Screening of Differential Genes for Endometriosis and Expression Validation of the Potential Target GAS6 / 中国生物化学与分子生物学报

Growth arrest specific protein 6 (GAS6) plays an important role in the occurrence and development of tumors, and its signal transduction is involved in cell proliferation, adhesion and migration, but its related functions and molecular mechanisms in endometriosis (EMs) are still unclear. In this study, we searched and downloaded the transcriptome datasets of EMs from GEO database and performed GEO online analysis, and then screened out the differentially expressed genes and performed cluster analysis based on GO and KEGG pathway. The mRNA levels of the differentially expressed genes shared by more than three datasets were verified by qRT-PCR in the endometrium of ten women with no endometriosis and no clear disease and the ectopic endometrium of 11 patients with ovarian chocolate cysts. Immunohistochemistry and qRT-PCR were used to verify the expression of GAS6 and epithelial mesenchymal transition (EMT) marker genes, and immunofluorescence was used to co-label GAS6 and E-cadherin in endometriosis clinical samples. In this study, a total of 47 differentially expressed genes were screened out of the four transcriptome datasets, which were mainly enriched in processes such as cell migration and related signal pathways such as MAPK, PI3K-AKT, and tight junction. The mRNA levels of the nine differentially expressed genes shared by more than three datasets in endometriosis patients were consistent with the results of bioinformatics analysis. GAS6 expression levels in ectopic endometrium of EMs patients are higher than the control group (P < 0. 05), and EMs patients have the characteristics of EMT in the ectopic endometrial tissue, that is, the expression of E-cadherin is down-regulated (P < 0. 05) and the expression of vimentin is up-regulated (P < 0. 01). The expression of E-cadherin in the ectopic endometrial glandular epithelial cells of EMs patients is low while the expression of GAS6 is up-regulated, suggesting that GAS6 may mediate the EMT process in endometriosis. In conclusion, this study reveals that GAS6 is highly expressed in endometriosis patients and may mediate the EMT process to participate in the occurrence and development of endometriosis, providing a potential target for clinical treatment of endometriosis.

Role of growth arrest-specific protein 6 in migration and osteogenic differentiation of human periodontal ligament cells / 北京大学学报(医学版)

OBJECTIVE@#To investigate the role of growth arrest-specific protein 6 (Gas6) in the process of the migration and osteogenic differentiation of human periodontal ligament cells (hPDLCs).@*METHODS@#After different concentrations of recombinant human Gas6 (rhGas6) were added to hPDLCs, cell prolife-ration experiment (CCK-8) was taken to observe the effect of rhGas6 on hPDLCs cell proliferation. Scratch test and cell migration test (Transwell) were taken to analyze the migratory ability of hPDLCs in different concentrations of rhGas6 groups. After osteogenic induction, real-time quantitative polymerase chain reaction (real-time PCR) was taken to detect the expression of the Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP). ALP staining was used to detect the amount of mineralized nodules.@*RESULTS@#After adding different concentrations of rhGas6, there were no statistically significant differences in hPDLCs cell proliferation among the experimental groups and the control group at 24, 48 and 72 hours (P>0.05). After 24 h of scratch, the healing area in the 800 μg/L of the rhGas6 group was greater than that in the control group, but without statistically significant difference (31.06%±13.70% vs. 21.79%±9.51%, P>0.05). In the migration test, after 24 h, the number of hPDLCs cells which penetrated through the membrane in the 800 μg/L rhGas6 group was significantly higher than that in the control group (P < 0.01). After rhGas6 was added and osteogenic induction, Runx2 and ALP gene expressions of hPDLCs in the 800 μg/L group were significantly higher than those in the control group (1.60±0.30 vs. 0.91±0.10, 2.81±0.61 vs. 0.86±0.12, P < 0.01). After Gas6 was knocked down, the ALP expression of hPDLCs was significantly lower than that of the control group (0.39±0.07 vs. 0.92±0.14, P < 0.01). There was no significant change in Runx2 expression (P>0.05). After 7 days of osteogenic induction, the mineralized nodules formed in the Gas6 knockdown group were significantly less than those in control group (0.25±0.04 vs. 1.00±0.11, P < 0.001). After 14 days of induction, the staining degree of the Gas6 knockdown group was lower than that of the control group, but there was no significant difference (0.86±0.04 vs. 1.00±0.16, P>0.05).@*CONCLUSION@#After downregulation of Gas6 gene, mineralized nodule formation was reduced and ALP gene expressions were decreased in the early stage of osteogenic induction (7 days). After addition of rhGas6, Runx2 and ALP gene expressions were increased and the number of cell migration was increased, suggesting that Gas6 might play a promoting role in the migration and osteogenic differentiation of human periodontal ligament cells.

Vascular calcification and growth arrest specific protein 6 levels in chronic renal failure / Calcificación vascular y detención de crecimiento de proteína específica 6 niveles en insuficiencia renal crónica

INTRODUCTION: Cardiovascular disease is the main cause of mortality and morbidity in chronic renal failure. It's known that vascular calcification (VC) and carotid intima media thickness (CIMT) are strongly associated with cardiovascular diseases. Growth arrest specific protein 6 (Gas6) is a vitamin K-dependent protein and regulates various processes such as proliferation, cell survival, migration and inflammation. Gas6 is known to protect endothelial cells and vascular smooth muscle cells against apoptosis by inhibiting Bcl-2 induced Caspase 3 activation. The relationship between Gas6 and cardiovascular diseases has been demonstrated in many mouse models and cell cultures. However, there are conflicting reports whether Gas6 levels are increasing or decreasing in human studies of diabetic and/or chronic renal failure. In present study the aim was to examine plasma Gas6 levels and its relation with CIMT and coronary artery calcification score (CACS) in chronic kidney disease (CKD) patients. METHODS: Total of 137 patients of which 32 chronic hemodialysis and 105 predialysis patients as well as 73 healthy controls were enrolled in the study. Human Gas6 levels in serum samples were studied by ELISA method. CIMT was measured by ultrasonography. CACS was measured by multislice computed tomography. RESULTS: The mean age was 54.37±16.61 years in dialysis group, 55.20±14.80 years in predialysis group and 53.26±9.04 years in control group. Serum creatinine was 0.78±0.16 mg/dl in the control group and 1.96±1.64 mg/dl in the predialysis group and 5.94±1.55 mg/dl in the dialysis group. 24 hours urine protein levels were significally higher in the dialysis group than the predialysis and the control group. CIMT values were similar in predialysis and dialysis groups. These values were significantly higher than the control group. Although CACS was higher in dialysis group than predialysis and control group, the results were not statistically significant since the distribution range was very wide. Gas6 was 98.84±53.32 ng/mL in the control group and statistically higher than the dialysis (63.85±38.92 ng/mL) and the predialysis groups (54.96±38.49 ng/mL) (p=0.001). Gas6 levels were lower in diabetic patients than non-diabetics (53.69±35.26 ng/mL, 69.26±47.50 ng/mL, p=0.023, respectively). Negative correlation was detected between Gas6 and age, BMI, CACS, carotid IMT and proteinuria. In the logistic regression analysis, Gas6 remained significantly associated with BMI, CIMT and proteinuria. CONCLUSION: In our study, a negative correlation of Gas6 with BMI, CACS, CIMT and proteinuria and lower Gas6 levels in diabetic patients support that decreased Gas6 levels in chronic renal failure may have a role in vascular calcification through altered glucose tolerance, chronic inflammation, endothelial dysfunction and increased apoptosis. Our study has an importance because it is the first study showing a relation between Gas6 and proteinuria, CACS and carotid IMT in patients with chronic renal failure

INTRODUCCIÓN: La enfermedad cardiovascular es la principal causa de mortalidad y morbilidad en la insuficiencia renal crónica. Se sabe que la calcificación vascular (CV) y el grosor de la íntima-media de la carótida (CIMT, por sus siglas en inglés) están vinculados de forma muy estrecha con enfermedades cardiovasculares. La proteína específica del gen 6 de la detención de crecimiento (Gas6) es una proteína dependiente de la vitamina K y regula diversos procesos, como la proliferación, la supervivencia celular, la migración y la inflamación. La proteína Gas6 es conocida por proteger las células endoteliales y las células musculares lisas vasculares contra la apoptosis mediante la inhibición de la activación de la caspasa-3 inducida por la proteína Bcl-2. Se ha demostrado la relación entre la Gas6 y las enfermedades cardiovasculares en muchos modelos de ratones y cultivos celulares. Sin embargo, existen informes contradictorios acerca de si los niveles de Gas6 aumentan o disminuyen en estudios de humanos con insuficiencia renal crónica y/o diabética. En este estudio, el objetivo fue examinar los niveles plasmáticos de Gas6 y su relación con el CIMT y la puntuación de calcificación de las arterias coronarias (CACS, por sus siglas en inglés) en pacientes con enfermedad renal crónica (ERC). MATERIAL Y MÉTODOS: Un total de 137 pacientes fueron incluidos en el estudio, de los cuales 32 estaban en hemodiálisis crónica, 105 en prediálisis, y 73 pacientes representaban controles sanos. Se esudiaron los niveles de Gas6 en muestras de suero mediante el método ELISA. El CIMT se midió por medio de ecografía. La CACS se midió mediante tomografía computarizada multicorte. RESULTADOS: La edad media fue de 54,37 ± 16,61 años en el grupo de diálisis; 55,20 ± 14,80 años en el grupo de prediálisis, y 53,26 ± 9,04 años en el grupo de control. La creatinina sérica fue de 0,78 ± 0,16 mg/dl en el grupo de control; 1,96 ± 1,64 mg/dl en el de prediálisis, y 5,94 ± 1,55 mg/dl en el de diálisis. Las concentraciones de proteína en orina de 24 horas fueron significativamente más altas en el grupo de diálisis que en los de prediálisis y control. Los valores del CIMT fueron similares en los grupos de prediálisis y de diálisis. Estos valores fueron considerablemnete más altos que en el grupo de control. Aunque la CACS fue más alta en el grupo de diálisis que en los otros dos, los resultados no fueron estadísticamente significativos, ya que el rango de distribución fue muy amplio. La proteína Gas6 fue de 98,84 ± 53,32 ng/ml en el grupo de control y estadísticamente más alta que en los grupos de diálisis (63,85 ± 38,92 ng/ml) y de prediálisis (54,96 ± 38,49 ng/ml) (p = 0,001). Los niveles de Gas6 fueron más bajos en los pacientes diabéticos que en los no diabéticos (53,69 ± 35,26 ng/ml; 69,26 ± 47,50 ng/ml, [p = 0,023], respectivamente). Se detectó una correlación negativa entre la proteína Gas6 y la edad, el IMC, la CACS, el CIMT y la proteinuria. En el análisis de regresión logística, la Gas6 se mantuvo estrechamente relacionada con el IMC, el CIMT y la proteinuria. CONCLUSIÓN: En nuestro estudio, la correlación negativa de Gas6 con IMC, CACS, CIMT y proteinuria, y los niveles más bajos de Gas6 en pacientes diabéticos sustentan la idea de que la disminución de los niveles de Gas6 en la insuficiencia renal crónica puede jugar un papel en la calcificación vascular a través de la tolerancia alterada a la glucosa, la inflamación crónica, la disfunción endotelial y el aumento de la apoptosis. La importancia de nuestro estudio radica en que es el primero que muestra una relación entre la Gas6 y la proteinuria, la CACS y el CIMT en pacientes con insuficiencia renal crónica

Serum sex hormone and growth arrest-specific protein 6 levels in male patients with coronary heart disease / 亚洲男科学杂志(英文版)

Epidemiological studies have shown a high prevalence of low serum testosterone levels in men with cardiovascular disease. Moreover, the tyrosine kinase receptor Axl, the ligand of which is growth arrest-specific protein 6 (GAS6), is expressed in the vasculature, and serum GAS6 levels are associated with endothelial dysfunction and cardiovascular events. Testosterone regulates GAS6 gene transcription directly, which inhibits calcification of vascular smooth muscle cells and provides a mechanistic insight into the cardioprotective action of androgens. This study was designed to determine the correlation between serum GAS6 and testosterone levels in male patients with coronary heart disease (CHD). We recruited 225 patients with CHD and 102 apparently healthy controls. Serum concentrations of GAS6 and soluble Axl were quantified by an enzyme-linked immunosorbent assay. Levels of high-sensitivity C-reactive protein, testosterone, estradiol, and other routine biochemical markers were also measured. Testosterone decreased from 432.69 ± 14.40 to 300.76 ± 6.23 ng dl-1 (P < 0.001) and GAS6 decreased from 16.20 ± 0.31 to 12.51 ± 0.19 ng ml-1 (P < 0.001) in patients with CHD, compared with control subjects. Multiple linear regression analysis showed that serum testosterone and GAS6 levels were positively associated in male patients with CHD. Alterations in GAS6 levels may influence the development of CHD. Downregulation of GAS6/Axl signaling in the presence of low sex hormone levels during disease progression is a potential mechanism by which GAS6 affects CHD. This study provides novel results regarding the influence of sex hormones on serum GAS6 levels in patients with CHD.

Changes in serum growth arrest-specific protein 6 in patients with SLE and correlation between this change and the activity of SLE / 实用医学杂志

Objective To investigated the association of serum level of growth arrest-specific protein 6 (Gas6) with the disease activity in patients with systemic lupus erythematosus. Methods The expression levels of Gas6 were detected by enzyme linked immunosorbent assay in 102 SLE patients and 67 healthy persons; and statistical methods were used to analyze the expressions of Gas6 in the patients with different clinical symptoms. Results The expression of Gas6 was significantly higher in the SLE group [54.59 (2.41 ~ 614.93) ng/mL] than in the healthy control group [19.22 (15.06 ~ 384.93) ng/mL, P < 0.05]. And serum Gas6 levels were significantly higher in the patients with dsDNA (+) , renal disorder , rash and vasculitis than the patients without symptoms (P < 0.05). The expression of Gas6 was correlated with SLEDAI significantly (r = 0.569, P < 0.01). Conclusions Gas6 plays an important role in the pathogenesis of SLE and it is an effective biomarker for the disease activity in patients with systemic lupus erythematosus.

Detection of plasma protein S and growth-arrest specific protein 6 and their clinical significance in systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the significance of plasma protein S and growth-arrest specific protein 6 (Gas6) in systemic lupus erythematosus (SLE).Methods Sandwich enzyme-linked immunosorbent assay (ELISA) was used to measure the protein S and Gas6 in the plasma of 103 SLE patients and 40 healthy controls.Mann-Whitney U-test,x2-test and Spearman's rank correlation were used for statistical analysis.Results The plasma concentrations of protein S and Gas6 were both significantly [protein S:30.6 (27.2,33.6) μg/ml,37.8(35.4,46.7) μg/ml,Z=6.04,P＜0.01; Gas6:402.6(239.2,757.8) pg/ml,913.6(765.0,1 290.6) pg/ml,Z=4.26,P＜0.01] decreased in plasma of SLE patients than that in healthy controls.There was a positive correlation between levels of protein S and Gas6 (r=0.312,P=0.001).The level of protein S in SLE patients was positively correlated with hemoglobin (Hb),WBC,platelet blood platelet (PLT),C3 and C4 (r=0.209,0.264,0.264,0.362,0.280,P＜0.01 or P＜0.05).Plasma protein S level was also found to be negatively correlated with auto-antibodies such as anti-double strand DNA (anti-dsDNA) antibody,anticardiolipin (ACL),anti-nucleosome antibody (AnuA) or IgG (r=-0.197,-0.264,-0.226,-0.229,P＜0.01or P＜0.05).The plasma Gas6 level was positively associated with age,disease duration and C-reaction protein (CRP) (r=0.229,0.198,0.263,P＜0.01 or P＜0.05).Patients with decreased Gas6 level showed higher incidence of fever,rash and serositis.Patients with decreased WBC or positive for proteinuria also showed decreased Gas6 (P＜0.05).Conclusion Protein S levels are significantly decreased in plasma of SLE patients and is associated with a series of severe disease manifestations such as hematological involvement,decreased complements and the presence of auto-antibodies.Decreased Gas6 levels in SLE patients are observed and they are correlated with age,disease duration and certain clinical characteristics such as rash,renal involvement and inflammatory response.