Over-expression of gastrin inhibits apoptosis of gastric cancer cells via reactive oxygen species and annexin A2-mediated mitochondrial dysfunction / La sobreexpresión de gastrina inhibe la apoptosis de las células de cáncer gástrico a través de especies reactivas de oxígeno y disfunción mitocondrial mediada por anexina A2

SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.

La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.

Construction of annexin A2 plasmid and its role in the proliferation and migration of bladder cancer pumc-91 cells / 西安交通大学学报(医学版)

【Objective】 Annexin A2 (annexin A2, Anxa2) has been reported to regulate bioactivity in various tumors cells. The purpose of this study was to investigate the correlation between the expression of Anxa2 protein and the proliferation and migration abilities of bladder cancer pumc-91 cells. 【Methods】 The ANXA2 sequence was amplified and inserted into the pcDNA3.1(+) vector in order to prepare the pcDNA3.1(+)-ANXA2 plasmid. PcDNA3.1 (+)-ANXA2 was transiently transfected into pumc-91 bladder cancer cells by lipofectamine 2000. Western blotting assay was performed to detect the expression of Anxa2 protein in the blank group, the control group transfected with pcDNA3.1(+), and the experimental group transfected with pcDNA3.1(+)-ANXA2 plasmid. The proliferation ability of pumc-91 cells was detected using Cell Counting Kit-8(CCK8), and the migration level of pumc-91 cells was detected by transwell assay. Differences in detection data among the groups were compared using one-way ANOVA or repeated measures ANOVA. 【Results】 The plasmid construction was successful and the sequencing was absolutely correct. Western blotting assay showed elevated Anxa2 protein expression level in the experimental group compared to the blank and control groups. CCK8 assay suggested that the number of proliferating pumc-91 cells was significantly higher in the experimental group than in the blank group (P<0.001) and the control group (P=0.001). Transwell assay also showed that the number of pumc-91 cells crossing the membrane was significantly higher in the experimental group than in the blank group (P=0.011) and the control group (P=0.027). 【Conclusion】 Our findings suggested that up-expression of Anxa2 may play a critical role in regulating proliferation and migration of bladder cancer pumc-91 cells.

Advance on phosphorylation and function regulation of annexin A2 / 第二军医大学学报

Annexin A2 (AnxA2) is a multifunctional protein and has complex functions regulated by post-translational modifications. Protein phosphorylation is the most popular post-translational modification of AnxA2. Ser11, Ser25 and Tyr23 are the three most important phosphorylation sites. The possible impact of phosphorylation of AnxA2 function regulation has always been a hot topic. Recently, AnxA2 has been found in the exosomes of tumor cells, and the research of AnxA2 and its phosphorylation has been expanded from intracellular to extracellular. Remarkable achievements in the study of AnxA2 and its phosphorylation have been made in many clinical disciplines. This paper reviewed the advances on the impact of phosphorylation of Ser11, Ser25, Tyr23 sites on AnxA2 function regulation and the research about AnxA2 in exosomes.

Identification of natural compounds targeting Annexin A2 with an anti-cancer effect

Annexin A2, a multifunctional tumor associated protein, promotes nuclear factor-kappa B (NF-κB) activation by interacting with NF-κB p50 subunit and facilitating its nuclear translocation. Here we demonstrated that two ginsenosides Rg5 (G-Rg5) and Rk1 (G-Rk1), with similar structure, directly bound to Annexin A2 by molecular docking and cellular thermal shift assay. Both Rg5 and Rk1 inhibited the interaction between Annexin A2 and NF-κB p50 subunit, their translocation to nuclear and NF-κB activation. Inhibition of NF-κB by these two ginsenosides decreased the expression of inhibitor of apoptosis proteins (IAPs), leading to caspase activation and apoptosis. Over expression of K302A Annexin A2, a mutant version of Annexin A2, which fails to interact with G-Rg5 and G-Rk1, effectively reduced the NF-κB inhibitory effect and apoptosis induced by G-Rg5 and G-Rk1. In addition, the knockdown of Annexin A2 largely enhanced NF-κB activation and apoptosis induced by the two molecules, indicating that the effects of G-Rg5 and G-Rk1 on NF-κB were mainly mediated by Annexin A2. Taken together, this study for the first time demonstrated that G-Rg5 and G-Rk1 inhibit tumor cell growth by targeting Annexin A2 and NF-κB pathway, and G-Rg5 and G-Rk1 might be promising natural compounds for targeted cancer therapy.

Expressions of annexins and epidermal growth factor receptor in pediatric middle ear cholesteatoma / 中华实用儿科临床杂志

Objective To explore the expressions of annexin and epidermal growth factor receptor(EGFR) in pediatric middle ear cholesteatoma.Methods Twenty-three children with middle ear cholesteatoma and 26 children with normal skin of external auditory canal(control group) were selected from the children enrolled in the First Affiliated Hospital of Zhengzhou University from August 2014 to March 2016.The expressions of annexin A1 (AnxA1),AnxA2 and EGFR mRNA in cholesteatoma and normal tissues were analyzed by quantitative real-time PCR (qPCR).Protein expressions of AnxA1,AnxA2 and EGFR were evaluated by using Western blot and immunohistochemistry methods.Results The expressions of AnxA1,AnxA2 and EGFR mRNA were significantly increased in cholesteatoma compared with the control group (AnxA1:4.68 ± 1.77 vs.2.65 ± 0.96,U =111.5,P ＜ 0.001;AnxA2:3.89 ± 1.00 vs.2.4 7 ± 0.81,U =84.5,P ＜ 0.001;EG FR:4.97 ± 1.85 vs.3.50 ± 0.95,U =15 3.5,P =0.004).AnxA1 and AnxA2 mRNA expressions were positively correlated with EGFR mRNA (AnxA1 and EGFR:r2 =0.283 2,P =0.009;AnxA2 and EGFR:r2 =0.213 5,P =0.027).Compared with the control group,protein expressions of AnxA1,AnxA2 and EGFR were markedly enhanced (AnxA1:0.450 ±0.031 vs.0.320 ±0.026,U =102.4,P ＜0.001;AnxA2:0.568 ±0.024 vs.0.365 ±0.028,U =94.6,P ＜0.001;EGFR:0.397 ±0.021 vs.0.228 ±0.017,U =128.4,P ＜0.001).The results of immunohistochemistry analysis showed that the ratio of AnxA1,AnxA2 and EGFR positive cells were higher than those in the control group(AnxA1:65.22％ vs.38.46％,x2 =9.296,P =0.026;AnxA2:69.57％ vs.46.15％,x2 =8.378,P =0.039;EGFR:69.57％ vs.50.00％,x2 =10.574,P =0.014).Conclusions The expressions of AnxAl,AnxA2 and EGFR are upregulated in pediatric cholesteatoma,with AnxA1 and AnxA2 expressions positively correlated with EGFR.

Expressions of S100A6, annexin A2 and c-myc in multiple myeloma and their clinical significance / 肿瘤

Objective: To investigate the expressions of S100A6, annexin A2 (AnxA2) and c-myc in patients with multiple myeloma (MM) and their clinical significance. Methods: The expressions of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from 28 cases of MM before and after chemotherapy and 20 controls (whose peripheral blood white cell count and the platelet count were a little lower than the normal values, but the result of bone marrow aspiration was normal) were detected by real-time fluorescent quantitative PCR. The relationships among the expressions of S100A6, AnxA2 and c-myc mRNAs were analyzed. The expressions of S100A6, AnxA2 and c-myc mRNAs and proteins in MM U266 cells after transfection with S100A6 siRNA were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: The expression levels of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from patients with MM were higher than those from the controls (all P < 0.05). The expression levels of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from patients with MM before chemotherapy were higher than those after chemotherapy (all P < 0.05). The expression levels of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from MM patients with extramedullary metastasis were higher than those from MM patients not having extramedullary metastasis (all P < 0.05). The expression of S100A6 mRNA was positively correlated with the expressions of AnxA2 and c-myc mRNAs (r = 0.585, P = 0.001; r = 0.540, P =0.004). The expression levels of S100A6, AnxA2 and c-myc mRNAs and proteins in MM U266 cells after transfection with S100A6 siRNA were down-regulated (all P < 0.05). Conclusion: The expression level of S100A6 in patients with MM is higher, and it is positively associated with AnxA2 and c-myc. S100A6 may be involved in the development, progression and extramedullary metastasis of MM.

Effect of silencing Annexin A2 gene expression by siRNA on radiosensitivity of nasopharyngeal carcinoma cells / 中华放射肿瘤学杂志

Objective To investigate the effect of silencing Annexin A2 gene expression by small interfering RNA (siRNA) on the radiosensitivity of nasopharyngeal carcinoma cells CNE-2 (R743).Methods siRNA targeting the Annexin A2 gene was chemically synthesized and transfected into R743 cells by HiPerFect.The mRNA and protein levels of Annexin A2 before and after transfection were measured by RT-PCR and Western blot,respectively.The change in radiosensitivity of R743 cells was analyzed by colonyforming assay.Cell cycle distribution and apoptosis after X-ray irradiation were analyzed using flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay,respectively.Results The results from RT-PCR and Western blot showed that the expression of Annexin A2 was down-regulated after transfection.The colony-forming assay indicated that the D0,Dq,and SF2 in transfected cells were significantly lower than those in untransfected cells with radiation alone and in cells transfected with control siRNA.The sensitization enhancement ratios (D0 ratios) of transfected cells relative to untransfected and control siRNA transfected cells were 1.30 and 1.27,respectively.After X-ray irradiation,the proportion of cells in G2/M phase was significantly higher in the transfected cells thin in untransfected and control siRNA transfected cells (32.46％ vs.9.17％ and 9.42％,respectively;P =0.000 and 0.000).The apoptosis rate was also significantly higher in the transfected cells than in the untransfected and control siRNA transfected cells (35.20％ vs.10.87％ and 11.33％,respectively;P=0.000 and 0.000).Conclusions Silencing Annexin A2 gene expression by siRNA can increase the radiosensitivity of R743 cells,which may be associated with DNA damage repair and change in cell cycle distribution.

Annexin A2 silencing enhances apoptosis of human umbilical vein endothelial cells in vitro

OBJECTIVE@#To study the effects of inhibited Annexin A2 (ANXA2) on human umbilical vein endothelial cells (HUVECs) in vitro.@*METHODS@#Short hairpin RNA (shRNA) targeting ANXA2 was designed and cloned into double marked lentivirial vector GV248 for RNAi to generate the recombinant expression plasmids, which were stably transfected into HUVECs. The protein and mRNA expression levels of ANXA2 were analyzed by western blotting and real-time polymerase chain reaction, respectively. Cell proliferation (cell counting kit-8 assay), apoptosis (flow cytometry analysis), the expression (western blotting) and the activity of caspases (enzyme-linked immunosorbent assay) were used to assess the effects of silencing ANXA2 on HUVECs in vitro.@*RESULTS@#The plasmids to express ANXA2-specific shRNA were constructed and were infected into HUVEC resulting in the stably transfected experimental (ANXA2-shRNA), control (control-shRNA) and mock (no plasmid) cell lines, which were verified with western blot and real-time PCR. HUVEC/ANXA2-shRNA showed an inhibition rate 91.89% of ANXA2 expression compared to the mock HUVEC. ANXA2 silencing cell strain obviously presented a lower cell proliferation activity compared to the control and mock HUVECs, with an inhibition rate 82.35% on day 7 in vitro. FACS analysis indicated that the HUVEC/ANXA2-shRNA cells undergoing apoptosis increased by 102.61% compared to the mock HUVECs (P < 0.01). Moreover, the activity levels of caspase-3, caspase-8 and caspase-9 in HUVEC/ANXA2-shRNA cells were increased and the activated cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 were upregulated evidently compared with that of the control and mock HUVECs by 56.29%, 89.59% and 144.58% (P < 0.01).@*CONCLUSIONS@#shRNA-mediated silencing of ANXA2 could not only be able to suppress HUVECs proliferation but to upregulate the enzyme activity of caspases, which bring to an increase of cell apoptosis. This work suggested that ANXA2 may represent a useful target of future molecular therapies.

Annexin A2 silencing enhances apoptosis of human umbilical vein endothelial cells in vitro

Objective: To study the effects of inhibited Annexin A2 (ANXA2) on human umbilical vein endothelial cells (HUVECs) in vitro. Methods: Short hairpin RNA (shRNA) targeting ANXA2 was designed and cloned into double marked lentivirial vector GV248 for RNAi to generate the recombinant expression plasmids, which were stably transfected into HUVECs. The protein and mRNA expression levels of ANXA2 were analyzed by western blotting and real-time polymerase chain reaction, respectively. Cell proliferation (cell counting kit-8 assay), apoptosis (flow cytometry analysis), the expression (western blotting) and the activity of caspases (enzyme-linked immunosorbent assay) were used to assess the effects of silencing ANXA2 on HUVECs in vitro. Results: The plasmids to express ANXA2-specific shRNA were constructed and were infected into HUVEC resulting in the stably transfected experimental (ANXA2-shRNA), control (control-shRNA) and mock (no plasmid) cell lines, which were verified with western blot and real-time PCR. HUVEC/ANXA2-shRNA showed an inhibition rate 91.89% of ANXA2 expression compared to the mock HUVEC. ANXA2 silencing cell strain obviously presented a lower cell proliferation activity compared to the control and mock HUVECs, with an inhibition rate 82.35% on day 7 in vitro. FACS analysis indicated that the HUVEC/ANXA2-shRNA cells undergoing apoptosis increased by 102.61% compared to the mock HUVECs (P < 0.01). Moreover, the activity levels of caspase-3, caspase-8 and caspase-9 in HUVEC/ANXA2-shRNA cells were increased and the activated cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 were upregulated evidently compared with that of the control and mock HUVECs by 56.29%, 89.59% and 144.58% (P < 0.01). Conclusions: shRNA-mediated silencing of ANXA2 could not only be able to suppress HUVECs proliferation but to upregulate the enzyme activity of caspases, which bring to an increase of cell apoptosis. This work suggested that ANXA2 may represent a useful target of future molecular therapies.

Restrain of hypoxia on the expression of annexin A2 in mouse RGC-5 cells / 中华实验眼科杂志

Background Hypoxia is the main factor of retinal neovascularization and is closely associated with retinal ganglial cells (RGCs) degeneration.However, the study of retinal neural tissue lesions is rare.Objective This study was to investigate the influence of hypoxia environment on the expression of annexin A2 (ANXA2) in mouse RGC-5 cells and explore the mechanism of RGCs damage induced by hypoxia.Methods Immortalized mouse RGC-5 cells were cultured in high glucose DMEM with 10％ fetal bovine serum.The cells were identified by detecting the expression of Thy-1 ,a specific biomarker of RGCs.CoCl2 was added into the medium at the final concentrations of 50,100,200 and 300 μmol/L, and the cells without CoCl2 served as the control group.Cell viability (absorbance) was assayed by cell counting kit-8 (CCK-8) method in 12,24 and 48 hours after addition of CoCl2.The hypoxic cell models were established in DMEM with 100 μmol/L CoCl2 and divided into the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group,with the normal cultured cells as the normal control group.Apoptotic cells were determined by using hoechst 33342 stain.The expression levels of ANXA2 mRNA and protein in the cells were detected by real-time quantification PCR and Western blot,respectively.The expression and location of ANXA2 in the cells were examined by using immunofluorescence technique.Results The cultured cells grew well and showed the fusiform and polygonal shape,with positive expression of Thy-1 protein.Compared with the normal control group, the viabilities of the cells were insignificantly changed in the 50 μ mol/L CoCl2 group and 100 μmol/L CoCl2 group (all at P＞0.05) ,but the cell viabilities were significantly reduced in the 200 μμmol/L CoCl2 group and 300 μmol/L CoCl2 group in various time points (all at P＜0.05).Hoechst 33342 staining showed that the apoptotic cells with nuclear condensation and high green fluorescence intensity were obtained in the hypoxia groups.The relative expression levels of ANXA2 mRNA were significantly lower in the hypoxic groups than those in the normal control group (all at P ＜ 0.05).The relative expression levels of ANXA2 protein were significantly lower in the hypoxia 3-,6-, 12-and 24-hour group than those in the normal control group (all at P＜ 0.05).Apoptotic cells were seen in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group compared with the normal control group, showing the bright blue fluorescence in cellular nucleus for hoechst 33342.The relative expressing levels of ANXA2 mRNA in the cells were 0.80±0.14,0.67±0.33, 0.49±0.17 and 0.39±0.02 in the hypoxic 3-hour group,hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, which were significantly declined in comparison with the normal control group, with a statistically difference among the groups (F=434.354, P =0.000).The relative expression values of ANXA2 protein were 0.552 6±0.012 3,0.425 9± 0.033 4,0.344 9 ± 0.017 8 and 0.382 7 ± 0.022 1 in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group,which were remarkably lower than 0.602 1 ±0.001 4 in the normal control group, showing considerably difference among the groups (F =3.057, P =0.000).ANXA2 proteins were highly expressed in the cellular nucleus and less expressed in the cell membrane and cytoplasm in the normal cells.Compared with the normal control group, the ANXA2 protein showed weak expression in the hypoxia group and primarily in the cytoplasm.Conclusions The expression of ANXA2 down-regulates in hypoxic mouse RGC-5 cells,which may participate in the apoptosis process of RGCs in high glucose environment.

Expression characteristics of annexin A2 in dermal papilla cells with aggregative behavior / 中华皮肤科杂志

Objective To analyze the expression characteristics of annexin A2 in dermal papilla cells (DPCs) with aggregative behavior.Methods Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to measure the mRNA and protein expressions of annexin A2 respectively in DPCs with or without aggregative behavior.Results The mRNA expression level of annexin A2 was significantly higher in DPCs with aggregative behavior than in those without aggregative behavior (0.50 ± 0.15 vs.0.35 ± 0.19, t =8.26, P ＜ 0.05).Western blot showed that annexin A2 had two isoforms, including one isoform with a relative molecular mass of 40 000 and the other one with a relative molecular mass of 36 000.The annexin A2 isoform with a relative molecular mass of 40 000 was highly expressed in both DPCs with aggregative behavior and those without aggregative behavior, while the other isoform was only expressed in DPCs with aggregative behavior.Conclusion Annexin A2 may be closely related to the aggregative growth of DPCs.

Annexin A2 protein expression in liver fibrosis and hepatocellular carcinoma / 中华肝胆外科杂志

Objective To investigate the clinicopathological significance of the expression of annexin A2 (ANXA2) in liver fibrosis and hepatocellular carcinoma (HCC).Methods The expression level of ANXA2 in normal liver,liver cirrhosis and HCC were examined by Western blot.The correlation between ANXA2 expression and clinicopathological parameters in liver fibrosis and HCC were analyzed by immunohistochemistry.Results Compared with the normal liver tissue,ANXA2 protein expression level increased significantly in HCC and liver cirrhosis,with the highest expression in HCC (P =0.000).There was significantly positive relationship between ANXA2 protein expression and stages for liver fibrosis (P ＜ 0.01).The expression of ANXA2 protein in HCC was closely associated with HBV infection,differentiation degree and the recurrence (P ＜ 0.05).In some cases,ANXA2-positive cancer cells were often dispersed in the periphery of cancer nodules and were adjacent to stromal cells.Conclusion Overexpression of ANXA2 may be involved in liver fibrosis and play a role in the development of HCC,indicating ANXA2 may serve as a diagnostic biomarker for liver fibrosis and tumor differentiation in HCC.

Down-regulation of AnnexinA2 expression is associated with invasion and metastasis of prostate cancer / 中华泌尿外科杂志

Objective To analyze the impact and its mechanism of down-regulation of AnnexinA2 expression on prostate cancer(PCa) invasion and metastasis.Methods The expression of AnnexinA2 in three prostate cancer cell lines with different metastasis ability,including LNCaP (lower metastasis ability),PC3 (lower metastasis ability),C4-2B (higher metastasis ability) were detected by Western blot.The correlations between the expression of AnnexinA2 and the metastasis ability of prostate cancer cells were also evaluated.The siRNA was used in PC3 cells to down-regulate the expression of AnnexinA2,the cell proliferation assay was performed by MTT method,the cell apoptosis level was detected by flow cytometry,the expression of MMP-2 and MMP-9 were detected by Westrn blot,the in vitro invasiveness of PC3 was detected by transwell cabinet test,and the migration ability of PC3 cells was detected by scratch test,respectively.Results The grayscale value of AnnexinA2 expression in C4-2B cells is 0.22,in contrast with the internal reference,which was obviously lower than those of LNCaP and PC3 cells with lower metastasis potency(relative grey value is 0.93 and 0.95,respectively.P＜0.01).After RNAi was used in PC3 cells to down-regulate the expression of AnnexinA2,the growth became faster for PC3-ANXA2-siRNA cells than PC3,PC3-Lip and PC3-empty vector cells (P＜0.05).After RNAi was used in PC3 cells to down-regulate the expression of AnnexinA2,the ratio of apoptosis was detected by flowcytometry in PC3,PC3-Lip,PC3-empty vector and PC3-ANXA2-siRNA cells,and the apoptosis ratio in PC3-ANXA2-siRNA cells was the highest.However,the difference was not significant compare to others (P＞0.05).After RNAi was used in PC3 cells to downregulate the expression of AnnexinA2,the expression of AnnexinA2 in PC3-ANXA2-siRNA cells was decreased while the expression of MMP-2 and MMP-9 were increased.After RNAi was used in PC3 cells to down-regulate the expression of AnnexinA2,invasiveness of PC3-ANXA2-siRNA cells detected by transwell cabinet test was increased in vitro,and migration of PC3-ANXA2-siRNA cells detected by scratch test was increased in vitro.Conclusions The down-regulation of expression of AnnexinA2 could increase the invasive and metastatic ability of prostate cancer,and this may attribute to the up-regulation of MMP-2 and MMP-9 expression in prostate cancer.

Effect of Shoutai Pill on Uterine Decidual Protein Expression of Recurrent Spontaneous Abortion Mice Model / 中国中医药信息杂志

Objective To explore the mechanism action of Shoutai Pill in the embryo from the molecular level. Methods The model of normal pregnancy was established with the model of recurrent abortion CBA/J ×DBA/2. The recurrent abortion model CBA/J×DBA/2 in pregnant mice were randomly divided into model group, high-, medium-, low-dose group of Shoutai Pill. From the first day of gestation, mice were given medicine by gavage for 14 d, and then sacrificed. Immunohistochemistry was used to detect differences in protein HSP27,α-enolase, transferrin, annexin A2 protein expression. Results Compared with normal group, decidual HSP27 and α-enolase expression of model group increased significantly, the expression of transferrin and annexin A2 was significantly decreased, with significant differences (P0.05). Conclusion Through the protein expression, Shoutai Pill achieves the maintenance of pregnancy, reducing the rate of embryo resorption, which may be one of the mechanisms of Shoutai Pill preventing miscarriage effect.

Changes in expression of annexin A2 in lung tissues in rats with hepato-pulmonary syndrome / 中华麻醉学杂志

Objective To evaluate the changes in the expression of annexin A2 (ANXA2) in lung tissues in rats with hepato-pulmonary syndrome.Methods Healthy 3-4-month-old Sprague-Dawley rats of both sexes,weighing 220-250 g,were randomly divided into 3 groups:control group (group C,n =10),sham operation group (group S,n =10) and hepatopulmonary syndrome group (group HPS,n =20).The rats were anesthetized with intraperitoneal 3％ pentobarbital sodium 0.1 ml/100 g.Hepatopulmonary syndrome was produced by chronic ligation of the common bile duct.After 5 weeks,the rats were sacrificed and lungs were removed for determination of ANXA2 and smooth muscle actin α (SM-α-actin) mRNA and protein expression in lung tissues by RT-PCR and Western blot,respectively.Results There was no significant difference in the expression of ANXA2 and SM-α-actin mRNA and protein between groups C and S (P ＞ 0.05).The expression of ANXA2 and SM-α-actin mRNA and protein was significantly higher in group HPS than in groups C and S (P ＜ 0.05).Conclusion The expression of ANXA2 in lung tissues is up-regulated in rats with hepato-pulmonary syndrome.

Estudo proteômico da interação do Aspergillus fumigatus com células endoteliais da veia umbilical humana (HUVECs) / Proteomic study of the interaction of Aspergillus fumigatus with human umbilical vein endothelial cells (HUVECs)

O Aspergillus fumigatus é o principal agente etiológico da aspergilose invasiva, uma infecção fúngica oportunista que acomete, principalmente, pacientes de Unidades Hematológicas, como aqueles com neutropenia profunda e prolongada. Após a filamentação este fungo angioinvasivo é capaz de ativar e causar danos em células endoteliais de veia umbilical humana (HUVEC) que passam a expressar um fenótipo pró-trombótico. A ativação destas células, dependente de contato célulacélula, é mediada por TNF-α e caracterizada pela expressão de moléculas próinflamatórias, como citocinas, quimiocinas e moléculas de adesão. Recentemente, nosso grupo comparou a ativação endotelial de HUVECs desafiadas com cepas selvagens e uma cepa mutante para o gene UGM1. Nestes experimentos a cepa mutante Δugm1, que apresenta um fenótipo de maior produção de galactosaminogalactana (GAG) na parede celular, mostrou um fenótipo hiperadesivo e uma capacidade maior de ativar células endoteliais. Entretanto, os receptores e as vias de sinalização envolvidos nesta ativação permanecem desconhecidos. Assim, o objetivo deste trabalho foi verificar as proteínas envolvidas nestes processos através do estudo das proteínas diferencialmente expressas nas HUVECs após a interação com A. fumigatus, usando a técnica proteômica 2D-DIGE. Brevemente, as HUVECs foram infectadas com tubos germinativos da cepa selvagem (AF293) e da cepa Δugm1 de A. fumigatus. Em seguida, as proteínas foram marcadas com diferentes fluorocromos e separadas por eletroforese bidimensional. A análise quantitativa foi realizada utilizando o software DeCyder...

Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, the main opportunistic fungal infection of Hematologial Unity’s patients, especially those with long-term neutropenia. Upon filamentation, this angioinvasive fungus can activate and damage the human umbilical vein endothelial cells (HUVEC), which in response switch to a pro-thrombotic phenotype. HUVEC activation is mediated by TNF-α once cell-cell contact occurs. This activation is characterized by the expression of pro-inflammatory molecules such cytokines, chemokines and adhesion molecules. Recently, our group performed the comparison of HUVEC activation upon interaction with a wild type and the UGM1 mutant strains of A. fumigatus. The Δugm1 strain, which presents an increased production of the cell wall galactosaminogalactan, showed a hyper adherent phenotype and an increased capability to cause endothelial cell stimulation and activation, when compared with the wild type strain. The receptors involved in the pathogen-host interaction or the signaling pathways after endothelial activation by A. fumigatus remain unknown. Thus, the aim of this study was to investigate the differentially expressed proteins in HUVECs upon interaction with A. fumigatus, using the 2D-DIGE proteomic approach. Briefly, HUVECs were challenged with germlings of A. fumigatus wild type Af293 and Δugm1 strains and then submitted to protein extraction. The total HUVEC protein extracts were labeled with different CyDyes and fractionated by 2D electrophoresis. Quantitative analysis to determine the differences in protein abundance amongst interacted cells vs. control endothelial cells was performed using the software DeCyder. Five differentially expressed proteins were identified by MS/MS including galectin-1 and annexin A2, both overexpressed after the interaction. These two proteins are described elsewhere to be associated with host-pathogen interaction...

Annexin A2 expression in human esophageal squamous cell carcinoma and correlation with invasion and metastasis / 中华消化杂志

Objective To explore Annexin A2 expression in human esophageal squamous cell carcinoma (ESCC) and investigate the correlation of Annexin A2 expression with invasion and metastasis of human ESCC. Methods From 2000 to 2008, specimens of Xinjiang medical University First Affiliated Hospital were collected. Pathologically confirmed ESCC surgical specimens were set as experimental group, and the corresponding tumor adjacent tissues located more than 5 cm far from ESCC center were set as control group. 22 fresh and 175 paraffin-embeded ESCC specimens with corresponding adjacent tissues were randomly collected as study samples. With qRT-PCR, Western-blot and immunohistochemistry, the expression of Annexin A2 were detected at the mRNA and protein level. The correlation between Annexin A2 expression and clinicopathological parameters was analyzed. Results In 22 pairs of fresh ESCC and corresponding tumor adjacent tissues, the expression of Annexin A2 at mRNA level was significantly higher in tumor adjacent tissues (0. 06 ± 0. 06) than that in ESCC (0. 02 ±0. 02) (P＜0.05 ). Annexin A2 expression at protein level was also significantly higher in tumor adjacent tissues (0.95±0. 42) than ESCC (0.81±0. 36) (P＜0.05). In 175 paraffin-embeded ESCC specimens and corresponding adjacent tissues, the positive rate of Annexin A2 protein expression was 82. 3% (144/175) of the ESCC samples, which was lower than corresponding tumor adjacent tissues 92. 0% (161/175)(P＜0. 05). In addition, Annexin A2 expression was correlated with lymphoid node metastasis (P＜0.05) and pathological differentiation in patients with ESCC (P＜0.05). However, there was no apparent correlation with gross type (P＞0. 05). Conclusion The low expression of Annexin A2 in ESCC maybe played a potential role in the carcinogenesis, invasion and metastasis.

Role of ANX A2 in APL-induced expression of tissue factor in THP-1 cells / 中国病理生理杂志

AIM: To construct a lentiviral vector harboring RNAi sequence targeting human annexin A2 (ANX A2) and to investigate the functions of ANX A2 in antiphospholipid antibody (APL)-induced tissue factor (TF) expression in monocytes. METHODS: Four different short hairpin RNAs (shRNA) targeting ANX A2 gene were constructed and cloned into the pGCSIL-GFP vector. After identification with PCR and sequencing, the effective interference sequence was further determined by Western blotting analysis. The recombinant lentivirus LV-RNAi-ANX A2 harvested from 293T cells was transferred into THP-1 cells and the ANX A2 mRNA and protein expression on THP-1 cells were examined. The transferred-THP-1 cells were stimulated by APL/β_2GPI compound, and the TF mRNA and TF activity was assayed. RESULTS: The RNAi sequences targeting the human ANX A2 gene were successfully inserted into the lentiviral vector and the high-performance RNA interference sequence was sieved out. The recombinant lentivirus was harvested from 293T cells with a viral titer of 3×10~(12) TU/L. THP-1 cells infected with LV-RNAi-ANX A2 showed almost lockout of ANX A2 both at mRNA and protein levels. The TF expression was also significantly decreased in the transferred-THP-1 cells induced by APL/β_2GPI compound. CONCLUSION: The lentiviral vector constructed in the present study blocks the ANX A2 expression in THP-1 cells and further inhibits the TF expression induced by APL/β_2GPI compound, which indicates that ANX A2 do play an important role in APL-induced TF expression on monocytes.

The role of anti-annexin A2 antibodies in antiphospholipid syndrome / 中华风湿病学杂志

Objective This study has explored the role of antibody against annexin A2 in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Methods Using purified recombinant annexin A2, IgG anti-annexin A2 antibody was measured by ELISA in 101 APS patients, 41 SLE patients with thrombosis, 124 SLE patients without thrombosis and 120 healthy controls. Results The positive rate of IgG anti-annexin A2 antibody in APS patients and SLE patients with thrombosis was 21.8%, 26.8%, respectively, they were all significantly higher than in SLE patients without thrombosis (6.5%). IgG anti-annexin A2 antibody was associated with thrombosis and/or pregnancy morbidity (P<0.01). Conclusion Anti-annexin A2 antibody is associated with thrombosis and/or pregnancy mnrbidity. It suggests that anti-annexin A2 antibody may be helpful in identifying in some potential AIRS.