The mechanism of emodin inhibiting YAP1 and FOXD1 in gastric cancer AGS cells and its related study / 实用医学杂志

Objective To explore the possible mechanism of emodin in inhibiting proliferation,migration,and invasion of AGS cells and in suppressing the expressions of YAP1 and FOXD1.Methods Normal gastric cell GES-1 and gastric cancer cell AGS were cultured with different concentrations of emodin.CCK8 test,scratch test and Transwell assay were used to verify changes in the biological phenotype of AGS cells.TCGA database was applied to analyze expressions of HK2,YAP1 and FOXD1 in gastric cancer tissues and normal gastric tissues.Western blotting method was used to detect the impacts of emodin on HK2,YAP1 and FOXD1 proteins in AGS cells.Exogenous pyruvic acid was added to verify the changes in YAP1 and FOXD1.Results The IC50 of emodin was significantly higher in GES-1 cells than in AGS cells(P<0.05).CCK8 proliferation test,scratch test,and Transwell assay showed that emodin significantly inhibited the biological abilities of AGS(P<0.05 for comparisons).Analysis on the TCGA bioinformatics database found that the expression of key enzymes HK2 in the glycolysis pathway and oncogenes YAP1 and FOXD1 was significantly higher in gastric cancer tissues than in normal gastric tissues(P<0.05 for comparisons).Emodin significantly inhibited the protein expressions of key glycolytic enzymes HK2 and oncogenes YAP1 and FOXD1(P<0.05 for comparisons).With supplement of exogenous glycolytic metabolite pyruvate,the protein expressions of oncogenes YAP1 and FOXD1 significantly increased(P<0.05 for comparisons).Conclusions Emodin has a significant pharmacological inhibitory effect on gastric cancer AGS cells,markedly suppressing their biological phenotype.Emodin not only significantly inhibits the key enzyme HK2 in glycolysis metabolism,but also the protein expressions of oncogenes YAP1 and FOXD1.With the addition of exogenous pyruvate to enhance the glycolytic metabolic pathway,the protein expressions of oncogenes YAP1 and FOXD1 significantly increased.The above results suggest a close association of YAP1 and FOXD1 with glycolytic metabolism.Emodin may inhibit oncogenes YAP1 and FOXD1 through the glycolytic metabolism of gastric cancer AGS cells.

Effects of Polydatin on Proliferation and Apoptosis of Pulmonary Artery Smooth Muscle Cells in Neonatal Rats with Hypoxic Pulmonary Hypertension by Regulating YAP1/TAZ Signaling Pathway / 华中科技大学学报(医学版)

Objective To investigate the effects of polydatin(PD)on the proliferation and apoptosis of pulmonary artery smooth muscle cells(PASMCs)in hypoxic pulmonary hypertension(HPH)neonatal rats,and its mechanism of action.Methods Neonatal rats were randomly separated into six groups:control group,model group,low dose PD group,medium dose PD group,high dose PD group,and high dose PD+Hippo pathway inhibitor(high dose PD+XMU-MP-1)group,with 10 rats in each group.After 2 weeks of hypoxia treatment,the right ventricular systolic blood pressure(RVSP)and right ventricular hypertro-phy index(RVHI)of rats in each group were measured.Hematoxylin-eosin(HE)staining was applied to observe pathological changes in lung tissue,and the percentage of pulmonary artery wall thickness to total thickness(WT)and the percentage of wall area to total area(WA)were calculated.Neonatal rat PASMCs were separated from each group,which were divided into NC group,hypoxia group,low dose PD group,medium dose PD group,high dose PD group,and high dose PD+XMU-MP-1 group.Cell counting kit 8(CCK-8)and 5-ethynyl-2'-deoxyuridine(EdU)were applied to detect cell proliferation.Flow cytometry was applied to detect cell apoptosis.Western blot was applied to detect the expression of Yes-associated protein 1(YAP1),tran-scriptional coactivator with PDZ-binding motif(TAZ),mammalian sterile 20-like kinase 1(MST1),B-cell lymphoma 2(Bcl-2),and Bcl-2 associated protein(Bax)in lung tissue and PASMCs.Results Compared with the control group,the pulmonary artery wall in the model group was significantly thickened,lumen was narrowed,and protein expressions of RVSP,RVHI,WT％,WA％,YAP1,MST1 and TAZ were significantly increased(all P＜0.05).Compared with the model group,pulmonary artery thickening and lumen enlargement were observed in the low,medium and high dose PD groups,and the protein expressions of RVSP,RVHI,WT％,WA％,YAP1,MST1 and TAZ were significantly decreased,which showed a dose-dependent relationship(all P＜0.05).The effect could be reversed by XMU-MP-1.Compared with the NC group,the cell A450nm value,EdU positive rate,the protein expression of YAP1,MST1,TAZ and Bcl-2 in the hydropoxia group were significantly increased.The apoptosis rate and the expression of Bax protein were obviously reduced(all P＜0.05).Compared with the hypoxia group,the cell A450nm value,EdU positive rate,the protein expression of YAP1,MST1,TAZ and Bcl-2 in the low,medium and high dose PD groups were obviously reduced.The apoptosis rate and the expression of Bax were significantly increased,which showed a dose-depend-ent relationship(all P＜0.05).The effect could be reversed by XMU-MP-1.Conclusion PD may inhibit the proliferation of PASMCs in HPH neonatal rats and promote apoptosis by inhibiting YAP1/TAZ signaling pathway.

Metformin:A promising clinical therapeutical approach for BPH treatment via inhibiting dysregulated steroid hormones-induced prostatic epithelial cells proliferation / 药物分析学报

The occurrence of benign prostate hyperplasia(BPH)was related to disrupted sex steroid hormones,and metformin(Met)had a clinical response to sex steroid hormone-related gynaecological disease.How-ever,whether Met exerts an antiproliferative effect on BPH via sex steroid hormones remains unclear.Here,our clinical study showed that along with prostatic epithelial cell(PEC)proliferation,sex steroid hormones were dysregulated in the serum and prostate of BPH patients.As the major contributor to dysregulated sex steroid hormones,elevated dihydrotestosterone(DHT)had a significant positive rela-tionship with the clinical characteristics of BPH patients.Activation of adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)by Met restored dysregulated sex steroid hormone homeostasis and exerted antiproliferative effects against DHT-induced proliferation by inhibiting the formation of androgen receptor(AR)-mediated Yes-associated protein(YAP1)-TEA domain transcription factor(TEAD4)heterodimers.Met's anti-proliferative effects were blocked by AMPK inhibitor or YAP1 over-expression in DHT-cultured BPH-1 cells.Our findings indicated that Met would be a promising clinical therapeutic approach for BPH by inhibiting dysregulated steroid hormone-induced PEC proliferation.

Bone morphogenetic protein 7 attenuates renal fibrosis in diabetic kid-ney disease rats by down-regulating Ajuba / 中国病理生理杂志

AIM:Bone morphogenetic protein 7(BMP7)reduces the expression of Yes-related protein 1(YAP1)by down-regulating Ajuba level and decreasing extracellular matrix(ECM)deposition.This study aimed to inves-tigate the influence of these factors on modifying the degree of renal fibrosis in rats with diabetic nephropathy.METH-ODS:Eighteen Sprague-Dawley(SD)rats were randomly divided into three groups:the normal control(NC)group,the diabetes mellitus(DM)group,and the DM group treated with BMP7 overexpressing adeno-associated virus(DM+rAAV-BMP7).Each group consisted of six rats.Diabetic kidney disease(DKD)was established in the DM and DM+rAAV-BMP7 groups by injecting 55 mg/kg streptozotocin(STZ)via the tail vein.NRK-52E cells were divided into three groups:the normal glucose(NG)group,the high glucose(HG)group,and the high glucose group treated with recombinant hu-man BMP7(HG+rhBMP7)group.Pathological changes in renal tissues were observed using hematoxylin and eosin(HE)and Sirius red staining.Immunohistochemical staining was performed to examine the expression sites of Ajuba and YAP1 in the renal cortex.Western blot analysis was conducted to determine the expression levels of BMP7,Ajuba,YAP1,colla-gen type Ⅲ(Col-Ⅲ),and fibronectin(FN)in the rat renal cortex and NRK-52E cells.RT-qPCR was used to measure the mRNA levels of Ajuba and YAP1 in the rat renal cortex.RESULTS:Biochemical indices revealed significantly ele-vated levels of blood glucose,serum creatinine,triglycerides,total cholesterol,and 24-hour urinary protein in the DM group compared to the NC group(P<0.05).In the DM+rAAV-BMP7 group,the levels of serum creatinine,24-hour uri-nary protein,triglycerides,and total cholesterol were lower than those in the DM group(P<0.05).Pathological staining demonstrated that the renal interstitium of the DM group exhibited inflammatory cell infiltration,fibrous tissue,collagen fi-ber deposition,disordered renal tubule arrangement,atrophy,and vacuolar degeneration,which were ameliorated in the DM+rAAV-BMP7 group.Immunohistochemistry revealed that Ajuba and YAP1 were mainly expressed in the cytoplasm and nucleus,with high expression in the cytoplasm of the DM group,which was significantly decreased in the DM+rAAV-BMP7 group.Western blot results indicated that the protein levels of FN,Col-Ⅲ,Ajuba,and YAP1 were up-regulated in the DM and the HG groups(P<0.05),but significantly down-regulated in the DM+rAAV-BMP7 group(P<0.05).RT-qP-CR results demonstrated that the mRNA levels of Ajuba and YAP1 were higher in the DM group and significantly lower in the DM+rAAV-BMP7 group(P<0.05).CONCLUSION:The overexpression of BMP7 can ameliorate renal fibrosis in rats with DKD.This effect is likely mediated by the down-regulation of Ajuba,reduction of YAP1 expression,and subse-quent inhibition of ECM deposition.

Isolated ocular colobomas caused by a novel variant of the YAP1 gene / 中华眼底病杂志

Objective:To identify the causative gene and observe the phenotypic characteristics of a family with isolated microphthalmia-anophthalmia-coloboma (MAC).Methods:A retrospective clinical study. One patient (proband) and 3 family members of a family with MAC visited the Henan Eye Hospital from May 2019 to May 2022 were included in the study. The patient's medical history and family history were inquired in detail, and the best corrected visual acuity (BCVA), slit lamp microscope, fundus photography, optical coherence tomography (OCT), ophthalmological B mode ultrasound and axial length (AL) measurement were performed. The peripheral venous blood of the proband, his parents and brother was collected for Trio whole-exome sequencing and pathogenic gene screening. Fluorescence quantitative Polymerase chain reaction was used to verify the suspicious variations. The clinical features of the patient's ocular and systemic also were observed.Results:The proband, male, was 3 years old at the first visit. The horizontal pendular nystagmus was detected in both eyes. Vertical elliptical microcornea and keyhole-shaped iris colobomas were detected in both eyes. The objective refraction at first visit (3 years old) was -4.00 DS/-0.50 DC×105° (OD) and -3.50 DS/-1.25 DC×80° (OS). Refraction and BCVA at 6 years old: -6.50 DS/-2.00 DC×110°→0.05 (OD) and -6.00 DS/-1.50 DC×80°→0.2 (OS). The AL at 4 years and 10 months old was 24.62 mm (OD) and 23.92 mm (OS), respectively. The AL at 5 years and 7 months old was 25.24 mm (OD) and 24.36 mm (OS), respectively. Ultrasonography shows tissue defects in both eyes. Fundus photography showed the inferior choroidal coloboma involving optic disc. OCT showed the optic disc in both eyes was abnormal with colobomas around, and the retinal neurosensory layer in colobomas area was disordered and thin; the retinoschisis was visible in the left eye. The proband's parents and siblings have normal phenotypes. Whole exome sequencing reveals a denovo heterozygous deletion of YAP1 gene: YAP1, chr11: 10280247-102100671, NM_ 001130145, loss 1 (EXON: 6-9). The results of bioinformatics analysis were pathogenic variants. Parents and siblings were of the wild type. Conclusions:Loss of heterozygosity in exons 6-9 of YAP1 gene is the pathogenic variation in this family. It can cause abnormal development of anterior segment, chorioretinal colobomas, deepening of axial myopia, even severe macular colobomas and retinoschisis.

Hydrogen sulfide inhibits apoptosis of cardiomyocytes and ameliorates myocardial fibrosis in rats after myocardial infarction through downregulating Yes-related protein 1 and a transcriptional co-activator with a PDZ-binding motif / 中华老年医学杂志

Objective:To investigate the effects of exogenous hydrogen sulfide on myocardial fibrosis and apoptosis in rats after myocardial infarction and the underlying mechanisms.Methods:Forty-three Sprague Dawley(SD)rats were divided into 4 groups according to the random number table method: a control group(n=12), a myocardial infarction group(MI group, n=13), an hydrogen sulfide(H 2S)group(n=6)and an MI+ H 2S group(n=12). The rat model of acute myocardial infarction was established by intraperitoneal injections of isoproterenol(50 mg/kg, once a day, for 2 days). Electrocardiogram and troponin changes were recorded 48 h after the last drug administration to determine whether the rat model was successfully constructed.After successful establishment of the model, rats in the MI group and the MI+ H 2S group were intraperitoneally injected with sodium hydrosulfide(56 μmol/kg, once a day, for 6 weeks).6 weeks later, echocardiogram and Masson's trichrome staining were performed to assess changes in cardiac function and collagen volume fraction in each group.Terminal deoxynucleotidyl transferase(TdT)dUTP nick end labeling(TUNEL)was used to detect the myocardial apoptosis rate in each group, and Western-blot was used to detect protein expression of Yes-related protein 1(YAP1), WW domain containing transcriptional regulator1(TAZ), mammalian Ste20-like kinase 2(MST2), Bcl-2-associated X protein(Bax), cysteine protease 3(caspase-3), the ratio of matrix metalloproteinase 3(MMP3)/matrix metalloproteinase inhibitor 2(TIMP2), and B-cell lymphoma factor(Bcl-2). Results:Compared with the control group, myocardial collagen volume fraction was increased( P<0.05), the myocardial cell apoptosis rate was increased( P<0.05), and myocardial YAP1, TAZ, MST2, Bax, caspase-3 protein expression and MMP3/TIMP2 ratio were increased in the MI group(all P<0.05), while the expression of Bcl-2 protein was decreased( P<0.05). Compared with the MI group, collagen volume fraction and the cardiomyocyte apoptosis rate were significantly decreased in the MI+ H 2S group( P<0.05). Also, protein expression of YAP1(2.406±0.024 vs.2.830±0.063), TAZ(0.964±0.090 vs.1.329±0.018), MST2(0.780±0.082 vs.1.788±0.097), Bax(1.500±0.008 vs.0.613±0.003)and caspase-3(0.620±0.024 vs.0.780±0.012)and the MMP3/TIMP2 ratio were decreased(all P<0.05), while protein expression of Bcl-2 was increased( P<0.05)in myocardial tissue. Conclusions:H 2S can mitigate myocardial fibrosis after myocardial infarction, through inhibiting the activation of the YAP1/TAZ signaling pathway, thus reducing apoptosis of cardiomyocytes.

Dihydroartemisinin increased the abundance of Akkermansia muciniphila by YAP1 depression that sensitizes hepatocellular carcinoma to anti-PD-1 immunotherapy / 医学前沿

The effect of anti-programmed cell death 1 (anti-PD-1) immunotherapy is limited in patients with hepatocellular carcinoma (HCC). Yes-associated protein 1 (YAP1) expression increased in liver tumor cells in early HCC, and Akkermansia muciniphila abundance decreased in the colon. The response to anti-PD-1 treatment is associated with A. muciniphila abundance in many tumors. However, the interaction between A. muciniphila abundance and YAP1 expression remains unclear in HCC. Here, anti-PD-1 treatment decreased A. muciniphila abundance in the colon, but increased YAP1 expression in the tumor cells by mice with liver tumors in situ. Mechanistically, hepatocyte-specific Yap1 knockout (Yap1LKO) maintained bile acid homeostasis in the liver, resulting in an increased abundance of A. muciniphila in the colon. Yap1 knockout enhanced anti-PD-1 efficacy. Therefore, YAP1 inhibition is a potential target for increasing A. muciniphila abundance to promote anti-PD-1 efficacy in liver tumors. Dihydroartemisinin (DHA), acting as YAP1 inhibitor, increased A. muciniphila abundance to sensitize anti-PD-1 therapy. A. muciniphila by gavage increased the number and activation of CD8+ T cells in liver tumor niches during DHA treatment or combination with anti-PD-1. Our findings suggested that the combination anti-PD-1 with DHA is an effective strategy for liver tumor treatment.

Overview of recent advances in the classification of ependymomas in WHO CNS5 classification: Simplified approach to their integrated diagnosis

Ependymomas can arise along the entire neuraxis; however, they possess site-specific unique molecular alterations and a methylome pattern which is directly related with the prognostic outcomes. Since 2016, when the updated fourth edition of World Health Organization (WHO) classification of tumors of the central nervous system was published, it has been emphasized to classify ependymomas by anatomic site and molecular signatures associated genetic alterations so that classification of the disease reflects its underlying biology. In continuation, the fifth edition of the WHO classification of CNS tumors introduces major changes, including site-specific molecular profiles as the basis of classifying ependymomas. Furthermore, an integrated tier system of reporting is recommended for better clinical correlation and predicting outcomes. WHO grading can still be included in a specific tier, along with molecular markers.

Construction of an adenovirus vector expressing engineered splicing factor for regulating alternative splicing of YAP1 in neonatal rat cardiomyocytes / 南方医科大学学报

OBJECTIVE@#To construct an adenovirus vector expressing artificial splicing factor capable of regulating alternative splicing of Yap1 in cardiomyocytes.@*METHODS@#The splicing factors with different sequences were constructed against Exon6 of YAP1 based on the sequence specificity of Pumilio1. The PCR fragment of the artificially synthesized PUF-SR or wild-type PUFSR was cloned into pAd-Track plasmid, and the recombinant plasmids were transformed into E. coli DH5α for plasmid amplification. The amplified plasmids were digested with Pac I and transfected into 293A cells for packaging to obtain the adenovirus vectors. Cultured neonatal rat cardiomyocytes were transfected with the adenoviral vectors, and alternative splicing of YAP1 was detected using quantitative and semi-quantitative PCR; Western blotting was performed to detect the signal of the fusion protein Flag.@*RESULTS@#The transfection efficiency of the adenovirus vectors was close to 100% in rat cardiomyocytes, and no fluorescent protein was detected in the cells with plasmid transfection. The results of Western blotting showed that both the negative control and Flag-SR-NLS-PUF targeting the YAPExon6XULIE sequence were capable of detecting the expression of the protein fused to Flag. The results of reverse transcription-PCR and PCR demonstrated that the artificial splicing factor constructed based on the 4th target sequence of YAP1 effectively regulated the splicing of YAP1 Exon6 in the cardiomyocytes (P < 0.05).@*CONCLUSION@#We successfully constructed adenovirus vectors capable of regulating YAP1 alternative splicing rat cardiomyocytes.

Mild hypothermia alleviates renal ischemia-reperfusion injury by up-regulating the expression of cold-shock protein RBM3 / 器官移植

Objective To evaluate the effect of mild hypothermia on the renal ischemia-reperfusion injury (IRI), and the expression profile of RNA-binding motif protein 3(RBM3) and its downstream effector molecules during this process. Methods Eighteen healthy SD male rats were randomly divided into the normal control (NC) group, IRI group and mild hypothermia pretreat (MHP) group, with 6 rats in each group. Serum creatinine level was measured to evaluate the renal function. Hematoxylin-eosin (HE) staining was performed to assess the renal tissue injury. Western blot was used to determine the relative expression levels of RBM3, Yes-associated protein 1(YAP1), nuclear factor E2-related factor 2(Nrf2), B cell-lymphoma-2(Bcl-2) and Bcl-2-associated X protein (Bax) in the kidney tissues. Immunohistochemical staining was employed to further detect the expression levels of RBM3 and YAP1 proteins. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was adopted to detect the cell apoptosis of kidney tissues. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were evaluated to determine the oxidative stress level of kidney tissues. Results Compared with the NC group, the serum creatinine level, the pathological injury score of kidney tissues and the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably lower, the apoptosis rate was remarkably elevated, the MDA content was significantly increased and the SOD activity was dramatically reduced in the IRI and MHP groups (all P < 0.05). Compared with the IRI group, the serum creatinine level and the pathological injury score of kidney tissues were significantly decreased, the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably higher, the apoptosis rate was significantly decreased, the MDA content was significantly decreased and the SOD activity was considerably elevated in the MHP group (all P < 0.05). Conclusions Mild hypothermia may exert protective effect upon renal IRI and it could alleviate cell apoptosis and oxidative stress injury induced by IRI, probably by up-regulating the expression level of RBM3 and its downstream effector molecules of YAP1 and Nrf2.

The effect and mechanism of YAP1 expression on placental angiogenesis in preeclampsia exposed to hypoxia environment / 西安交通大学学报(医学版)

【Objective】 To investigate the effect and mechanism of inhibiting Yes-associated protein1 (YAP1) expression by verteporfin on proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) exposed to hypoxia environment and the possible mechanisms that further affect placental angiogenesis in preeclampsia. 【Methods】 MTT method was used to detect the cell viability of HUVECs at different concentrations (0, 4, 8, 12 and 16 μg/mL) after 12 h and 24 h treatment with verteporfin under hypoxia and calculate the IC50 value to select the subsequent experimental drug concentration. Flow cytometry was made to analyze verteporfin’s effect on HUVEC apoptosis in hypoxic environment. The wound healing assay and Transwell invasion assay were used to determine the effect of verteporfin on HUVEC cell migration and invasion abilities under hypoxic environment. Angiogenesis test was used to detect the effect of verteporfin on the angiogenesis of HUVECs under hypoxic environment. The effects of verteporfin on the expression levels of YAP1 and TEAD1 in Hippo signaling pathway under normoxia and hypoxia were determined by Western blotting. 【Results】 Under hypoxic environment, verteporfin could inhibit the proliferation of HUVECs by calculating the IC50 value, the subsequent experimental group selected 16 μg/mL verteporfin to treat cells. Flow cytometry showed that verteporfin induced the apoptosis rate of HUVECs under hypoxia (P<0.01). The results of wound healing, Transwell invasion and the angiogenesis experiments confirmed that compared with the control group, verteporfin could inhibit the migration, invasion and angiogenesis of HUVECs in hypoxic environment (P<0.05). Western blotting assay indicated that under normoxia and hypoxia, the expressions of YAP1 and TEAD1 were reduced (P<0.01). 【Conclusion】 In hypoxic environment, verteporfin inhibits the proliferation of HUVECs by inhibiting the expressions of YAP1 and TEAD1, and reduces the migration, invasion and angiogenesis of HUVECs. It is confirmed that the Hippo-YAP1 signaling pathway may affect the placental angiogenesis of preeclampsia and participate in the occurrence of preeclampsia by regulating the proliferation and invasion of vascular endothelial cells.

Correlation between Notch signaling pathway and YAP1/TAZ in breast cancer / 西安交通大学学报(医学版)

Objective: To investigate the relationship of Notch1 expression with YAP1/TAZ expression in breast cancer and its possible mechanism. Methods: MDA-MB-231 and MCF-7 breast cancer cells were selected, and lentiviruses were used to construct stable infected cell lines with different expression levels of Notch1 and YAP1. Western blotting, RT-qPCR and co-immunoprecipitation were used to study the relationship between Notch1 and YAP1/TAZ expressions. Results: On the gene expression profiling interactive analysis (GEPIA) website, we analyzed the breast cancer data in the Cancer Gene Atlas Database (TCGA) and found that the expressions of Notch1 and YAP1/TAZ were positively correlated. Compared with those in the control group, the protein and mRNA levels of YAP1/TAZ in the shNotch1 group were reduced (P0.05). Co-immunoprecipitation showed that TAZ protein interacted with Notch1 and β-TrCP protein. Compared with those in the control group, the mRNA and protein levels of Notch1 and JAG1 in shYAP1 group were reduced (P<0.05), while those in YAP1 group were increased (P<0.05). TEAD family was predicted to be a JAG1 transcription factor on the JASPAR 2018 website. TEAD1/3/4 siRNA could effectively inhibit TEAD1/3/4 expression, and JAG1 expression decreased too (P<0.05). Conclusion: There is a positive feedback loop between Notch1 and YAP1/TAZ in breast cancer. YAP1/TAZ-TEAD can activate the Notch1 signaling pathway by regulating JAG1 expression. Notch1 protein can affect the degradation of YAP1/TAZ protein.

Establishment of YAP1 knockdown system induced by tetracycline and its effect on the function of gastric cancer cells / 西安交通大学学报(医学版)

Objective: To establish a tetracycline-induced gene knockdown system and study the effect of YAP1 on the function of gastric cancer cells. Methods: We constructed pLKO.1-tetON-YAP1 knock-down lentivirus and detected the vector by enzyme digestion and sequencing. Gastric cancer cell lines SGC-7901 and MKN-28 were infected with lentivirus, and YAP1 knocked-down gastric cancer cell lines induced by DOX were established. The mRNA level of YAP1 was detected by RT-qPCR, and the protein level of YAP1 was detected by Western blotting. Cell proliferation was detected by plate cloning experiment, and cell migration was detected by scratch-healing assay and Transwell assay. Results: The results of double enzyme digestion showed two bands at 6 000 bp and 3 000 bp, and that the sequencing results were consistent with the designed shRNA sequence. In the DOX-induced group, the mRNA and protein levels of YAP1 in gastric cancer cells infected with pLKO.1-tetON-YAP1 lentivirus significantly decreased compared with those in non-induced group. In the plate cloning experiment, the number of clones in shYAP1 groups decreased significantly after DOX induction, but there was no significant change in the non-induced group. Scratch-healing assay and Transwell assay showed that after DOX induction, the cell migration ability of shYAP1 groups was inhibited, but without significant change in the non-induced group. Results: We have successfully established a tetracycline-induced lentivirus system, and knocked down YAP1 gene of gastric cancer cells with this system. The proliferation and migration of gastric cancer cells are inhibited by YAP1 in this tetracycline induced lentivirus system.

Mechanism of reversing cisplatin resistance by Xiaoyan Decoction in lung cancer / 中草药

Objective: To investigate the possible mechanism of reversal of cisplatin resistance in lung cancer by Xiaoyan Decoction. Methods: The migration ability of cancer cells was observed by the scratch test, and the MTT was used to test the mobility of cancer cells. The siRNA transfection was applied to demonstrate the possible molecular mechanisms. The mRNA and protein expression levels were detected by real-time quantitative reverse transcription PCR and Western Blot. Results: MTT showed that A549/DDP could significantly inhibit cell proliferation at later points of time. Similarly, cisplatin combined with Xiaoyan Decoction could significantly inhibit cell proliferation at 48, 72 and 96 h. Scratch test showed that the migration distance was shorter in A549/DDP/siRNA-Beclin1 cell line than NC. MTT showed the proliferation of A549/DDP/siRNA-Beclin1 cell line was decreased when compaired with NC. Western blot also showed that the protein expression of P-gp and LRP was decreased. MTT also detected that cisplatin and Xiaoyan Decoction significantly inhibited cell proliferation of A549/DDP/siRNA-Beclin1 cell at later points of time. Cisplatin combined with Xiaoyan Decoction significantly inhibited cell proliferation in the whole course. The results showed that protein expression level of YAP1, P-gp and LRP in A549/DDP/siRNA-Beclin1 cell line was significantly lower than those in NC. Conclusion: Xiaoyan Decoction may improve the sensitivity to cisplatin may via Beclin 1-YAP1 pathway.

Long non-coding RNA FAM99A modulated YAP1 to affect trophoblast cell behaviors in preeclampsia by sponging miR-134-5p

Preeclampsia (PE) is a complex pregnancy syndrome. Convincing evidence indicates that long non-coding RNAs (lncRNAs) are involved in the pathogenesis of PE. This research mainly investigated the mechanism of family with sequence similarity 99 member A (FAM99A) in PE. The expressions of FAM99A, miR-134-5p, and YAP1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell apoptosis, migration, and invasion were detected by flow cytometry or transwell assay. The interaction between miR-134-5p and FAM99A or YAP1 was confirmed by dual-luciferase reporter assay. The protein expression of YAP1 was determined by western blot assay. FAM99A and YAP1 were significantly up-regulated, and miR-134-5p was significantly down-regulated in PE tissues (n=30). miR-134-5p was verified as a candidate of FAM99A and YAP1. FAM99A promoted cell metastasis, but reduced apoptosis in HTR8/SVneo cells by regulating miR-134-5p. miR-134-5p down-regulated YAP1 expression to suppress cell metastasis, while it induced apoptosis in HTR8/SVneo cells. FAM99A positively modulated YAP1 expression by sponging miR-134-5p. FAM99A modulated YAP1 to accelerate cell migration and invasion, and inhibited cell apoptosis in PE cells by sponging miR-134-5p. The novel regulatory network may shed light on the pathogenesis of PE.

Effects of miR-200a on the proliferation of lung cancer cells by inhibiting YAP1 / 中国肿瘤临床

Objective: To investigate the effects of miR-200a on the proliferation of lung cancer cells and to identify its direct target genes. Methods:Real-time PCR was performed to analyze the miR-200a expression in 15 paired clinical specimens of non-small cell lung cancer and adjacent noncancerous tissues, human lung cancer cell lines (A549, NCI-H520, and SK-MES-1), and one human normal lung bronchial epithelial cell line (16HBE). The effects of miR-200a on the proliferation of A549 lung cancer cells were detected through CCK-8 method. The candidate target genes of miR-200a were identified by bioinformatics screening and then verified by dual luciferase reporter gene assay, real-time PCR, and Western blot. The effects of YAP1 downregulation on the proliferation of A549 lung cancer cell line were also observed through CCK-8 method. Results:The miR-200a expression in non-small cell lung cancer tissues and lung cancer cell lines was significantly decreased (P<0.01). The upregulation of miR-200a expression could significantly inhibit the pro-liferation of A549 lung cancer cells (P<0.01). Dual luciferase reporter gene indicated that miR-200a could directly affect the 3′-untrans-lated region of the YAP1 gene to inhibit luciferase activity (P<0.01). Real-time PCR and Western blot revealed that the upregulation of miR-200a expression could significantly reduce the mRNA and protein expression levels of YAP1 in A549 lung cancer cells (P<0.01). CCK-8 method indicated that the downregulation of YAP1 could significantly prevent the proliferation of A549 lung cancer cells (P<0.01). Conclusion:MiR-200a inhibits the proliferation of lung cancer cells by targeting YAP1. Thus, miR-200a elicits tumor suppression effects.

Investigation of the Possible Role of the Hippo/YAP1 Pathway in Asthma and Allergy

PURPOSE: Several lines of evidence indicate that the Hippo/Yes-associated protein 1 (YAP1) pathways might play a role in the pathogenesis of asthma. To investigate the possible role of the Hippo/YAP1 pathway in the pathogenesis of asthma or its phenotypes. METHODS: The levels of gene expressions of the members of the Hippo/YAP1 were compared. The presence of the proteins of the YAP1 and FRMD6 were analyzed with Western blot in induced sputum of 18 asthmatic subjects and 10 control subjects. Fourteen single nucleotide polymorphisms (SNPs) in the YAP1 gene were genotyped in 522 asthmatic subjects and 711 healthy controls. The results were evaluated with traditional frequentist methods and with Bayesian network-based Bayesian multilevel analysis of relevance (BN-BMLA). RESULTS: The mRNA of all the members of the Hippo/YAP1 pathway could be detected in the induced sputum of both controls and cases. A correlation was found between YAP1 mRNA levels and sputum bronchial epithelial cells (r=0.575, P=0.003). The signal for the FRMD6 protein could be detected in all sputum samples while the YAP1 protein could not be detected in the sputum samples, of the healthy controls and severe asthmatics, but it was detectable in mild asthmatics. The rs2846836 SNP of the YAP1 gene was significantly associated with exercise-induced asthma (odds ratio [OR]=2.1 [1.3-3.4]; P=0.004). The distribution of genotypes of rs11225138 and certain haplotypes of the YAP1 gene showed significant differences between different asthma severity statuses. With BN-BMLA, 2 SNPs, genetic variations in the FRMD6 gene proved to be the most relevant to exercise-induced asthma and allergic rhinitis. These 2 SNPs through allergic rhinitis and exercise-induced asthma were in epistatic interaction with each other. CONCLUSIONS: Our results provided additional evidence that the FRMD6/Hippo/YAP1 pathway plays a role in the pathogenesis of asthma. If additional studies can confirm these findings, this pathway can be a potential novel therapeutic target in asthma and other inflammatory airway diseases.

Yap1 plays a protective role in suppressing free fatty acid-induced apoptosis and promoting beta-cell survival

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.

Effect of YAP1 on erlotinib-resistance in lung adenocarcinoma cells / 军事医学

Objective To explore the effect of Yes-associated protein 1 (YAP1) and potential mechanism on erlotinib ( ER) resistance in lung adenocarcinoma.Methods In PC-9 cells and acquired ER resistant PC-9 ( PC-9/ER) cells, the expression changes in YAP1 gene were measured by quantitative real-time PCR( RT-qPCR) and Western blot.Indirect immunofluorescence was adopted to observe the location of YAP1.PC-9/ER cells were treated with the verteporfin ( VP, YAP1 inhibitor) for 24 h and 48 h, respectively.Expression changes in mRNA and proteins of YAP1, AKT and p-AKT were detected in the presence or absence of VP.The effect of VP was analyzed by drug resistance index using Cell Counting Kit 8(CCK-8) assay.Results The resistance index of PC-9/ER cells was (99.80 ±25.81).Compared with PC-9 cells, the expression levels of YAP1 mRNA and protein were increased in PC-9/ER.The inhibitory efficiency of VP was (50.96 ±5.86)%, and the levels of AKT and p-AKT proteins were down-regulated by the inhibition of YAP1 simultaneously.The half maximal inhibitory concentration (IC50) of PC-9/ER decreased from (11.10 ±2.72) to (1.47 ± 0.32)μmol/L (P =0.024).Resistance index was reduced to one eighth of the original.Conclusion These results indicate that the YAP1 mediates ER resistance in lung adenocarcinoma.Suppression of YAP1 can reduce the resistance through PI3K/AKT signaling pathway.Therefore, YAP1 may be a potential target for lung cancer gene therapy.