MiR-145-5p modulates lipid metabolism and M2 macrophage polarization by targeting PAK7 and regulating ß-catenin signaling in hyperlipidemia.

The present study aims to explore the role of microRNA 145-5p (miR-145-5p) in hyperlipidemia. Using bioinformatics tools and a wide range of function and mechanism assays, we attempted to understand the specific function and potential mechanism of miR-145-5p in hyperlipidemia. A cholesterol-enriched diet induced an increase of serum cholesterol and triacylglycerol but a decrease of serum high-density lipoprotein. MiR-145-5p level was decreased in hyperlipidemia rat models. MiR-145-5p regulated lipid metabolism by antagonizing the alteration of high-density lipoprotein, cholesterol, and triacylglycerol in serum mediated by a cholesterol-enriched diet. In mechanism, miR-145-5p directly bound with p21 protein (RAC1)-activated kinase 7 (PAK7) and negatively regulated mRNA and protein levels of PAK7 in THP-1 cells. Furthermore, miR-145-5p level was negatively associated with PAK7 level in rat cardiac tissues. Finally, overexpression of PAK7 reversed the effects of miR-145-5p on ß-catenin activation and M2 macrophages polarization in THP-1 cells. In conclusion, MiR-145-5p modulated lipid metabolism and M2 macrophage polarization by targeting PAK7 and regulating ß-catenin signaling in hyperlipidemia, which may provide a potential biomarker for the treatment of hyperlipidemia-induced cardiovascular diseases.

Carboxypeptidase E-ΔN promotes migration, invasiveness, and epithelial-mesenchymal transition of human osteosarcoma cells via the Wnt-ß-catenin pathway.

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents, and metastatic OS is the major cause of OS-related death. Carboxypeptidase E (CPE) is known to be highly expressed in some cancer types, and its N-terminal truncated form, CPE-ΔN, is implicated in tumor metastasis and poor prognosis. In this study, we investigated the effect of CPE-ΔN on cell migration, invasiveness, and the epithelial-mesenchymal transition (EMT) of OS cells, and illustrated the molecular mechanisms. We first constructed CPE-ΔN overexpressing human OS cell lines (143B and U2OS cells), and found that ectopic CPE-ΔN expression in OS cells enhanced cell migration and invasiveness, and promoted the EMT process. Further, overexpression of CPE-ΔN increased the levels of c-myc and nuclear ß-catenin in OS cells, which suggested the CPE-ΔN promotes activation of the Wnt-ß-catenin pathway in OS cells. Treatment with ß-catenin small interfering RNA (siRNA) inhibited the migration and invasiveness of CPE-ΔN-overexpressing cells, and reduced the expression of E-cadherin. Together, these results suggest that CPE-ΔN promotes migration, invasiveness, and the EMT of OS cells via the Wnt-ß-catenin signaling pathway.

Alteration of Bcl11b upon stimulation of both the MAP kinase- and Gsk3-dependent signaling pathways in double-negative thymocytes.

B-cell lymphoma/leukemia 11B (Bcl11b) is a transcription factor critical for thymocyte development. We have previously characterized the kinetic post-translational modifications (PTMs) of Bcl11b in double-positive (DP) thymocytes during stimulation of the T cell receptor-activated MAP kinase pathway. However, the PTMs of Bcl11b in thymocytes from other developmental stages in the thymus, primarily double-negative (DN) cells, have not been previously identified. We found that kinetic modifications of Bcl11b in DN cells are somewhat different than the patterns observed in DP cells. Distinct from DP thymocytes, phosphorylation and sumoylation of Bcl11b in DN cells were not oppositely regulated in response to activation of MAP kinase, even though hyper-phosphorylation of Bcl11b coincided with near complete desumoylation. Additionally, prolonged stimulation of the MAP kinase pathway in DN cells, unlike DP thymocytes, did not alter Bcl11b levels of sumoylation or ubiquitinylation, or stability. On the other hand, activation of Wnt-Gsk3-dependent signaling in DN cells resulted in composite dephosphorylation and sumoylation of Bcl11b. Moreover, stimulation of MAP kinase and (or) Wnt signaling pathways differentially affects gene expression of some Bcl11b target and maturation-associated genes. Defining the signaling pathways and regulation of sequence-specific transcription factors by PTMs at various stages of thymopoiesis may improve our understanding of leukemogenesis.

Effects of metformin on apoptosis and α-synuclein in a rat model of pentylenetetrazole-induced epilepsy.

The present study was designed to examine the possible neuroprotective and antiepileptic effects of metformin (Metf) in a rat model of pentylenetetrazole (PTZ)-induced epilepsy and its possible underlying mechanisms. Forty male albino rats were assigned to 4 groups of equal size: (1) normal control (NC) group, (2) Metf group: daily treatment with Metf (200 mg/kg, i.p.) for 2 weeks, (3) PTZ group: treatment with PTZ (50 mg/kg, i.p.) every other day for 2 weeks, and (4) Metf + PTZ group: daily treatment with PTZ and metformin (200 mg/kg, i.p.) for 2 weeks. Administration of PTZ caused a significant increase in seizure score and duration, induced a state of oxidative stress (high malondialdehyde, low reduced glutathione and catalase activity), and led to the upregulation of ß-catenin, caspase-3, and its cleavage products, Hsp70 and α-synuclein, in hippocampal regions as well as a significant reduction in seizure latency. While Metf treatment significantly ameliorated PTZ-induced seizures, attenuated oxidative stress, and upregulated α-synuclein and ß-catenin expression, it also inhibited caspase-3 activation and the release of the cleavage product and caused more upregulation in Hsp70 expression in hippocampal regions (p < 0.05). In conclusion, the antiepileptic and neuroprotective effects of Metf in PTZ-induced epilepsy might be due to the inhibition of apoptosis, attenuation of oxidative stress and α-synuclein expression, and upregulation of Hsp70.

Knockdown of the prolyl isomerase Pin1 inhibits Hep-2 cell growth, migration, and invasion by targeting the ß-catenin signaling pathway.

There is increasing evidence indicating that peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) plays a decisive role in a variety of cancers. Nevertheless, its function in laryngeal squamous cell carcinoma (LSCC) has not been elaborated. The aim of this study is to determine the role of Pin1 in LSCC. Here, we established stably transfected Hep-2 cells with low expression of Pin1. Intriguingly, cell proliferation, migration, and invasion was significantly inhibited in Pin1-silenced Hep-2 cells. Similarly, knockdown of Pin1 induced apoptosis of Hep-2 cells, as evidenced by increased expression of cleaved-caspase-3, cleaved-PARP, and bax, and decreased expression of bcl2. We also demonstrated that silencing of Pin1 down-regulated ß-catenin and cyclin D1 expression. Inversely, over-expression of ß-catenin reversed the inhibiting effect of Pin1 silencing on Hep-2 cells. Moreover, we proved that knockdown of Pin1 inhibited tumorigenesis of Hep-2 cells in vivo. Taken together, we demonstrate that silencing of Pin1 effectively suppresses the growth of Hep-2 cells through ß-catenin, indicating that Pin1 possess the potential to serve as a therapeutic target for the treatment of LSCC.

Knockdown of aquaporin-5 sensitizes colorectal cancer cells to 5-fluorouracil via inhibition of the Wnt-ß-catenin signaling pathway.

Aquaporin-5 (AQP5), a water channel protein, has been reported to possess oncogenic potential in multiple types of malignancies, including colorectal cancer (CRC). However, its effect on the chemosensitivity of CRC cells remains elusive. Hence, this study investigated the effect of AQP5 silencing in CRC cells on 5-fluorouracil (5-FU) sensitivity and attempted to elucidate the underlying mechanisms. A short hairpin RNA construct targeting AQP5 was transfected into HCT116 or HT29 cells to generate stable AQP5-silenced cell lines. The effects of AQP5 knockdown on cell viability, apoptosis, tumor growth, and 5-FU chemoresistance were evaluated. Relative protein levels of Wnt-ß-catenin pathway effectors were also measured. The results showed that silencing of AQP5 increased the chemosensitivity of CRC cells to 5-FU, facilitated 5-FU-mediated apoptosis, suppressed tumor growth, and reduced 5-FU chemoresistance in vivo. Furthermore, the effect of AQP5 on 5-FU chemosensitivity was mediated by the Wnt-ß-catenin pathway. Silencing of AQP5 inhibited Wnt-ß-catenin signaling, whereas overexpression of the degradation-resistant mutant of ß-catenin (S33Y) reversed apoptosis induced by AQP5 silencing. Taken together, these results suggest that AQP5 silencing enhances the sensitivity of CRC cells to 5-FU, and the underlying mechanism is related to inhibition of the Wnt-ß-catenin pathway. AQP5 could be a useful therapeutic target for CRC treatment.

Defining the regulatory role of programmed cell death 4 in laryngeal squamous cell carcinoma.

Programmed cell death 4 (PDCD4) is decreased in many different kinds of malignant tumors. EMT endows tumor cells invasive and metastatic properties. However, few studies have determined the role of PDCD4 in the regulation of EMT in the context of laryngeal carcinoma. We examined the relationship between PDCD4 and EMT-associated proteins E-cadherin and N-cadherin using laryngeal carcinoma tissues. Gene manipulation was used to define the regulatory capacity of PDCD4. We report that PDCD4 and E-cadherin/N-cadherin expression were significantly changed in the carcinoma tissues, and their expression was associated with pathological grade, metastatic state, and clinical stage. The suppression of PDCD4 (and consequently, E-cadherin) was concomitant with increased proliferation and G2-phase arrest, decreased apoptosis, and increased cell invasion. PDCD4 upregulation reversed the above-mentioned results. In nude mice, PDCD4 knockdown increased tumor growth and pathological features, confirming the tumorigenic role of PDCD4. Finally, PDCD4 silencing was associated with dysregulation of the carcinogenic Wnt-ß-catenin and the STAT3-miR-21 signaling pathways. This study revealed a dynamic regulatory relationship between PDCD4 and critical factors for EMT, establishing a broad, functional role for PDCD4 in laryngeal carcinoma, which may be propagated by the STAT3-miR-21 pathway. These findings provide new information on an EMT-associated target that may lead to a novel therapy.

Frizzled gene expression and negative regulation of canonical WNT-ß-catenin signaling in mouse F9 teratocarcinoma cells.

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-ß-catenin pathway. The upregulation of Wnt6 and activation of ß-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-ß-catenin signaling, thereby allowing PE cells to differentiate.