Immune response induced by 14-3-3.3 recombinant protein vaccine of Taenia solium in mice / 中华地方病学杂志

Objective:To explore the potential of Taenia solium (Ts) 14-3-3.3 protein as a candidate molecule for cysticercosis vaccine. Methods:Sixty Kunming mice with the body weight of 18 - 22 g were selected and divided into 3 groups according to their body weight via the random number table method, including normal saline control group (control group), Ts14-3-3.3 recombinant protein vaccine group (vaccine group), and Ts14-3-3.3 recombinant protein vaccine + adjuvant group (vaccine + adjuvant group), with 20 mice in each group. The multi-point subcutaneous injection method was adopted. After the first immunization at 0 week, the booster immunization was carried out twice, a total of 3 times, with an interval of 2 weeks. Four mice in the three groups were killed at 0, 2, 4, 6 and 8 weeks after the first immunization, and the blood of eyeballs and spleen were collected aseptically for serum separation and preparation of spleen lymphocytes suspension [treatment: cell suspension, antigen-stimulate and concanavalin (Con) A-stimulate], respectively. The levels of mouse serum specific immunoglobulin (Ig) G, IgG2a, IgG1 and IgE were detected by indirect enzyme-linked immunosorbent assay (ELISA). The proliferation level of mouse spleen lymphocytes was detected via the CCK-8 method. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-13 and IL-10 in culture supernatant of mouse spleen lymphocytes were determined by double-antibody sandwich ELISA.Results:The IgG, IgG2a, and IgG1 levels of the vaccine and vaccine + adjuvant groups immunized for 2 to 8 weeks were higher than those of the control group, and the above indicators of the vaccine + adjuvant group were higher than those of the vaccine group ( P < 0.05). With the same treatment between the groups, the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13, and IL-10 in the culture supernatant after 2 - 8 weeks of immunization were statistically significantly different ( P < 0.05); the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13 and IL-10 in the culture supernatant of the vaccine and vaccine + adjuvant groups immunized for 2 to 8 weeks were higher than those of the control group, and the above indicators of the vaccine + adjuvant group were higher than those of the vaccine group ( P < 0.05). When treatment was different in the group, the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13 and IL-10 in the culture supernatant of the antigen-stimulate and ConA-stimulate were higher than those of the cell suspension, and the above indicators of the ConA-stimulate were higher than those of the antigen-stimulate ( P < 0.05). Conclusion:The recombinant protein vaccine of Ts14-3-3.3 can induce an effective immune response in mice.

Construction of prokaryotic expression system of Taenia solium 14-3-3.2 and its expression at the stages of Taenia solium adult and cysticercus / 中华地方病学杂志

Objective:To establish the prokaryotic expression system of Taenia solium (Ts) 14-3-3.2, and observe the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus. Methods:Based on the Ts14-3-3.2 gene sequence obtained by the Department of Parasitology, Zunyi Medical University in the previous study, the whole gene was synthesized by PCR-based accurate synthesis (PAS) method. After double digestion with restriction enzymes Nde Ⅰ and Xba Ⅰ, the plasmid pCzn1 was ligated to construct a recombinant plasmid pCzn1-Ts14-3-3.2. Then it was transformed into Escherichia coli ArcticExpress competent cells to induce the expression of Ts14-3-3.2 protein. The expression products were analyzed and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie blue staining. The purified Ts14-3-3.2 recombinant protein was obtained by Ni-affinity chromatography. New Zealand rabbits were immunized with the recombinant protein to produce Ts14-3-3.2 polyclonal antibody. Western blotting was used to detect the expression of Ts14-3-3.2 protein at the stages of Ts adult and cysticercus. Results:The recombinant plasmid pCzn1-Ts14-3-3.2 was successfully constructed. After induced expression, Ts14-3-3.2 target protein bands appeared in the supernatant and precipitated at the relative molecular weight of about 29.31 × 10 3. The purified Ts14-3-3.2 recombinant protein with His label could be recognized by anti-His monoclonal antibody, and the Ts14-3-3.2 polyclonal antibody with titer of 1 ∶ 512 000 was obtained. Western blotting showed that Ts14-3-3.2 protein was expressed at the stages of Ts adult and cysticercus. Conclusions:The prokaryotic expression system of Ts14-3-3.2 is successfully established, and the Ts14-3-3.2 polyclonal antibody with relatively higher purity and titer is obtained. The Ts14-3-3.2 protein is expressed at the stages of Ts adult and cysticercus.

The Adaptational Regulation Mechanism of the Apoptosis in Aged Rats’ Striatum in Response to Regular Aerobic Exercise Based on Carbonylation Proteomics / 中国生物化学与分子生物学报

This paper investigated the effects of regular aerobic exercise on protein oxidative stress and apoptosis in aging rat striatum, and further analyzed its target proteins and mechanism based on differential carbonylation proteomics. Totally 24 specific pathogen-free (SPF) 23-month-old male Sprague-Dawley (SD) rats were randomly divided into aged sedentary control group (Con-SED, n = 12) and aged regular aerobic exercise runner group (Aero-EXE, n = 12). The medium intensity of regular aerobic exercise model: The intensity of maximum oxygen consumption (VO

Tetramethylpyrazine attenuates doxorubicin induced cardiotoxicity through 14-3-3γ/Bcl-2 / 中国临床药理学与治疗学

AIM: To investigate the effect of tetramethylpyrazine (TMP) on doxorubicin (Dox) induced cardiotoxicity and the role of 14-3-3γ/Bcl-2 protein expression. METHODS: Primary cultured cardiomyocytes were randomly divided into Control group, Dox group, Dox+TMP group and Dox+TMP+pAD/14-3-3γ-shRNA group. After 48 hours, the cell viability was detected by MST, the activity of LDH in culture medium, the activities of Caspase-3, SOD, GSH-Px and the content of MDA were detected; the expression of 14-3-3γ and mitochondrial Bcl-2 was detected by Western blot; ROS generation, mitochondrial membrane potential and mPTP opening were detected by flow cytometry; apoptosis was detected by TUNEL method. RESULTS: After Dox exposed for 48 hours, the viability of cardiomyocytes decreased significantly, the activity of LDH in culture medium increased, the activities of SOD and GSH-Px decreased, the content of MDA increased, ROS generation increased; the mitochondrial membrane potential decreased, mPTP continued to open, caspase-3 activity and apoptosis increased. TMP pretreatment significantly upregulated the expression of 14-3-3γ and mitochondrial Bcl-2, and reversed the above changes simultaneously; pAD/14-3-3γ-shRNA not only downregulated the expression of 14-3-3γ, but also decreased the expression of Bcl-2 in mitochondria. CONCLUSION: TMP pretreatment upregulates the expression of 14-3-3γ and mitochondrial Bcl-2, inhibits oxidative stress, maintains mitochondrial function and reduces Dox induced apoptosis.

Progress of effects of 14-3-3 protein on gastric cancer and its mechanism / 解放军医学杂志

14-3-3 proteins are a highly conserved family of cellular proteins, and widely expressed in all eukaryotes. Because they can bind to a variety of functional signal proteins, so be regarded as "bridge proteins" in protein interaction. Studies have shown that the expression level of 14-3-3 protein family in malignant tumors is closely related to tumor size, depth of invasion and metastasis. Some other studies have shown that 14-3-3 protein plays a pivotal role in cell signal transduction, control of cell cycle process and regulation of apoptosis, and plays an important role in the development of gastric cancer, proliferation, invasion, prognosis and drug resistance, so is expected to become a novel therapeutic target for gastric cancer. The present paper aims to expound the role and relevant mechanisms of 14-3-3 protein in gastric cancer.

14-3-3 proteins mediate the localization of Centrin2 to centrosome

14-3-3e and 14-3-3c localize to the centrosome and regulate centrosome duplication, by inhibiting cdc25C function. As14-3-3c and 14-3-3e form a complex with centrosomal proteins, we asked if this ability was required to regulate centrosomeduplication. The results in this report demonstrate that 14-3-3e and 14-3-3c form a complex with Centrin2 and that thebinding site is located in the N-terminal EF hand in Centrin2, EF1. A Centrin2 mutant that does not form a complex with14-3-3 proteins displays a punctate cytoplasmic localization and does not localize to the centrosome. These results suggestthat in addition to negatively regulating centrosome duplication as previously reported, 14-3-3 proteins might also berequired for centriole biogenesis by regulating the localization of Centrin2 at the centrosome.

14-3-3γ Haploinsufficient Mice Display Hyperactive and Stress-sensitive Behaviors

14-3-3γ plays diverse roles in different aspects of cellular processes. Especially in the brain where 14-3-3γ is enriched, it has been reported to be involved in neurological and psychiatric diseases (e.g. Williams-Beuren syndrome and Creutzfeldt-Jakob disease). However, behavioral abnormalities related to 14-3-3γ deficiency are largely unknown. Here, by using 14-3-3γ deficient mice, we found that homozygous knockout mice were prenatally lethal, and heterozygous mice showed developmental delay relative to wild-type littermate mice. In addition, in behavioral analyses, we found that 14-3-3γ heterozygote mice display hyperactive and depressive-like behavior along with more sensitive responses to acute stress than littermate control mice. These results suggest that 14-3-3γ levels may be involved in the developmental manifestation of related neuropsychiatric diseases. In addition, 14-3-3γ heterozygote mice may be a potential model to study the molecular pathophysiology of neuropsychiatric symptoms.

Dexamethasone Downregulates Expressions of 14-3-3β and γ-Isoforms in Mice with Eosinophilic Meningitis Caused by Angiostrongylus cantonensis Infection

Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infections. The mechanism steroids act on eosinophilic meningitis remains unclear. In this mouse experiments, expressions of 14-3-3 isoform β and γ proteins significantly increased in the CSF 2–3 weeks after the infection, but not increasedin the dexamethasone-treated group. Expression of 14-3-3 β, γ, ɛ, and θ isoforms increased in brain meninges over the 3-week period after infection and decreased due to dexamethasone treatment. In conclusion, administration of dexamethasone in mice with eosinophilic meningitis decreased expressions of 14-3-3 isoform proteins in the CSF and in brain meninges.

Screening of 14-3-3τ protein inhibitors from natural products based on fluorescence spectroscopy, surface plasmon resonance and molecular docking technique / 国际药学研究杂志

Objective:To study and establish a method for the efficient screening of 14-3-3τ protein inhibitors from natural products, and analyze the sites of their interactions with the 14-3-3τ protein. Methods: The binding activity of natural compounds with the 14-3-3τ protein was tested by the liquid chromatography-fluorescence spectroscopy, and the binding activity of the potential compounds was further verified by the surface plasmon resonance(SPR)technique. The binding sites of the active compounds were predicted by the molecular docking technique. Furthermore, the key binding sites were selected and then validated using amino acid site-directed mutants. Results: A total of 17 different type compounds with potential 14-3-3τ binding activity were screened out from 82 natural products. The binding activi- ties of 10 compounds were verified by the SPR experiments. Then the binding sites of interactions between the 14-3-3τ protein and the 10 compounds were predicted by the molecular docking technology to be mainly at the Arg56, Arg127 and Y128A, dem- onstrate that the binding of five of the 10 compounds with the target protein was associated with the three sites Arg56, Arg127 and Tyr128. Conclusion: The established method is accurate and efficient, which could be used for rapid screening of small molecule 14-3-3τ inhibitors from natural products. The present study provides a reference and a new approach for the rapid screening of 14-3-3τ inhibitors for the new breast cancer therapeutic drugs.

Effect of Marsdenia tenacissima extract on G2/M cell cycle arrest by upregulating 14-3-3σ and downregulating c-myc in vitro and in vivo / 中草药·英文版

Objective: Marsdenia tenacissima extract (MTE) is a traditional Chinese herbal medicine with anti-cancer activity. In some previous studies, different mechanism actions of the anti-cancer effect of MTE have been revealed. In this study, we first observed that MTE exhibited G2/M cell cycle arrest on two different human breast cancer cell lines, MDA-MB-231 and MCF-7 by mediating 14-3-3σ and c-myc. Methods: The effect of MTE on G2/M cell cycle arrest was evaluated in MDA-MB-231 and MCF-7 cell lines. MTT assay was done for evaluation of cell viability. Flow cytometry was employed for cell cycle analysis. Western blotting analysis and immunohistochemistry were performed to analyze the expression of G2/M cell cycle-related key protein in cells and tissue samples. Animal studies have been conducted to elucidate the anti-tumor effect of MTE. Results: Cell cycle is the backbone for developing cancer. Cell cycle proteins play a major role in the progression of cell cycle and cell proliferation. However, some key protein directly or indirectly modulate the action of cell cycle protein that highly affect cell cycle regulation. In order to investigate cellular proliferation of cancer, we observed that MTE induced the upregulation of 14-3-3σ and downregulation of c-myc, and then reduced the expression of G2/M cell cycle associated key protein, leading to the inhibition of cellular entry into mitosis phase. We also confirmed that MTE exerted a significant antitumor effect on the MDA-MB-231 xenograft model in vivo. Conclusion: G2/M cell cycle arrest occurred by the action of MTE, mediated by the upregulation of 14-3-3σ as well as downregulation of c-myc in MDA-MB-231 and MCF-7 cell lines.

14-3-3ζ protein mediates gemcitabine resistance in NK/T-cell lymphoma / 中华血液学杂志

Objective: To explore the molecular mechanisms of 14-3-3ζ in gemcitabine resistance in extranodal NK/T-cell lymphoma, nasal type (ENKTL) . Methods: The effects of cell proliferation and invasion were detected by cell counting kit-8 (CCK-8) assay and transwell assay. YTS cells were exposed to gradually increased concentrations of gemcitabine to establish gemcitabine-resistant YTS cells (YTS-gem) in vitro. 14-3-3ζ specific siRNA lentiviral vector was transfected into YTS and YTS-gem cells to downregulate 14-3-3ζ expression, and stable transfected cell clones were screened. The protein expression was determined by Western blot. Results: ①14-3-3ζ expression was significantly up-regulated in gemcitabine resistant YTS-gem cells, comparing with that of YTS cells (P<0.05) . ②The results of CCK-8 and transwell assay showed that downregulation of 14-3-3ζ significantly reduced the cell proliferation and invasion abilities (P<0.05) . ③Downregulation of 14-3-3ζ could restore gemcitabine sensitivity in gemcitabine resistant YTS-gem cells (P<0.05) . ④Western blotting results showed that knockdown of 14-3-3ζ significantly upregulated pro-apoptotic Bax, and downregulated anti-apoptotic Bcl-2, Caspase-3, cleaved caspase-3, Cyclin D1 in gemcitabine-resistant YTS-gem cells (P<0.05) . There was no significant difference in p53 ang P-gp expression levels. Conclusions: 14-3-3ζ was upregulated in gemcitabine resistant YTS cells. Overexpression of 14-3-3ζ promoted cell proliferation and enhanced cell migration. 14-3-3ζ contributed to gemcitabine resistance to ENKTL through anti-apoptosis.

14-3-3ζ protein mediates gemcitabine resistance in NK/T-cell lymphoma / 中华血液学杂志

Objective@#To explore the molecular mechanisms of 14-3-3ζ in gemcitabine resistance in extranodal NK/T-cell lymphoma, nasal type (ENKTL) .@*Methods@#The effects of cell proliferation and invasion were detected by cell counting kit-8 (CCK-8) assay and transwell assay. YTS cells were exposed to gradually increased concentrations of gemcitabine to establish gemcitabine-resistant YTS cells (YTS-gem) in vitro. 14-3-3ζ specific siRNA lentiviral vector was transfected into YTS and YTS-gem cells to downregulate 14-3-3ζ expression, and stable transfected cell clones were screened. The protein expression was determined by Western blot.@*Results@#①14-3-3ζ expression was significantly up-regulated in gemcitabine resistant YTS-gem cells, comparing with that of YTS cells (P<0.05) . ②The results of CCK-8 and transwell assay showed that downregulation of 14-3-3ζ significantly reduced the cell proliferation and invasion abilities (P<0.05) . ③Downregulation of 14-3-3ζ could restore gemcitabine sensitivity in gemcitabine resistant YTS-gem cells (P<0.05) . ④Western blotting results showed that knockdown of 14-3-3ζ significantly upregulated pro-apoptotic Bax, and downregulated anti-apoptotic Bcl-2, Caspase-3, cleaved caspase-3, Cyclin D1 in gemcitabine-resistant YTS-gem cells (P<0.05) . There was no significant difference in p53 ang P-gp expression levels.@*Conclusions@#14-3-3ζ was upregulated in gemcitabine resistant YTS cells. Overexpression of 14-3-3ζ promoted cell proliferation and enhanced cell migration. 14-3-3ζ contributed to gemcitabine resistance to ENKTL through anti-apoptosis.

Differential expression of 14-3-3 gene in different stages of Taenia solium / 中华地方病学杂志

Objective To investigate the expression difference of 14-3-3 gene in different stages of Taenia solium (Ts),and to provide basic information for exploring the regulation mechanism of Ts14-3-3 gene in growth and development of Ts.Methods Adult worms were collected from patients with taeniasis solium in the taeniasis epidemic area of Yajiang County,Ganzi Tibetan Autonomous Prefecture,Sichuan Province.After determining the species through morphological observation under the light microscope and transcribed spacer 1 (ITS1) method,it was identified as Ts,then piglets were infected to obtain the Cysticercus cellulosae of Ts (larvae).Reverse transcription PCR was used to detect the expression of Ts14-3-3 gene in the adult stage and larval stage of Ts,and the relative expression levels of each were detected by real-time fluorescent quantitative PCR.Results Under light microscope,it could be seen that the collected adult worm scolex consisted of 4 suckers,rostellum and hooks,which was consistent with the typical characteristics of the Ts scolex;the worm sample could amplify the target fragment which was consistent with the length of the ITS1 gene by reverse transcription PCR,and was determined to be Ts.The Ts14-3-3 gene family members Ts14-3-3.1,Ts14-3-3.2,Ts14-3-3.3,Ts14-3-3.4,Ts14-3-3.5 and Ts14-3-3.6 were expressed in the Ts adult stage and larval stage.Compared with the larval stage,the expression levels of Ts14-3-3.1 (0.47 ± 0.09 vs 1.01 ± 0.23),Ts14-3-3.2 (0.31 ± 0.09 vs 1.05 ± 0.14),Ts14-3-3.3 (0.64 ± 0.23 vs 1.26 ± 0.23) and Ts14-3-3.4 (0.30 ± 0.09 vs 0.79 ± 0.23) were significantly decreased in the adult stage (t =-3.816,-7.093,-3.377,-3.481,P ＜ 0.01 or ＜ 0.05);the expression levels of Ts14-3-3.5 (3.59 ± 0.09 vs 0.99 ± 0.12) and Ts14-3-3.6 (5.74 ± 2.76 vs 1.03 ± 0.60) were significantly increased (t =30.714,9.718,P ＜ 0.01).Conclusion The expression of Ts14-3-3 gene has stage specificity and the Ts14-3-3 gene may play a special regulatory role in different stages of Ts.

14-3-3ζ Overexpression is Associated with Poor Prognosis in Ovarian Cancer

PURPOSE: 14-3-3ζ regulates cell signaling, cell cycle progression, and apoptosis, and its overexpression is associated with disease recurrence and poor clinical outcomes in some solid tumors. However, its clinicopathological role in ovarian cancer is unknown. Our goal was to investigate whether 14-3-3ζ is associated with ovarian cancer prognosis. MATERIALS AND METHODS: We examined 14-3-3ζ expression by immunohistochemistry in ovarian cancer tissues obtained from 88 ovarian cancer patients. The examined tissues were of various histologies and stages. 14-3-3ζ expression was also analyzed by western blot in seven ovarian cancer cell lines and a primary ovary epithelial cell line. Cell viability was measured using an MTS-based assay following cisplatin treatment. RESULTS: Among the ovarian cancer samples, 53.4% (47/88) showed high 14-3-3ζ expression, and 14-3-3ζ overexpression was positively correlated with more advanced pathologic stages and grades. 14-3-3ζ overexpression was also significantly associated with poor disease-free survival (DFS) and overall survival (OS) of ovarian cancer patients. Median DFS and OS were 1088 and 3905 days, respectively, in the high 14-3-3ζ expression group, but not reached in the low 14-3-3ζ expression group (p=0.004 and p=0.033, log-rank test, respectively). Downregulating 14-3-3ζ by RNA interference in ovarian cancer cells led to enhanced sensitivity to cisplatin-induced cell death. CONCLUSION: 14-3-3ζ overexpression might be a potential prognostic biomarker for ovarian cancer, and the inhibition of 14-3-3ζ could be a therapeutic option that enhances the antitumor activity of cisplatin.

Diagnostic value of protein markers in plasma exosomes of lung squamous cell carcinoma / 中华肿瘤杂志

Objective@#To investigate the tumor-associated protein molecules carried by plasma exosomes of patients with lung squamous cell carcinoma before treatment and analyze their value as clinical markers.@*Methods@#Exosomes from 2 patients with lung squamous cell carcinoma before treatment and 2 healthy controls were collected by ultracentrifugation. Proteomics was applied to analyze the protein expression profiles of exosomes. Candidate molecules were verified in another 30 exosomes samples from lung squamous cell carcinoma and healthy controls using enzyme-linked immunosorbent assay (ELISA).@*Results@#Electron microscopy and particle-counting assay showed that high-quality exosomes were collected. The number of exosomes distributed from 45 to 135 nm in 2 cases of lung cancer patients were 7.89×1011/ml and 9.71×1011/ml, respectively, significantly higher than 2.76×1011/ml and 1.41×1011/ml in healthy controls. Proteomic analysis showed that proteins of exosomes in lung squamous cell carcinoma patients were very different from those of healthy controls, and some proteins are related to important functions in tumor progression. 14-3-3ζ from exosomes was selected and further verified as a marker, and the area under the receiver operating characteristic curve (ROC) was 0.68. The sensitivity and specificity of 14-3-3 ζ from exosomes were 60.0% and 80.0%, respectively, suggested that it could be used as a diagnostic marker for lung squamous cell carcinoma.@*Conclusion@#The exosome counts in plasma and the protein molecules from exosomes, such as 14-3-3ζ, are closely related to the tumorigenesis, which can be used to assist clinical diagnosis of lung squamous cell carcinoma patients.

Silencing Filamin A Inhibits the Invasion and Migration of Breast Cancer Cells by Up-regulating 14-3-3σ / 华中科技大学学报（医学）（英德文版）

Filamin A and 14-3-3-σ are closely associated with the development of breast cancer.However,the exact relationship between them is still unknown.The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer.RNA interference technology was employed to silence filamin A in MDA-MB-231 cells.Real-time PCR and Western blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels,respectively.Double immunofluorescence was applied to show their colocalization morphologically.Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells.The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ.In addition,double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells.Silencing filamin A led to the enhanced fluorescence of 14-3-3σ.Furthermore,cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro.In conclusion,silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.

Silencing Filamin A Inhibits the Invasion and Migration of Breast Cancer Cells by Up-regulating 14-3-3σ / 华中科技大学学报（医学）（英德文版）

Filamin A and 14-3-3-σ are closely associated with the development of breast cancer.However,the exact relationship between them is still unknown.The present study aimed to examine the interaction of filamin A with 14-3-3-σ in the invasion and migration of breast cancer.RNA interference technology was employed to silence filamin A in MDA-MB-231 cells.Real-time PCR and Western blotting were used to detect the expression of filamin A and 14-3-3-σ at mRNA and protein levels,respectively.Double immunofluorescence was applied to show their colocalization morphologically.Wound healing assay and Trans-well assay were used to testify the migration and invasion of MDA-MB-231 cells in filamin A-silenced cells.The results showed that silencing filamin A significantly increased the mRNA and protein levels of 14-3-3σ.In addition,double immunofluorescence displayed that filamin A and 14-3-3σ were predominantly colocalized in the cytoplasm of MDA-MB-231 cells.Silencing filamin A led to the enhanced fluorescence of 14-3-3σ.Furthermore,cell functional experiments showed that silencing filamin A inhibited the migration and invasion of MDA-MB-231 cells in vitro.In conclusion,silencing filamin A may inhibit the invasion and migration of breast cancer cells by upregulating 14-3-3σ.

Expression analysis on 14-3-3 proteins in regenerative liver following partial hepatectomy

Abstract 14-3-3 proteins play a vital part in the regulation of cell cycle and apoptosis as signaling integration points. During liver regeneration, the quiescent hepatocytes go through hypertrophy and proliferation to restore liver weight. Therefore, we speculated that 14-3-3 proteins regulate the progression of liver regeneration. In this study, we analyzed the expression patterns of 14-3-3 proteins during liver regeneration of rat to provide an insight into the regenerative mechanism using western blotting. Only four isoforms (γ, ε, σ and τ/θ) of the 14-3-3 proteins were expressed in regenerative liver after partial hepatectomy (PH). The dual effects, the significant down-regulation of 14-3-3ε and the significant up-regulation of 14-3-3τ/θ at 2 h after PH, might play particularly important roles in S-phase entry. The significant peaks of 14-3-3σ at 30 h and of ε and τ/θ at 24 h might be closely related not only to the G2/M transition but also to the size of hepatocytes. Possibly, the peak of 14-3-3ε expression seen at 168 h plays critical roles in the termination of liver regeneration by inhibiting cellular proliferation.

Effect of 14-3-3ε protein on localization of Cdc25B during meiotic resumption of mouse oocytes / 吉林大学学报(医学版)

Objective:To explore the effect of 14-3-3εprotein on the localization of Cdc25B protein during the meiotic resumption of mouse oocytes,and to pay foundation for the further study on the molecular mechanism of 14-3-3εprotein in regulating the development of mouse oocytes.Methods:The Kunming genealology female mice aged 3 weeks were used to obtain the germinal vesicle (GV)-stage oocytes after superovulation.The GV-stage oocytes were divided into non-injection group,control siRNA injection group and 14-3-3εsiRNA injection group. The pmax-FP-Red-HA-14-3-3εexpression vector was constructed.Indirect immunofluorescence was used to observe the colocalization of 14-3-3εprotein and Cdc25B protein in the mouse oocytes;direct immunofluorescence was used to observe the subcellular localization of 14-3-3εprotein and Cdc25B protein in the mouse oocytes;14-3-3εsiRNA was microinj ected into the GV-stage oocytes;the morphology was observed under phase-contrast microscope;the germinal vesicle breakdown (GVDB)rates of the mouse oocytes were calculated;the expression level of 14-3-3εprotein and the relative expression level of Cdc2-pTyr15 protein were observed by Western blotting method;the matuation-promoting factor (MPF)activity in the oocytes was measured by autoradiography.Results:The indirect immunofluorescence and direct immunofluorescence results showed that the 14-3-3εprotein and wild Cdc25B protein were co-localized in the cytoplasm;Cdc25B was translocated from the cytoplasm to the nucleus shortly before GVBD.When the Ser321 of Cdc25B protein turned into Ala,the expression level of 14-3-3εprotein was decreased. None of the oocytes in non-injection group and control siRNA injection group were able to undergo GVBD until at least 24 h after injection,there was no significant differences in the rate of GVBD between non-injection group and control siRNA injection group (P>0.05);the GVBD rates of oocytes in 14-3-3εsiRNA injection group at 22 and 24 h after injection were significantly higher than those in non-injection group and control siRNA injection group (P<0.01);the rate of oocytes progressed to metaphaseⅡ (MII)in 14-3-3εsiRNA injection group at 24 h after injection was significantly higher than those in non-injection group and control siRNA injection group (P<0.01). Conclusion:Ser321 might be involved in the process of regulating the subcellular localization of Cdc25B by 14-3-3εprotein in the meiotic resumption of mouse oocytes.

Clinical significance of serum 14-3-3η protein and auto-antibodies detection in rheumatoid arthritis / 中国免疫学杂志

Objective:To explore the clinical value of detecting serum 14-3-3η and auto-antibodies in rheumatoid arthritis (RA),and compare their performance in RA diagnosis.Methods: Serum samples of 134 RA patients,90 non-RA inflammatory arthropathy patients,70 of whom with osteoarthritis(OA)and 20 with ankylosing spondylitis(AS),and 40 healthy controls from the second affiliated hospital of Nanchang University were collected.Concentrations of 14-3-3η,anti-CCP,anti-RA33,anti-Sa were detected with enzyme linked immunosorbent assay(ELISA),with RF detected by immunonephelometry.Diagnostic utilities of them for RA were evaluated and compared then.Results:① Serum levels of 14-3-3η,anti-CCP,anti-RA33,anti-Sa and RF were significantly higher in patients with RA than non-RA inflammatory arthropathy patients and healthy controls,the differences between groups were statistically significant;② ROC curves were conducted according to the serum levels detected.The AUC of 14-3-3η,anti-CCP,anti-RA33,anti-Sa and RF were 0.831(95% CI:0.782-0.881),0.852(95% CI:0.802-0.901),0.615(95% CI:0.546-0.684),0.706(95% CI:0.643-0.770)and 0.739(95% CI:0.676-0.802)respectively,with P values<0.01.Among all index,only anti-CCP and 14-3-3η were of moderate diagnostic value,at the threshold of 24.10 U/ml and 2.59 ng/ml individually;③anti-Sa was of highest specificity and RF was of highest sensitivity among all indexes detected;the specificity of 14-3-3η was merely moderately inferior to anti-Sa and anti-RA33,but its sensitivity was superior to them both.Conclusion:Serume14-3-3η,anti-CCP,anti-RA33,anti-Sa and RF levels increased remarkably in patients with RA,and contributed to RA diagnosis.Meanwhile,14-3-3η was advantageous,to some extent,in the sensitivity and specificity over auto-antibodies,and can be utilized as a reference index in diagnosing RA.