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Objective:To evaluate the role of Yes-associated protein 1 (YAP1) in acute lung injury (ALI) and the relationship with ferroptosis in septic mice.Methods:Twenty-four male wild-type mice and 24 YAP1 conditional knockout mice, aged 9-10 weeks, weighing 22-25 g, were divided into 2 groups ( n=12 each) using a random number table method: wild-type sham operation group (WT+ Sham group) and wild-type sepsis-induced ALI group (WT+ ALI group); YAP1 conditional knockout sham operation group (CKO+ Sham group) and YAP1 conditional knockout sepsis-induced ALI group (CKO+ ALI group). The sepsis-induced ALI model was developed by cecal ligation and perforation (CLP) in anesthetized animals.The bronchoalveolar lavage fluid (BALF) was collected at 24 h after CLP to determine the protein concentration (by bicinchoninic acid method) and concentrations of interleukin-1beta (IL-1β) and tumor necrosis factor-α (TNF-α) (by enzyme-linked immunosorbent assay). Mice were then sacrificed, and the lung tissues were obtained for examination of ultrastructure (using a transmission electron microscope) and for determination of wet/dry lung weight ratio (W/D ratio), contents of Fe 2+ , malondialdehyde (MDA) and glutathione (GSH) (by colorimetric assay), and expression of YAP1, glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4) and solute carrier family 7 member 11 (SLC7A11) (by Western blot). Results:Compared with WT+ Sham group, the concentrations of protein in BALF, IL-1β and TNF-α were significantly increased, W/D ratio and contents of Fe 2+ and MDA were increased, GSH contents were decreased, the expression of GPX4 and SLC7A11 was down-regulated, ACSL4 expression was up-regulated ( P<0.05), alveolar epithelial cells showed characteristic changes of ferroptosis with mitochondrial shrinkage and decreased mitochondrial cristae in WT+ ALI group.Compared with WT+ CLP and CKO+ Sham groups, the concentrations of protein in BALF, IL-1β and TNF-α were significantly increased, W/D ratio and contents of Fe 2+ and MDA were increased, GSH contents were decreased, the expression of GPX4 and SLC7A11 was down-regulated, ACSL4 expression was up-regulated ( P<0.05), and the mitochondria in alveolar epithelial cells in lung tissues shrank obviously, and the mitochondrial cristae were reduced or even disappeared in CKO+ CLP group ( P<0.05). Conclusions:YAP1 is involved in the endogenous protective mechanism against ALI, which is related to inhibition of ferroptosis in septic mice.
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Objective:To investigate the mechanism of neurofibromin 1 (NF1) in gallbla-dder cancer.Methods:The experimental study was conducted. Human gallbladder cancer cell lines, including GBC-SD, NOZ, SGC996, EH-GB1, ZJU0428, human embryonic kidneys cell line 293T and human cervical cancer cell line HELA, were cultured. The recombinant plasmids (mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA) were constructed for co-immunoprecipitation experiment. The truncated Yes associated protein 1(YAP1) and NF1 recombinant proteins were purified in vitro. The interaction between NF1 and YAP1 in vitro or in vivo were verified by isothermal titration calori-metry (ITC) assay, GST pull-down experiment, co-immunoprecipitation, immunofluorescence, laser confocal microscopy, and the expression of NF1 protein in different gallbladder cancer cell lines was verified by Western blot experiments. Observation indicators: (1) interaction between NF1 and YAP1 in vitro; (2) interaction between NF1 and YAP1 in cells; (3) expression of NF1 protein in different human gallbladder cancer cell lines. The dissociation constants were exported from ITC 200 software and represented as Mean± SD. Count data were represented as absolute numbers. Results:(1) Interaction between NF1 and YAP1 in vitro. ① Results of ITC assay showed that there was interac-tion between PPQY and YAP1-WW1, between PPQY and YAP1 (Amino acid residues 162?275), and the dissociation constants between PPQY and YAP1-WW1, between PPQY and YAP1(Amino acid residues 162?275) were (0.42±0.06)mmol/L, (0.69±0.14)mmol/L, respectively. ② GST pull-down results indicated that the target protein His-Sumo-YAP1 WW1 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW1, relative to the reaction system between GST protein and His-Sumo-YAP1 WW1. The target protein His-Sumo-YAP1 WW2 was obviouly observed in protein lane of reaction system between GST-PPQY recombinant protein and His-Sumo-YAP1 WW2, relative to the reaction system between GST protein and His-Sumo-YAP1 WW2. (2) Interaction between NF1 and YAP1 in cells. ① Co-immunoprecipitation results indica-ted that NF1 protein was observed in cell lysis solution which was incubated by FLAG gel beads and cotransfected with mRFP-YAP1 FL-FLAG and eGFP-MYC-NF1 2650?2750-HA. ② Immuno-fluorescence and laser confocal microscopy results indicated that YAP1 and NF1 with obvious fluorescence were co-localized in the cytoplasm of human gallbladder cancer NOZ cells. However, YAP1 with obvious fluorescence was localized in the nucleus of human gallbladder SGC996 cells and NF1 showed weak fluorescence. (3) Expression of NF1 protein in different human gallbladder cancer cell lines. Western blot results showed that with the expression level of NF1 protein in HELA cell line as the standard, the relative expression levels of NF1 protein in EH-GB1, GBC-SD, NOZ, SGC996, ZJU0428 cell lines were 1.28, 0, 1.01, 0, 0, respectively. Conclusion:NF1 affects the gallbladder cancer by directly acting on YAP1 protein.
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Objective To investigate the role of PKCι, YAP1 and high-risk HPV infection in the local immune microenvironment of cervical cancer. Methods We chose 80 cases of normal tissue of the cervix (NCT), cervical low-grade squamous intraepithelial lesion (LSIL), cervical high-grade squamous intraepithelial lesion (HSIL) and early cervical squamous cell carcinoma (SCC) each. Four groups were collected.The infection rate of high-risk HPV in four groups was determined by real-time fluorescence PCR method. The expression levels of PKCι, YAP1, CD4 and CD8 in four groups were measured and correlated by IHC and clinicopathologic features were also analyzed. Results The differences of high-risk HPV infection rate and PKCι, YAP1, CD4, CD8 positive rate among groups of NCT, LSIL, HSIL and SCC had statistical significance (P < 0.05). The level of cervical lesions was positively associated with high-risk HPV infection and positive PKCι, YAP1, CD8 expression (P < 0.05), while negatively associated with positive CD4 expression (P < 0.05). HPV infection and positive PKCι, YAP1, CD8 expression were positively correlated with each other in SCC, while were all negatively correlated with positive CD4 expression(P < 0.05). The differences of HPV infection, PKCι, YAP1 and CD8 positive expression were significant in different levels of differentiation and vascular invasion of SCC (P < 0.05). Conclusion The patients with cervical lesions are often accompanied by high-risk HPV infection and abnormal expression of PKCι, YAP1, CD4 and CD8, which may have synergistic effects on each other, causing the local immunosuppression microenvironment of SCC. It provides a possible strategy for the study of pathogenesis, diagnosis and treatment of cervical cancer.
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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.
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Objective To investigate the clinical significances of the expressions of Yes-associated protein 1 (YAP1) and collagen triple helix repeat containing 1 (CTHRC1) in triple-negative breast cancer (TNBC) and their correlations with expression of E-cadherin. Methods Immunohistochemical analysis (SABC) was performed to detect expressions of YAP1 and CTHRC1 in 73 specimens of TNBC and adjacent cancer tissues collected from patients in Dandong First Hospital from January 2006 to December 2017. The correlations between the expressions of YAP1 and CTHRC1 and clinicopathologic features and E-cadherin expression were analyzed. Results The expression rates of YAP1 and CTHRC1 in TNBC were 71.23％(52/73) and 79.45％(58/73), and 13.70％(10/73) and 27.40％(20/73) in adjacent cancer tissues, respectively, and the differences were statistically significant (χ2 values were 49.452 and 39.748, both P< 0.01). The expressions of YAP1 and CTHRC1 were related to tumor grade, clinical stage and lymphatic metastasis (YAP1:χ2 values were 10.244, 8.754, and 6.914, all P<0.05;CTHRC1:χ2 values were 12.582, 13.172, and 6.400, all P< 0.05), but they were not related to patient's age, tumor diameter and menopausal status (all P>0.05). The expressions of YAP1 and CTHRC1 were negatively correlated with expression of E-cadherin in TNBC (r=-0.371, P=0.001;r=-0.323, P=0.005). Conclusion YAP1 and CTHRC1 is closely related to the occurrence and development of TNBC and may participate in the invasion of TNBC through epithelial mesenchymal transition.
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Objective: To investigate the effects of Yes-associated protein 1 (YAP 1) gene knockdown in cancer-associated fibroblast (CAF) on the migration and invasion of breast cancer MDAMB- 231 cells. Methods: The expression of YAP1 in breast primary CAF and paired normal fibroblast (NF) in mRNA microarray was analyzed by bioinformatics methods. To verify the authenticity and accuracy of the microarray results, the expressions of YAP1 mRNA and protein in primary and immortalized NF and CAF were detected by real-time fluorescent quantitative PCR and Western blotting. A co-cultured system including breast cancer MDA-MB-231 cells and the conditioned medium of NF or CAF was used, and the effect of fibroblasts on the invasion ability of breast cancer cells was detected by Transwell chamber assay. CAF cells were transfected with the plasmids carrying the specific shRNA targeting YAP 1 gene, then the expressions of YAP1 mRNA and protein in CAF were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The effects of YAP 1 gene interference in CAF on the migration and invasion of MDA-MB-231 cells cultured with CAF conditioned medium were detected by scratch wound healing and Transwell chamber assay, respectively. CAF cells were treated with Hippo pathway inhibitor XMU-MP-1, then the expressions of total YAP1 protein and phosphorylated YAP1 protein in CAF were detected by Western blotting, as well as the invasion ability of MDA-MB-231 cells co-cultured with the CAF conditioned medium was measured by Transwell chamber assay. The expressions of transforming growth factor-beta 1 (TGF -β 1) and interleukin 6 (IL 6), two target genes of YAP1 and the cytokines associated with cell migration and invasion, in CAF with YAP 1 gene interference were detected by real-time fluorescent quantitative PCR. The effect of YAP1-knocked CAF on the extracellular matrix remodeling was tested by Collagen gel contraction assay. Results: Compared with primary NF, the expression of YAP 1 gene was significantly upregulated in primary CAF of breast cancer according mRNA microarray (P < 0.05). The higher expressions of YAP1 mRNA and protein in primary and immortalized CAF were confirmed by real-time fluorescent quantitative PCR (P < 0.05) and Western blotting (P < 0.01). The invasion ability of breast cancer MDA-MB-231 cells was significantly promoted in the coculture system with the conditioned medium of CAF as compared with NF (P < 0.01). The CAF cell line transfected with YAP1 shRNA was successfully constructed, and the expressions of YAP1 mRNA and protein were successfully stably knocked down in CAF (both P < 0.01). Compared with the YAP1-unknocked CAF control group, the migration (P < 0.05) and invasion (P < 0.01) abilities of breast cancer MDA-MB-231 cells were significantly weakened after culture with the conditioned medium of YAP1-knocked CAF. The phosphorylation level of YAP1 protein was decreased in CAF treated with Hippo pathway inhibitor XMU-MP-1 (P < 0.01), while the invasion ability of MDA-MB-231 cells was significantly enhanced after coculture with XMU-MP-1-treated CAF (P < 0.05). The expression levels of TGF-β1 and IL6 mRNAs were down-regulated in YAP1-knocked CAF (both P < 0.05), and the collagen gel shrinking ability of extracellular matrix was significantly decreased in YAP1-knocked CAF group (P < 0.05). Conclusion: The stable knockdown of YAP1 expression in CAF can inhibit the migration and invasion of breast cancer MDA-MB-231 cells through down-regulating the expressions of TGF-β1 and IL6 and remodeling the extracellular matrix.
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OBJECTIVE@#To investigate the effects of Yes-associated protein 1 (YAP1) knockdown on the proliferation, migration and invasion in human nasopharyngeal carcinoma (NPC) cells.@*METHODS@#We detected the expression of YAP1 mRNA and protein in different NPC cell lines and an immortalized nasopharyngeal epithelial cell line using RT-PCR and Western blotting. Two YAP1-targeting small interfering RNAs (siRNA) were transfected into NPC cell lines S26 and S18, and the knockdown efficiency was confirmed by RT-PCR and Western blotting. The effect of YAP1 knockdown on the proliferation of the NPC cells was determined by cell counting and colony formation assay; wound healing assay and Transwell assay were used to analyze the changes in the cell migration and invasion abilities in each group. Western blotting was used to analyze the changes in the expressions of c-myc, E-cadherin, N-cadherin and vimentin in the NPC cells after YAP1 knockdown.@*RESULTS@#YAP1 was highly expressed in the NPC cell lines. Compared with the negative control group, the NPC cell lines with YAP1 knockdown showed significantly lowered YAP1 expressions at both the mRNA and protein levels ( < 0.05). YAP1 knockdown significantly suppressed the growth, cloning formation, migration and invasion of the NPC cells as compared with control cells ( < 0.01). YAP1 knockdown obviously decreased the expression levels of c-myc, N-cadherin and vimentin and increased E-cadherin expression in the NPC cells.@*CONCLUSIONS@#YAP1 knockdown siRNA suppresses the proliferation, migration and invasion of NPC cells , suggesting that YAP1 may serve as a therapeutic target for NPC.
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Humans , Adaptor Proteins, Signal Transducing , Genetics , Metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Nasopharyngeal Carcinoma , Genetics , Neoplasm Invasiveness , Phosphoproteins , Genetics , MetabolismABSTRACT
Objective@#To investigate the role of Yes-associated protein 1 (YAP1)-JUN in promoting the proliferation and epithelial mesenchymal transitions (EMT) transformation of laryngeal squamous cell carcinoma.@*Methods@#The expression and subcellular localization of YAP1 were detected by tissue immunofluorescence assay. The effects of YAP1 on the proliferation of laryngeal squamous cell were examined by cell clone formation experiment. Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the effect of YAP1 on the expression and transcriptional level of EMT-related molecular markers. The interaction between YAP1 and JUN was detected by immunocoprecipitation (CoIP) and Western blot assay, which regulated the expression of downstream genes to control the EMT process.@*Results@#The expression of YAP1 in laryngeal squamous cell carcinoma tissue increased and transferred from cytoplasm to nucleus. The number of clones increased significantly after YAP1 up regulation (P<0.01). The expression level of key gene E-cadherin in epithelial cells was significantly inhibited after YAP1 up (P<0.01), while the expression level of the key genes of interstitial cells, β-catenin, vimentin and N-cadherin was significantly up (P<0.01). YAP1 was interacted with nuclear transcription factor JUN, and the proliferation ability of YAP1 decreased significantly (P<0.01) after the inhibition of JUN expression (P<0.01), and the expression of EMT related molecular markers decreased significantly (P<0.01).@*Conclusions@#YAP1 combined with JUN gene promotes the proliferation and EMT transformation of human laryngeal squamous cell carcinoma cells.
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Objective To investigate the role of Yes-associated protein 1 (YAP1)-JUN in promoting the proliferation and epithelial mesenchymal transitions (EMT) transformation of laryngeal squamous cell carcinoma.Methods The expression and subcellular localization of YAP1 were detected by tissue immunofluorescence assay.The effects of YAP1 on the proliferation of laryngeal squamous cell were examined by cell clone formation experiment.Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the effect of YAP1 on the expression and transcriptional level of EMT-related molecular markers.The interaction between YAP1 and JUN was detected by immunocoprecipitation (CoIP) and Western blot assay,which regulated the expression of downstream genes to control the EMT process.Results The expression of YAP1 in laryngeal squamous cell carcinoma tissue increased and transferred from cytoplasm to nucleus.The number of clones increased significantly after YAP1 up regulation (P ＜0.01).The expression level of key gene E-cadherin in epithelial cells was significantly inhibited after YAP1 up (P ＜0.01),while the expression level of the key genes of interstitial cells,β-catenin,vimentin and N-cadherin was significantly up (P ＜ 0.01).YAP1 was interacted with nuclear transcription factor JUN,and the proliferation ability of YAP1 decreased significantly (P ＜0.01) after the inhibition of JUN expression (P ＜ 0.01),and the expression of EMT related molecular markers decreased significantly (P ＜0.01).Conclusions YAP1 combined with JUN gene promotes the proliferation and EMT transformation of human laryngeal squamous cell carcinoma cells.
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Yes-associated protein 1 (YAP1), one of the main effectors in the Hippo signaling pathway, is significantly correlated with high tumor grade, lymph node metastasis, advanced stage, and poor prognosis in patients with non-small cell lung cancer (NSCLC). Re-cently, several studies have shown that YAP1 can increase proliferation, invasiveness, and migration of NSCLC cells; promote self-re-newal and angiogenic mimicry of cancer stem cells; and decrease sensitivity to chemotherapy, radiotherapy, and epidermal growth fac-tor receptor tyrosine kinase inhibitors. Moreover, the relationship between YAP1 and PD-L1 revealed the important role of YAP1 in im-munotherapy against NSCLC. Further, preclinical studies reported roles of verteporfin, one drug targeting YAP1, such as inhibition of NSCLC cell proliferation, reduction in cancer stem cell activity, and restoration of treatment sensitivity. This review summarizes the above advancements, discussing the importance and necessity of target therapy against YAP1 in NSCLC.
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Objective To explore the role of Yes-associated protein 1 (YAP1) in the proliferation, cell cycle, migration and invasion of human non-small cell lung cancer (NSCLC) cells. Methods A lenti-siRNA targeting YAP1 (si-YAPl) was used to inhibit the expression of YAP1 gene of human NSCLC cell line A549 cells. CCK-8 assay and flow cytometry were used to determine the effects of silencing of YAP1 expression on A549 cells proliferation and cell cycle, respectively; Transwell assay was used to observe the effect of silencing of YAP1 expression on A549 cell migration and invasion. Results After infection with si-YAPl, the expressions of YAP1 mRNA and protein in A549 cells were significantly down-regulated (P<0. 01). YAP1 silencing significantly inhibited A549 cell proliferation, increased the percentage of cells in G0/G1 phase (P
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Objective To explore the role of Yes-associated protein 1 (YAP1) in the proliferation,cell cycle,migration and invasion of human non-small cell lung cancer (NSCLC) cells.Methods A lenti-siRNA targeting YAP1 (si-YAP1) was used to inhibit the expression of YAP1 gene of human NSCLC cell line A549 cells.CCK-8 assay and flow cytometry were used to determine the effects of silencing of YAP1 expression on A549 cells proliferation and cell cycle,respectively;Transwell assay was used to observe the effect of silencing of YAP1 expression on A549 cell migration and invasion.Results After infection with si-YAP1,the expressions of YAP1 mRNA and protein in A549 cells were significantly down-regulated (P＜0.01).YAP1 silencing significantly inhibited A549 cell proliferation,increased the percentage of cells in G0/G1 phase (P＜0.01),and significantly decreased the migration and invasion of A549 cells (P＜0.01).Conclusion YAP1 silencing can inhibit malignant biological characteristics of NSCLC,which suggests that YAP 1 gene may serve as an important target in the gene therapy of lung cancer.
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Objective To investigate the expressions of Yes-associated protein-1 (YAP1) in gallbladder mucosal epithelium of normal persons,in patients with simple/calculous cholecystitis,and in patients with gallbladder carcinoma;and to study the mechanism of YAP1 in gallbladder carcinoma development.Methods Immunohistochemistry was used to detect the expression and distribution of YAP1 protein in 50 persons with normal gallbladder,101 patients with simple cholecystitis/calculous cholecystitis and 100 patients with gallbladder carcinoma.RT-PCR and western-blot were used to detect the mRNA and protein levels of YAP1 in normal and malignant gallbladder mucosal epithelium cells.siRNA was used to shut down the expression of YAP1 in SGC996 cells.MTT was used to test cell vitality.Flow cytometry was used to measure cell cycle.Results Immunohistochemistry revealed the expression rates of YAP1 in the gallbladder carcinoma group,the cholecystitis/gallstone group and the control group to be 87.0％ (87/100),56.4％ (57/101) and 5.0％ (1/20),respectively (P ＜ 0.01).The YAP1 protein levels were higher in gallbladder carcinoma tissues and cells when compared to normal tissues and cells.RT-PCR showed the mRNA levels of gallbladder carcinoma cells to be 12.5 ± 1.2 times of normal gallbladder mucosal epithelial cells (P ＜ 0.05).After using siRNA to shut down the YAP1 expression,EMT associated proteins were down-regulated,cell vitality was decreased,and cell cycle was arrested in the S-phase.Conclusions YAP1 is closely related to cell proliferation and metastasis of gallbladder carcinoma.It may promote tumor progression through epithelial-mesenchymal transition.transition;Tumor progress