基于CRISPR/Cas9系统构建精准调控Wnt信号通路的表达载体及其效果的验证

目的:构建基于CRISPR/cas9系统调控Wnt信号通路的载体,并在细胞水平验证其调控基因表达的效率。方法:选取Wnt信号中负性调控分子,设计并合成能够表达靶向上述分子gRNA的互补DNA克隆序列,BsmBI限制性内切酶酶切载体后,采用分子克隆的方法将上述序列克隆至目的载体lenti-sgRNA-Ms2-zeo,测序正确的克隆通过Lipofectamine2000与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共同转染入293细胞;转染24h后收集细胞,qRt-PCR检测目的基因的表达。结果:筛选了Wnt信号通路中已知的19个负性调控基因;针对每个基因设计了两对gRNA序列,并构建了能够表达gRNA和MS2融合序列的载体,测序结果显示重组质粒的DNA序列与预期完全相符。随机挑选了4个表达载体与lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine共转进入细胞,qPCR结果显示构建的目的载体联合lenti-Ms2-P65-HSF1-Hygro和lenti-dcas9-VP64-blastine载体可以协同促进靶分子表达。结论:本研究成功构建了基于CRISPR/cas9基因编辑系统调控Wnt信号的载体。     

Long non‐coding RNA LINC01225 promotes proliferation,invasion and migration of gastric cancer via Wnt/β‐catenin signalling pathway

Emerging evidence has classified the aberrant expression of long non‐coding RNAs (lncRNAs) as a basic signature of various malignancies including gastric cancer (GC). LINC01225 has been shown to act as a hepatocellular carcinoma‐related gene, with its expression pattern and biological function not clarified in GC. Here, we verified that LINC01225 was up‐regulated in tumour tissues and plasma of GC. Analysis with clinicopathological information suggested that up‐regulation of LINC01225 was associated with advanced disease and poorer overall survival. Receiver operating characteristic (ROC) analysis showed that plasma LINC01225 had a moderate accuracy for diagnosis of GC. In addition, knockdown of LINC01225 led to retardation of cell proliferation, invasion and migration, and overexpression of LINC01225 showed the opposite effects. Mechanistic investigations showed that LINC01225 silencing inhibited epithelial‐mesenchymal transition (EMT) process and attenuated Wnt/β‐catenin signalling of GC. Furthermore, ectopic expression of Wnt1 or suppression of GSK‐3β abolished the si‐LINC01225‐mediated suppression against EMT, thereby promoting cell proliferation, invasion and migration of GC. In conclusion, LINC01225 promotes the progression of GC through Wnt/β‐catenin signalling pathway, and it may serve as a potential target or strategy for diagnosis or treatment of GC.     

Ginsenoside Rb1 ameliorates CKD‐associated vascular calcification by inhibiting the Wnt/β‐catenin pathway

Vascular calcification (VC) is a pathological process underpinning major cardiovascular conditions and has attracted public attention due to its high morbidity and mortality. Chronic kidney disease (CKD) is a common disease related to VC. Ginsenoside Rb1 (Rb1) has been reported to protect the cardiovascular system against vascular diseases, yet its role in VC and the underlying mechanisms remain unclear. In this study, we established a CKD‐associated VC rat model and a β‐glycerophosphate (β‐GP)‐induced vascular smooth muscle cell (VSMC) calcification model to investigate the effects of Rb1 on VC. Our results demonstrated that Rb1 ameliorated calcium deposition and VSMC osteogenic transdifferentiation both in vivo and in vitro. Rb1 treatment inhibited the Wnt/β‐catenin pathway by activating peroxisome proliferator‐activated receptor‐γ (PPAR‐γ), and confocal microscopy was used to show that Rb1 inhibited β‐catenin nuclear translocation in VSMCs. Furthermore, SKL2001, an agonist of the Wnt/β‐catenin pathway, compromised the vascular protective effect of Rb1. GW9662, a PPAR‐γ antagonist, reversed Rb1's inhibitory effect on β‐catenin. These results indicate that Rb1 exerted anticalcific properties through PPAR‐γ/Wnt/β‐catenin axis, which provides new insights into the potential theraputics of VC.     

MicroRNA‐488 inhibits endometrial glandular epithelial cell proliferation,migration, and invasion in endometriosis mice via Wnt by inhibiting FZD7

Endometriosis is a chronic inflammatory syndrome and nearly 6%‐10% of women are affected by it during the reproductive period. Previous studies have proved that microRNAs (miRNAs) are implicated in the pathogenesis of ovarian endometriosis. In this study, we aimed to investigate that restored miR‐488 would effectively inhibit the development of endometriosis. The microarray‐based data analysis was performed to screen endometriosis‐related differentially expressed genes (DEGs). The mouse model in endometriosis syndrome was established by being subcutaneously injected with Estradiol benzoate, and the ectopic endometrial tissues and normal endometrial tissues were collected. Additionally, the endometrial glandular epithelial cells were extracted from the endometrial glandular epithelial tissues from normal and endometriosis mice. In order to examine the role of miR‐488 in mice with endometriosis, we measured miR‐488 expression and expression levels of Frizzled‐7 (FZD7), cyclinD1, β‐catenin, and c‐Myc in vivo and in vitro. Finally, we detected the effect of miR‐488 on cell proliferation, apoptosis, migration and invasion in vitro. FZD7 was upregulated in human endometriosis. The data showed higher expression levels of FZD7, β‐catenin, c‐Myc and cyclinD1, and lower miR‐488 expression in mouse endometrial tissues. FZD7 was the target gene of miR‐488. Furthermore, elevated miR‐488 in isolated mouse endometrial glandular endometrial cells inhibited FZD7, the translocation of β‐catenin to nucleus, the activation of Wnt pathway, and the cell proliferation, migration and invasion. Collectively, these findings indicated that up‐regulated miR‐488 may reduce the proliferation, migration and invasion of endometrial glandular epithelial cells through inhibiting the activation of Wnt pathway by down‐regulating FZD7.     

(Pro)renin receptor accelerates development of sarcopenia via activation of Wnt/YAP signaling axis

To extend life expectancy and ensure healthy aging, it is crucial to prevent and minimize age‐induced skeletal muscle atrophy, also known as sarcopenia. However, the disease's molecular mechanism remains unclear. The age‐related Wnt/β‐catenin signaling pathway has been recently shown to be activated by the (pro)renin receptor ((P)RR). We report here that (P)RR expression was increased in the atrophied skeletal muscles of aged mice and humans. Therefore, we developed a gain‐of‐function model of age‐related sarcopenia via transgenic expression of (P)RR under control of the CAG promoter. Consistent with our hypothesis, (P)RR‐Tg mice died early and exhibited muscle atrophy with histological features of sarcopenia. Moreover, Wnt/β‐catenin signaling was activated and the regenerative capacity of muscle progenitor cells after cardiotoxin injury was impaired due to cell fusion failure in (P)RR‐Tg mice. In vitro forced expression of (P)RR protein in C2C12 myoblast cells suppressed myotube formation by activating Wnt/β‐catenin signaling. Administration of Dickkopf‐related protein 1, an inhibitor of Wnt/β‐catenin signaling, and anti‐(P)RR neutralizing antibody, which inhibits binding of (P)RR to the Wnt receptor, significantly improved sarcopenia in (P)RR‐Tg mice. Furthermore, the use of anti‐(P)RR neutralizing antibodies significantly improved the regenerative ability of skeletal muscle in aged mice. Finally, we show that Yes‐associated protein (YAP) signaling, which is coordinately regulated by Wnt/β‐catenin, contributed to the development of (P)RR‐induced sarcopenia. The present study demonstrates the use of (P)RR‐Tg mice as a novel sarcopenia model, and shows that (P)RR‐Wnt‐YAP signaling plays a pivotal role in the pathogenesis of this disease.     

Population and Single-Cell Analysis of Human Cardiogenesis Reveals Unique LGR5 Ventricular Progenitors in Embryonic Outflow Tract

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4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis,biological activity,and molecular docking studies

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC₅₀ values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC₅₀ values of 2.0 and 0.9?μM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100?μg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.     

Synthesis and biological evaluation of BMS-986120 and its deuterated derivatives as PAR4 antagonists

BMS-986120 is a PAR4 antagonist that is being investigated as an antiplatelet agent in phase I clinical trial. An improved synthesis of BMS-986120 has been developed. Based on the novel synthetic approach to BMS-986120, a series of deuterated derivatives of BMS-986120 have been synthesized and biologically evaluated to search for more potent antiplatelet agents. The in vitro antiplatelet assay by turbidimetry demonstrated that PC-2 and PC-6 had IC₅₀ values of 6.30?nM and 6.97?nM, respectively, versus BMS-986120 with an IC₅₀ of 7.80?nM. The result of in vitro metabolic stability study showed that all of the deuterated compounds had similar half-life (T_1/2) and intrinsic clearance (Clint) in comparison with BMS-986120. Further probing the metabolic profile of BMS-986120 is worth being conducted.