Genotyping Zebrafish Point Mutant by Allele-Specific Blocking PCR.

Genotyping zebrafish carrying wild-type, heterozygous, or homozygous copies of a mutant allele is often required for investigating gene specific functions, and is routinely performed to differentiate point mutants. In this study, we describe a modified allele-specific PCR method using an additional blocking primer to promote target sequence amplification while suppressing sequences with single mismatch. Using the tp53m214k point mutant as an example, we show that wild-type, heterozygous, and homozygous zebrafish can be easily distinguished using this simple PCR method, which could be widely adapted for genotyping zebrafish with point mutations or small nucleotide insertions/deletions.

The impact of the energy-consuming right trading system on corporate environmental performance: Based on empirical evidence from panel data of industrial enterprises listed in pilot regions.

The energy-consuming right trading system (ECRTS) is a significant institutional innovation in China to address the increasingly severe energy crisis and environmental issues. Identifying the policy effects of energy consumption rights on corporate environmental performance (CEP) is conducive to achieving a win-win situation for China's economic growth and carbon neutrality. This study aims to analyze the impact of energy-consuming right trading system on corporate environmental performance and provide empirical evidence and policy implications for the full implementation of future policies. Using data from Chinese listed industrial enterprises from 2012 to 2019 and adopting the difference-in-differences method and mediation analysis, we empirically analyze the policy effects of energy-consuming right trading system. We find that the energy-consuming right trading system significantly promotes the improvement of corporate environmental performance, and the conclusion remains valid after a series of robustness tests. Further mechanism examinations indicate that the system mainly enhances environmental performance by affecting corporate green technological innovation. Heterogeneity tests suggest that the energy-consuming right trading system has a stronger impact on companies in economically developed regions, non-state-owned enterprises, and those with higher asset flexibility. Our research results can aid in the green transformation of enterprises and provide practical evidence for China to accelerate the comprehensive construction of the energy consumption rights trading market.

Performance of ATT and UDFF in the diagnosis of non-alcoholic fatty liver: An animal experiment.

Objective: To establish a Bama minipigs model with Non-Alcoholic Fatty Liver (NAFL) induced by a high-fat diet and investigate the application of attenuation coefficient (ATT) and ultrasound-derived fat fraction (UDFF) in the diagnosis of NAFL. Methods: Six-month-old male Bama minipigs were randomly divided into normal control and high-fat groups (n = 3 pigs per group), and fed with a control diet and high-fat diet for 32 weeks. Weight and body length were measured every four weeks, followed by quantitative ultrasound imaging (ATT and UDFF), blood biochemical markers, and liver biopsies on the same day. Using the Non-Alcoholic Fatty Liver Disease (NAFLD) Activity Score (NAS) as a reference, we analyzed the correlation between ATT, UDFF, and their score results. Results: Compared with the normal control group, the body weight, body mass index (BMI), and serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the High-fat group were significantly different at Week 12 (P < 0.05). Spearman correlation analysis showed that the ATT value was significantly correlated with NAS score (r = 0.76, P < 0.001), and the UDFF value was significantly correlated with NAS score (r = 0.80, P < 0.001). The optimal cut-off value of ATT and UDFF were 0.59 dB/cm/MHz and 5.5%, respectively. These values are optimal for diagnosis of NAFL in Bama minipig model. Conclusion: ATT and UDFF have a high correlation with steatosis, and can be used as a non-invasive method for early screening of hepatic steatosis, which can dynamically monitor the change of disease course.

Mutant analysis of Kcng4b reveals how the different functional states of the voltage-gated potassium channel regulate ear development.

The voltage gated (Kv) slow-inactivating delayed rectifier channel regulates the development of hollow organs of the zebrafish. The functional channel consists of the tetramer of electrically active Kcnb1 (Kv2.1) subunits and Kcng4b (Kv6.4) modulatory or electrically silent subunits. The two mutations in zebrafish kcng4b gene - kcng4b-C1 and kcng4b-C2 (Gasanov et al., 2021) - have been studied during ear development using electrophysiology, developmental biology and in silico structural modelling. kcng4b-C1 mutation causes a C-terminal truncation characterized by mild Kcng4b loss-of-function (LOF) manifested by failure of kinocilia to extend and formation of ectopic otoliths. In contrast, the kcng4b-C2-/- mutation causes the C-terminal domain to elongate and the ectopic seventh transmembrane (TM) domain to form, converting the intracellular C-terminus to an extracellular one. Kcng4b-C2 acts as a Kcng4b gain-of-function (GOF) allele. Otoliths fail to develop and kinocilia are reduced in kcng4b-C2-/-. These results show that different mutations of the silent subunit Kcng4 can affect the activity of the Kv channel and cause a wide range of developmental defects.

Dynamics of Chromosome Aberrations and Cell Death in Zebrafish Embryos Exposed to 137Cs Total-Body Irradiation.

Ionizing radiation exposure induces significant DNA damage and cell death in aquatic species. Accurate sensing and quantification play pivotal roles in environmental monitoring and surveillance. Zebrafish (Danio rerio) is a well-suited animal model for research into this aspect, especially with recent development of cytogenetic and transgenic tools. In this study, we present time-course studies of chromosome aberrations and cell death in zebrafish embryos exposed to 2 Gy 137Cs total-body irradiation. Using a cytogenetic approach, we quantified chromosome and chromatid aberrations in irradiated embryos at 6, 14, 20, and 24 h postirradiation. Metaphases with aberrations showed rapid declining kinetics, accompanied by incomplete karyotypes and irregular chromatin contents. Using an apoptosis-reporting transgenic zebrafish, we found increasing cell death along these time points, with the embryonic eyes and brain contributing the majority of the cell death volumes. We provide evidence that self-proliferating progenitor cells form the underlying linkage between the two kinetics and their positions define radiosensitive niches in zebrafish embryos. Our results provide detailed chromosome aberration and cell death dynamics in 137Cs-irradiated zebrafish embryos and unveil the appropriate timeline and tissue positions for accurate sensing and quantification of radiation-induced damages in zebrafish embryos.

Imaging the radioprotective effect of amifostine in the developing brain using an apoptosis-reporting transgenic zebrafish.

PURPOSE: Normal tissue radioprotectants alleviate radiation-induced damages and preserve critical organ functions. Investigating their efficacy in vivo remains challenging, especially in enclosed organs like the brain. An animal model that enables direct visualization of radiation-induced apoptosis while possessing the structural complexity of a vertebrate brain facilitates these studies in a precise and effective manner. MATERIALS AND METHODS: We employed a secA5 transgenic zebrafish expressing secreted Annexin V fused with a yellow fluorescent protein to visualize radiation-induced apoptosis in vivo. We developed a semi-automated imaging method for standardized acquisition of apoptosis signals in batches of zebrafish larvae. Using these approaches, we studied the protective effect of amifostine (WR-2721) in the irradiated zebrafish larval brain. RESULTS: Upon 2 Gy total-body 137Cs irradiation, increased apoptosis could be visualized at high resolution in the secA5 brain at 2, 24, and 48 hour post irradiation (hpi). Amifostine treatment (4 mM) during irradiation reduced apoptosis significantly at 24 hpi and preserved Wnt active cells in the larval brain. When the 2 Gy irradiation was delivered in combination with cisplatin treatment (0.1 mM), the radioprotective effect of amifostine was also observed. CONCLUSIONS: Our study reveals the radioprotective effect of amifostine in the developing zebrafish larval brain, and highlights the utility of secA5 transgenic zebrafish as a novel system for investigating normal tissue radioprotectants in vivo.

In Vivo Imaging of Radiation-Induced Apoptosis at Single-Cell Resolution in Transgenic Zebrafish Embryos.

Among the various types of cell death induced by ionizing radiation, apoptosis is a highly regulated and well-characterized form. Investigating radiation-induced apoptosis in an intact organism offers advantages in capturing the dynamics of apoptosis under preserved physiology, although high resolution imaging remains challenging. Owing to their optical transparency and genetic amenability, zebrafish is an ideal animal model for research into this aspect. In this study, we present a secA5 transgenic zebrafish expressing genetically encoded secreted ANNEXIN V fused with mVenus, a yellow fluorescent protein that enables reporting of radiation-induced apoptosis. Using in vivo imaging approach, we show that after 2 Gy total-body irradiation, apoptosis could be visualized at single-cell resolution in different cell types throughout the embryo. Elevated apoptosis could be imaged and quantified in the neuroepithelium of the embryonic brain, as well as the proliferative zone and parenchyma of the larval brain. In addition, clearance of apoptotic cells by microglia, the professional phagocytes residing in the brain, could be imaged at single-cell resolution in irradiated larvae. These results establish transgenic secA5 zebrafish as a useful and versatile in vivo system for investigating the dynamic process of radiation-induced apoptosis.

The position of entry point in total knee arthroplasty is associate with femoral bowing both in coronal and sagittal planes.

Objective: To investigate the femoral entry point of the intramedullary (IM) guiding rod applied to total knee arthroplasty (TKA) in Chinese subjects and the relationship with femoral bowing in the coronal and sagittal planes through three-dimensional (3D) validation methods. Methods: Computed tomography (CT) images of 80 femurs in Chinese subjects were imported into Mimics 19.0 to construct 3D models. All operations were conducted by Rhinoceros software 5.0. The position of the IM rod entry point was assessed by calculating the distance between the entry point and the apex of the intercondylar notch (AIN) in the coronal and sagittal planes. The coronal femoral bowing angle (cFBA) and sagittal femoral bowing angle (sFBA) were also measured. Results: The average optimal entry point was 0.17 mm medial and 12.37 mm anterior to the AIN in males, while it was 0.02 mm lateral and 16.13 mm anterior to the AIN in females. There was a significant difference between males and females in the sagittal plane (t = -6.570, p = 0.000). The mean cFBA was 1.68 ± 2.29°, and the mean sFBA was 12.66 ± 1.98°. The sFBA was strongly correlated with the anterior distance of the proper entry point, and the cFBA was moderately correlated with the lateral distance of the proper entry point. Conclusions: There was a strong correlation between the position of the entry point and the femoral bowing angle in both the coronal and sagittal planes. Thus, to achieve better alignment, the position of the entry point should be measured individually based on femoral bowing.

A New Reference Axis for Tibial Component Rotation in Total Knee Arthroplasty: A Three-dimensional Computed Tomography Analysis.

The purpose of this study was to introduce a new reference axis for tibial rotation in total knee arthroplasty (TKA) and verify its reliability. A consecutive series of 80 knees that underwent TKA from 2018 to 2020 as well as 80 healthy knees were analyzed using a three-dimensional tibial model. A coordinate system was established based on the standard TKA tibial cut. The line connecting the lateral-tibial eminence and the medial 1/3rd of the tibial tubercle or the medial border of the tibial tubercle was identified as the lateral eminence line (LE line) and the medial lateral eminence line (MLE line), respectively. To evaluate the reliability of the new reference axis, Akagi's line, the medial third of the tibial tubercle (1/3 line) was compared with the LE and MLE lines by measuring the angle between the lines and the Z-axis. In the coronal view, the intersection angle (TPA), which is composed of the line connecting the center of the medial and lateral tibial plateau with the Z-axis, was measured. The mean angle between Akagi's line and the Z-axis in the healthy group and the osteoarthritis (OA) group was 87.57 ± 3.48° and 87.61 ± 3.47°, respectively. The mean angle between the LE line and Z-axis in the healthy and OA groups was 87.15 ± 4.13° and 86.78 ± 3.95°, respectively. A weak correlation was found between the TPA and Akagi's line and the 1/3 line. A moderate correlation was observed between the TPA and LE lines. There were no significant differences between the healthy and OA groups (P > 0.05) in any of the four reference axes. The LE line showed excellent intra- and inter-observer reliability and reproducibility. The novel and easily drawn LE line is a preferable option for tibial component rotational alignment in TKA.

The PARP1 Inhibitor Niraparib Represses DNA Damage Repair and Synergizes with Temozolomide for Antimyeloma Effects.

Purpose: Poly(ADP-ribose) polymerase 1 (PARP1) is necessary for single-strand break (SSB) repair by sensing DNA breaks and facilitating DNA repair through poly ADP-ribosylation of several DNA-binding and repair proteins. Inhibition of PARP1 results in collapsed DNA replication fork and double-strand breaks (DSBs). Accumulation of DSBs goes beyond the capacity of DNA repair response, ultimately resulting in cell death. This work is aimed at assessing the synergistic effects of the DNA-damaging agent temozolomide (TMZ) and the PARP inhibitor niraparib (Nira) in human multiple myeloma (MM) cells. Materials and Methods: MM RPMI8226 and NCI-H929 cells were administered TMZ and/or Nira for 48 hours. CCK-8 was utilized for cell viability assessment. Cell proliferation and apoptosis were detected flow-cytometrically. Immunofluorescence was performed for detecting γH2A.X expression. Soft-agar colony formation assay was applied to evaluate the antiproliferative effect. The amounts of related proteins were obtained by immunoblot. The combination index was calculated with the CompuSyn software. A human plasmacytoma xenograft model was established to assess the anti-MM effects in vivo. The anti-MM activities of TMZ and/or Nira were evaluated by H&E staining, IHC, and the TUNEL assay. Results: The results demonstrated that cotreatment with TMZ and Nira promoted DNA damage, cell cycle arrest, and apoptotic death in cultured cells but also reduced MM xenograft growth in nude mice, yielding highly synergistic effects. Immunoblot revealed that TMZ and Nira cotreatment markedly increased the expression of p-ATM, p-CHK2, RAD51, and γH2A.X, indicating the suppression of DNA damage response (DDR) and elevated DSB accumulation. Conclusion: Inhibition of PARP1 sensitizes genotoxic agents and represents an important therapeutic approach for MM. These findings provide preliminary evidence for combining PARP1 inhibitors with TMZ for MM treatment.

The Presence of Cartilage Affects Femoral Rotational Alignment in Total Knee Arthroplasty.

OBJECTIVE: To assess the difference between the posterior condylar angle (PCA) and the mechanical lateral distal femoral angle (mLDFA) in the osseous and cartilaginous contours in a non-arthritic Chinese population. METHODS: Computed tomography (CT) and magnetic resonance imaging (MRI) were obtained from 83 patients with knee injuries before arthroscopy, and femur and distal femoral cartilage three-dimensional (3D) models were constructed. The 3D cartilage model was arranged to share physical space with the 3D femoral model, and then PCA and mLDFA were measured on the osseous and cartilaginous contours, respectively. The differences between the measurements with and without cartilage were evaluated. RESULTS: The average PCA with cartilage was 2.88 ± 1.35° and without was 2.73 ± 1.34°. The difference was significant in all patients and females but not in males. The average mLDFA with cartilage was 84.73 ± 2.15° and without cartilage was 84.83 ± 2.26°, but the difference was statistically insignificant in all groups. CONCLUSION: PCA on the osseous and cartilaginous contours significantly differed with and without cartilage in the female group, suggesting that cartilage thickness should be considered during preoperative femoral rotational resection planning.

Metaphase-Based Cytogenetic Approach Identifies Radiation-Induced Chromosome and Chromatid Aberrations in Zebrafish Embryos.

Metaphase-based cytogenetic methods based on scoring of chromosome aberrations for the estimation of the radiation dose received provide a powerful approach for evaluating the associated risk upon radiation exposure and form the bulk of our current knowledge of radiation-induced chromosome damages. They mainly rely on inducing quiescent peripheral lymphocytes into proliferation and blocking them at metaphases to quantify the damages at the chromosome level. However, human organs and tissues demonstrate various sensitivity towards radiation and within them, self-proliferating progenitor/stem cells are believed to be the most sensitive populations. The radiation-induced chromosome aberrations in these cells remain largely unknown, especially in the context of an intact living organism. Zebrafish is an ideal animal model for research into this aspect due to their small size and the large quantities of progenitor cells present during the embryonic stages. In this study, we employ a novel metaphase-based cytogenetic approach on zebrafish embryos and demonstrate that chromosome-type and chromatid-type aberrations could be identified in progenitor cells at different cell-cycle stages at the point of radiation exposure. Our work positions zebrafish at the forefront as a useful animal model for studying radiation-induced chromosome structural changes in vivo.

Early Life Irradiation-Induced Hypoplasia and Impairment of Neurogenesis in the Dentate Gyrus and Adult Depression Are Mediated by MicroRNA- 34a-5p/T-Cell Intracytoplasmic Antigen-1 Pathway.

Early life radiation exposure causes abnormal brain development, leading to adult depression. However, few studies have been conducted to explore pre- or post-natal irradiation-induced depression-related neuropathological changes. Relevant molecular mechanisms are also poorly understood. We induced adult depression by irradiation of mice at postnatal day 3 (P3) to reveal hippocampal neuropathological changes and investigate their molecular mechanism, focusing on MicroRNA (miR) and its target mRNA and protein. P3 mice were irradiated by γ-rays with 5Gy, and euthanized at 1, 7 and 120 days after irradiation. A behavioral test was conducted before the animals were euthanized at 120 days after irradiation. The animal brains were used for different studies including immunohistochemistry, CAP-miRSeq, Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and western blotting. The interaction of miR-34a-5p and its target T-cell intracytoplasmic antigen-1 (Tia1) was confirmed by luciferase reporter assay. Overexpression of Tia1 in a neural stem cell (NSC) model was used to further validate findings from the mouse model. Irradiation with 5 Gy at P3 induced depression in adult mice. Animal hippocampal pathological changes included hypoplasia of the infrapyramidal blade of the stratum granulosum, aberrant and impaired cell division, and neurogenesis in the dentate gyrus. At the molecular level, upregulation of miR-34a-5p and downregulation of Tia1 mRNA were observed in both animal and neural stem cell models. The luciferase reporter assay and gene transfection studies further confirmed a direct interaction between miR-43a-5p and Tia1. Our results indicate that the early life γ-radiation-activated miR-43a-5p/Tia1 pathway is involved in the pathogenesis of adult depression. This novel finding may provide a new therapeutic target by inhibiting the miR-43a-5p/Tia1 pathway to prevent radiation-induced pathogenesis of depression.

A Simple Method to Establish Metaphase Chromosomes from Individual Zebrafish Embryos.

Cytogenetic approach based on metaphase chromosomes established from dividing cells enables direct microscopic visualization of individual chromosomes, a powerful technique to investigate aneuploidy, chromosome aberrations, and genomic instability. In this study, we describe a simple method based on direct blocking of metaphases in individual zebrafish embryo and dropping slides with temperature changes, water vapor, and acetic acid treatment to increase the metaphase diameters. We demonstrate that well-separated metaphases could be established from single zebrafish embryos using this method. Our method could be further adapted for the analyses of DNA damage, chromosome aberrations, and genomic instability using zebrafish and other teleost models.

Hypothalamic long noncoding RNA AK044061 is involved in the development of dietary obesity in mice.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been implicated in various important biological processes, however, its role in energy balance and obesity remains largely unknown. METHODS: Differentially expressed lncRNAs in the hypothalamus of diet-induced obesity (DIO) mice versus chow-fed mice were identified by RNA sequencing. Lentivirus-mediated overexpression and knockdown of a novel lncRNA, AK044061, were used to assess its role in energy balance and the development of DIO. RNA immunoprecipitation (RIP) and pull down assays were carried out to analyze the interaction between lncRNA AK044061 and RelA, an NF-κB subunit. RESULTS: LncRNA AK044061 was upregulated in the hypothalamus of DIO mice. Acute intracerebroventricular (i.c.v.) infusion of glucose reduced the expression of lncRNA AK044061, whereas an overnight of fasting enhanced its expression. RNA in situ hybridization data showed that AK044061 was expressed in the neurons of the arcuate nucleus (ARC). Lentivirus-mediated overexpression of AK044061 in ARC cells, or in the neurons of the ARC nucleus led to an obesity-like phenotype and related metabolic disorders. Furthermore, knockdown of lncRNA AK044061 in Agouti-related peptide (AgRP)-expressing neurons mitigated DIO and its related metabolic dysregulations. In mechanism, we showed that lncRNA AK044061 was associated with RelA and could enhance the NF-κB reporter activity. The effect of lncRNA AK044061 on energy balance is mediated by NF-κB. CONCLUSIONS: Our findings suggest that excessive lncRNA AK044061 in the ARC nucleus leads to energy imbalance and obesity. LncRNA AK044061 expressed in the AgRP neurons is important in the development of dietary obesity in mice.

Low-Intensity Focused Ultrasound-Augmented Multifunctional Nanoparticles for Integrating Ultrasound Imaging and Synergistic Therapy of Metastatic Breast Cancer.

The metastasis of breast cancer is believed to have a negative effect on its prognosis. Benefiting from the remarkable deep-penetrating and noninvasive characteristics, sonodynamic therapy (SDT) demonstrates a whole series of potential leading to cancer treatment. To relieve the limitation of monotherapy, a multifunctional nanoplatform has been explored to realize the synergistic treatment efficiency. Herein, we establish a novel multifunctional nano-system which encapsulates chlorin e6 (Ce6, for SDT), perfluoropentane (PFP, for ultrasound imaging), and docetaxel (DTX, for chemotherapy) in a well-designed PLGA core-shell structure. The synergistic Ce6/PFP/DTX/PLGA nanoparticles (CPDP NPs) featured with excellent biocompatibility and stability primarily enable its further application. Upon low-intensity focused ultrasound (LIFU) irradiation, the enhanced ultrasound imaging could be revealed both in vitro and in vivo. More importantly, combined with LIFU, the nanoparticles exhibit intriguing antitumor capability through Ce6-induced cytotoxic reactive oxygen species as well as DTX releasing to generate a concerted therapeutic efficiency. Furthermore, this treating strategy actives a strong anti-metastasis capability by which lung metastatic nodules have been significantly reduced. The results indicate that the SDT-oriented nanoplatform combined with chemotherapy could be provided as a promising approach in elevating effective synergistic therapy and suppressing lung metastasis of breast cancer.

A comparison study of MnO2 and Mn2O3 as zinc-ion battery cathodes: an experimental and computational investigation.

The high specific capacity, low cost and environmental friendliness make manganese dioxide materials promising cathode materials for zinc-ion batteries (ZIBs). In order to understand the difference between the electrochemical behavior of manganese dioxide materials with different valence states, i.e., Mn(iii) and Mn(iv), we investigated and compared the electrochemical properties of pure MnO2 and Mn2O3 as ZIB cathodes via a combined experimental and computational approach. The MnO2 electrode showed a higher discharging capacity (270.4 mA h g-1 at 0.1 A g-1) and a superior rate performance (125.7 mA h g-1 at 3 A g-1) than the Mn2O3 electrode (188.2 mA h g-1 at 0.1 A g-1 and 87 mA h g-1 at 3 A g-1, respectively). The superior performance of the MnO2 electrode was ascribed to its higher specific surface area, higher electronic conductivity and lower diffusion barrier of Zn2+ compared to the Mn2O3 electrode. This study provides a detailed picture of the diversity of manganese dioxide electrodes as ZIB cathodes.

Calycosin reduces infarct size, oxidative stress and preserve heart function in isoproterenol-induced myocardial infarction model.

Calycosin (CC) is a phytoestrogen, isolated from Radix astragali a well-known Chinese herb and used for treating various pathological conditions. The current study was projected to elucidate the cardio-preservative property of CC in isoproterenol (ISO) induced cardiac injury model (MI) in rats. Male SD rats (n=48) were equally divided into 4 groups which include normal rats (Control; n=12), ISO-MI rats (n=12) which were injected with 85 mg/kg of ISO for 2 days. ISO+CC rats (n=12) were pre and post-treated with CC (30 mg/kg). CC alone rats (n=12) were injected with only CC (30 mg/kg). Pre and post-treatment with CC after and before ISO exposure showed strong cardioprotective property through significant reduction (p<0.05) in the mean values of cardiac infarct size, serum cardiac markers, inflammatory markers, apoptotic markers, lipid peroxidation (oxidative stress) by improving antioxidant status as well as reversing all those histopathological changes. Based on the results, we suggest that CC might be useful against MI if consumed along with standard MI medication to lower cardiac dysfunction and its related complications. However, further studies are needed to justify the above statement.

Formononetin inhibits osteosarcoma cell proliferation and promotes apoptosis by regulating the miR-214-3p/phosphatase and tensin homolog pathway.

BACKGROUND: Phytoestrogens have a similar molecular structure to estrogens which can produce either estrogenic or anti-estrogenic effects. It is generally believed that phytoestrogens combine with the estrogen receptor of osteosarcoma cells, affecting a variety of signal transduction pathways and cell metabolism, resulting in altered cell proliferation, differentiation, apoptosis, invasion and migration ability. Formononetin (FN) is the active ingredient of traditional Chinese medicine astragalus, angelica, and Pueraria lobate. Our study aims to detect the role of FN on MG-63 cell viability and apoptosis through regulating phosphatase and tensin homolog (PTEN) expression via MicroRNA-214-3p (miR-214-3p). METHODS: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Caspase 3 assay evaluated cell viability and apoptosis, respectively. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blot evaluated the mRNA and protein expressions, respectively. The binding site of miR-214-3p/PTEN was detected via dual luciferase assay. RESULTS: FN suppressed cell viability and induced apoptosis, and decreased miR-214-3p level and promoted PTEN expression. PTEN was then regarded as a target of miR-214-3p, and FN improved PTEN level via inhibiting miR-214-3p. Further analysis showed that overexpressed miR-214-3p improved cell viability and suppressed apoptosis of MG-63 cells by inhibiting PTEN expression. CONCLUSIONS: Finally, our results revealed that FN inhibited cell viability and induced apoptosis by regulating miR-214-3p. FN acted as a new treatment for MG-63 cells via increasing PTEN level by inhibiting the miR-214-3p level.

Oleuropein induces apoptosis via abrogating NF-κB activation cascade in estrogen receptor-negative breast cancer cells.

Oleuropein is one of the most abundant phenolic compounds found in olives. Epidemiological studies have indicated that an increasing intake of olive oil can significantly reduce the risk of breast cancer. However, the potential effect(s) of oleuropein on estrogen receptor (ER)-negative breast cancer is not fully understood. This study aims to understand the anticancer effects and underlying mechanism(s) of oleuropein on ER-negative breast cancer cells in vitro. The effect of oleuropein on the viability of breast cancer cell lines was examined by mitochondrial dye-uptake assay, apoptosis by flow cytometric analysis, nuclear factor-κB (NF-κB) activation by DNA binding/reporter assays and protein expression by Western blot analysis. In the present report, thiazolyl blue tetrazolium bromide assay results indicated that oleuropein inhibited the viability of breast cancer cells, and its effects were more pronounced on MDA-MB-231 as compared with MCF-7 cells. It was further found that oleuropein increased the level of reactive oxygen species and also significantly inhibited cellular migration and invasion. In addition, the activation of NF-κB was abrogated as demonstrated by Western blot analysis, NF-κB-DNA binding, and luciferase assays. Overall, the data indicates that oleuropein can induce substantial apoptosis via modulating NF-κB activation cascade in breast cancer cells.