whose root is long used as medicinal part. In our previous study, the aqueous extract of DY could inhibit the Wnt/β-catenin pathway and showed great antitumor effect against CRC. The Wnt/β-catenin pathway is involved in CRC chemoresistance and metastasis. However, there is little study on the antitumor and antimetastatic effects of DY on resistant CRC cells. The aim of this study is to explore the effect of aqueous extract of DY on the growth and metastasis of 5-fluorouracil (5-FU) sensitive and resistant CRC, and to elucidate the underlying molecular mechanism. MethodologyIn this study, cell viability, cell colony formation and apoptosis analyses were performed to verify the in vitro antitumor effect of DY on 5-FU-sensitive and -resistant CRC cells. Next, transwell assays were used to test the inhibition activity of DY on CRC migration and invasion. Western Blotting assays were carried out to identify the molecular mechanism underlying the efficacy of DY extract. Xenograft CRC nude mice model and tumor metastasis model were used to confirm the in vivo antitumor and antimetastatic effects of DY. ResultsDY inhibited cell proliferation and apoptosis via the upregulation of Bax, cleaved-caspase3 and cleaved-PARP proteins and downregulation of Bcl-2 protein. DY also inhibited cell migration and invasion via the downregulation of N-cadherin, vimentin and snail proteins and upregulation of E-cadherin protein, demonstrating that DY suppressed cell metastasis by reversing epithelial-to-mesenchymal transition (EMT) procession. Moreover, the protein expression levels of β-catenin in whole cell, cytoplasm and nucleus were decreased after DY treatment. Taken together, DY suppressed CRC cell growth and metastasis via inhibition of the Wnt pathway. Additionally, DY also demonstrated effective antitumor and anti-metastasis activities in vivo. ConclusionsIn conclusion, DY suppressed the growth and metastasis of 5-FU-sensitive and -resistant CRC via inhibition of the Wnt pathway, which indicated that DY could be a potential drug to treat CRC patients and improve clinic outcome.

Fractions of Shen-Sui-Tong-Zhi Formula Enhance Osteogenesis Via Activation of β-Catenin Signaling in Growth Plate Chondrocytes.

Background: Shen-sui-tong-zhi formula (SSTZF) has been used to treat osteoporosis for decades and shows excellent clinical efficacy. This article aims to explore the optimal anti-osteoporotic ingredient and its precise mechanisms in mice models. Methods: In this study, we first screened the optimal anti-osteoporosis fraction of SSTZF extract in vivo, and then further explored the mechanism of its effects both in vivo and in vitro. Ten-week-old female C57BL/6J mice were administrated with each fraction of SSTZF. At 10 weeks after ovariectomy (OVX), femurs were collected for tissue analyses, including histology, micro-CT, biomechanical tests, and immunohistochemistry for ALP, FABP4, and β-catenin. Additionally, we also evaluated the mRNA expression level of ALP and FABP4 and the protein expression level of β-catenin after being treated with SSTZF extract in C3H10T1/2 cells. Moreover, we investigated the anti-osteoporosis effect of SSTZF extract on mice with β-catenin conditional knockout in growth plate chondrocytes (β-catenin Gli1ER mice) through μCT, histology, and immunohistochemistry analyzes. Results: At 10 weeks after treatment, osteoporosis-like phenotype were significantly ameliorated in SSTZF n-butanol extract (SSTZF-NB) group mice, as indicated by increased trabecular bone area and ALP content, and decreased lipid droplet area and FABP4 content. No such improvements were observed after being treated with other extracts, demonstrating that SSTZF-NB is the optimal anti-osteoporosis fraction. Additionally, the elevated β-catenin was revealed in both OVX mice and C3H10T1/2 cells with SSTZF-NB administered. Furthermore, a significant osteoporosis-like phenotype was observed in β-catenin Gli1ER mice as expected. However, SSTZF-NB failed to rescue the deterioration in β-catenin Gli1ER mice, no significant re-upregulated ALP and downregulated FABP4 were observed after being treated with SSTZF-NB, demonstrating that SSTZF-NB prevents bone loss mainly via β-catenin signaling. Conclusion: SSTZF-NB enhances osteogenesis mainly via activation of β-catenin signaling in growth plate chondrocytes. SSTZF-NB is the optimal anti-osteoporosis fraction of SSTZF and it can be considered a salutary alternative therapeutic option for osteoporosis.

Changes in SLIT2 expression are associated with the migration of human ovarian clear cell carcinoma cells.

Ovarian clear cell carcinoma (OCCC) is characterized by a poor survival of patients, which is mainly due to metastasis and treatment failure. Slit guidance ligand 2 (SLIT2), a secreted protein, has been reported to modulate the migration of neural cells and human cancer cells. However, the effect of changes in SLIT2 expression on the regulation of cell migration in OCCC remains unknown. The present study examined alterations in SLIT2 expression using OCCC cell models, including low- and high-mobility SKOV3 cells, as well as OCCC tissues. DNA methylation analysis suggested that promoter hypermethylation was responsible for the low expression levels of SLIT2 in OCCC cells. The demethylating agent 5-Aza-deoxycytosine was able to restore SLIT2 expression at both the mRNA and protein levels in high-mobility SKOV3 cells that harbored the relevant methylated promoter. Overexpression of SLIT2 inhibited the migration of high-mobility OCCC cells, as well as decreased the protein expression levels of β-catenin, phosphorylated (p)AKT and snail family transcriptional repressor 1 (SNAI1). On the other hand, knockdown of SLIT2 increased the migration of low-mobility OCCC cells, and enhanced the protein expression levels of β-catenin, pAKT and SNAI1. Overall, the results of the present study provided evidence that low expression levels of SLIT2 were associated with increased OCCC cell migration, and that SLIT2 may act as a suppressor gene of cancer cell migration.

The platelet derived growth factor BB promotes osteogenic differentiation of periodontal ligament stem cells via the Wnt/β-catenin signaling pathway

ObjectiveTo determine the role of platelet derived growth factor BB in the regulation of cell cycle, migration and differentiation of stem cells. DesignThe gene was overexpressed in periodontal ligament stem cells using lentiviral vectors. Normal stem cells and empty lentiviral vectors-transfected were used as controls. Real time-PCR, western blotting, Cell Counting Kit-8 assay, flow cytometry, cell scratch test, Alkaline phosphatase activity assay, cell cycle analyses were conducted to assess the biological properties of stem cells. In addition, the effect of platelet derived growth factor BB on the Wnt/β-catenin signaling pathway were assessed by western blotting and immunofluorescent staining. ResultsThe gene was successfully overexpressed in periodontal ligament stem cells. The Cell Counting Kit-8, scratch test and cell cycle experiments proved that platelet derived growth factor BB promoted stem cells proliferation, migration and cell cycle progression. The Real time-PCR results showed that the Osterix (OSX) and Bone Morphogenetic Protein 2 (BMP2) genes in the overexpression group were significantly higher than those in the control group, but the Peroxisome Proliferators-activated Receptors (PPARγ) and Glycogen Synthase Kinase-3β (GSK-3β) gene were lower than that in the control group. Western blotting results also indicated that the Collagen Type 1 (COL-1), BMP2, Wnt1 and β-catenin proteins were increased in the overexpression group. In addition, the expression level of β-catenin protein in the cell nuclei was higher than that in the control group. ConclusionsIn conclusion, overexpression of platelet derived growth factor BB promoted cell proliferation, migration, cell cycle progression and decreased adipogenic differentiation. Furthermore, platelet derived growth factor BB regulated osteogenic differentiation of stem cells through the Wnt/β-catenin signaling pathway.

Knockdown of TRIM15 inhibits the proliferation, migration and invasion of esophageal squamous cell carcinoma cells through inactivation of the Wnt/β-catenin signaling pathway.

TRIM15 is a member of tripartite motif-containing protein (TRIM) protein family, which plays important roles in several cancers. The aim of the present study was to evaluate the role of TRIM15 in esophageal squamous cell carcinoma (ESCC). Our results showed that TRIM15 was upregulated in human ESCC tissues and cell lines. In vitro studies showed that knockdown of TRIM15 significantly inhibited the proliferation, migration, and invasion of ESCC cells. Knockdown of TRIM15 caused a significant increase in E-cadherin expression, as well as decreases in expression of N-cadherin and Vimentin proteins. Moreover, in vivo assay proved that tumor growth was suppressed by knockdown of TRIM15. Furthermore, the protein expression levels of β-catenin, C-myc, and CyclinD1 were markedly decreased in sh-TRIM15-infected ESCC cells. Additionally, treatment with LiCl reversed the inhibitory effects of TRIM15 knockdown on ESCC cells. In conclusion, these findings indicated that knockdown of TRIM15 blocked the growth and metastasis of ESCC in part through inhibiting the Wnt/β-catenin signaling pathway. Thus, TRIM15 might serve as a promising therapeutic target for ESCC.

ZEB1 serves an oncogenic role in the tumourigenesis of HCC by promoting cell proliferation, migration, and inhibiting apoptosis via Wnt/β-catenin signaling pathway.

Zinc finger E-box-binding homeobox 1 (ZEB1), a functional protein of zinc finger family, was aberrant expressed in many kinds of liver disease including hepatic fibrosis and Hepatitis C virus. Bioinformatics results showed that ZEB1 was abnormally expressed in HCC tissues. However, to date, the potential regulatory role and molecular mechanisms of ZEB1 are still unclear in the occurrence and development of HCC. This study demonstrated that the expression level of ZEB1 was significantly elevated both in liver tissues of HCC patients and cell lines (HepG2 and SMMC-7721 cells). Moreover, ZEB1 could promote the proliferation, migration, and invasion of HCC cells. On the downstream regulation mechanism, ZEB1 could activate the Wnt/β-catenin signaling pathway by upregulating the protein expression levels of β-catenin, c-Myc, and cyclin D1. Novel studies showed that miR-708 particularly targeted ZEB1 3'-UTR regions and inhibited the HCC cell proliferation, migration, and invasion. Furthermore, results of nude mice experiments of HCC model indicated that miR-708 could inhibit tumor growth and xenograft metastasis model was established to validate that miR-708 could inhibit HCC cell metastasis through tail-vein injection in vivo. Together, the study suggested that ZEB1 modulated by miR-708 might be a potential therapeutic target for HCC therapy.