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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.
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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.
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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.
Subject(s)
Animals , Rats , Adenoviridae/metabolism , Alternative Splicing , Animals, Newborn , Escherichia coli/metabolism , Genetic Vectors , Myocytes, Cardiac/metabolism , Plasmids , RNA Splicing Factors/metabolism , TransfectionABSTRACT
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 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.
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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.
Subject(s)
Humans , Female , Pregnancy , Adult , Pre-Eclampsia/genetics , RNA, Long Noncoding/genetics , Trophoblasts , Cell Movement/genetics , MicroRNAsABSTRACT
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.
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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.
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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.
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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.
Subject(s)
Asthma , Asthma, Exercise-Induced , Blotting, Western , Epithelial Cells , Gene Expression , Genetic Variation , Genetics , Genotype , Haplotypes , Hypersensitivity , Multilevel Analysis , Phenotype , Polymorphism, Single Nucleotide , Rhinitis , Rhinitis, Allergic , RNA, Messenger , SputumABSTRACT
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.
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Animals , Humans , Mice , Rats , Actins , Metabolism , Adaptor Proteins, Signal Transducing , Genetics , Metabolism , Apoptosis , Physiology , Bridged Bicyclo Compounds, Heterocyclic , Pharmacology , Cell Line, Tumor , Connective Tissue Growth Factor , Genetics , Metabolism , Pharmacology , Cytochalasin D , Pharmacology , Fatty Acids, Nonesterified , Pharmacology , HEK293 Cells , Immunohistochemistry , Insulin-Secreting Cells , Cell Biology , Metabolism , Microscopy, Fluorescence , Palmitic Acid , Pharmacology , Phosphoproteins , Genetics , Metabolism , RNA Interference , RNA, Small Interfering , Metabolism , Recombinant Proteins , Genetics , Metabolism , Pharmacology , Thiazolidines , PharmacologyABSTRACT
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.