Deep proteomic analysis of obstetric antiphospholipid syndrome by DIA-MS of extracellular vesicle enriched fractions.

Proteins in the plasma/serum mirror an individual's physiology. Circulating extracellular vesicles (EVs) proteins constitute a large portion of the plasma/serum proteome. Thus, deep and unbiased proteomic analysis of circulating plasma/serum extracellular vesicles holds promise for discovering disease biomarkers as well as revealing disease mechanisms. We established a workflow for simple, deep, and reproducible proteome analysis of both serum large and small EVs enriched fractions by ultracentrifugation plus 4D-data-independent acquisition mass spectrometry (4D-DIA-MS). In our cohort study of obstetric antiphospholipid syndrome (OAPS), 4270 and 3328 proteins were identified from large and small EVs enriched fractions respectively. Both of them revealed known or new pathways related to OAPS. Increased levels of von Willebrand factor (VWF) and insulin receptor (INSR) were identified as candidate biomarkers, which shed light on hypercoagulability and abnormal insulin signaling in disease progression. Our workflow will significantly promote our understanding of plasma/serum-based disease mechanisms and generate new biomarkers.

BRCA1 and BRCA2 germline mutations in Chinese Hakka breast cancer patients.

OBJECTIVE: To investigate the prevalence of BRCA1/2 gene variants and evaluate the clinical and pathological characteristics associated with these variants in Chinese Hakka breast cancer patients. METHODS: A total of 409 breast cancer patients were analyzed based on next-generation sequencing results, with 337 categorized as non-carriers and 72 as carriers of BRCA1/2 variants. Data on the patients' BRCA1/2 gene mutation status, clinical and pathological characteristics, as well as menstrual and reproductive information, were collected, analyzed, compared, and tabulated. Logistic regression analysis was performed to explore the relationship between clinical characteristics and pathogenic variants. RESULTS: Among the patients, 72 were identified as carriers of pathogenic or likely pathogenic variants in BRCA1/2, while 337 had likely benign or benign mutations. The BRCA1 c.2635G > T (p. Glu879*) variant was detected at a high frequency, accounting for 12.5% (4/32) of the BRCA1 mutations, while the c.5164_5165del (p.Ser1722Tyrfs*4) variant was common among the BRCA2 mutations, accounting for 17.5% (7/40). It was observed that a higher proportion of BRCA1 carriers had the triple-negative breast cancer subtype, whereas more BRCA2 carriers exhibited estrogen receptor (ER) + and progesterone receptor (PR) + subtypes. Multivariate logistic regression analysis revealed that a family history of cancer (OR = 2.36, 95% CI = 1.00-5.54), bilateral cancer (OR = 4.78, 95% CI 1.61-14.20), human epidermal growth factor receptor 2 (HER2)- (OR = 8.23, 95% CI 3.25-20.84), and Ki67 ≥ 15% (OR = 3.88, 95% CI 1.41-10.65) were associated with BRCA1/2 mutations, with the age at diagnosis, age at menarche, and premenopausal status serving as covariates. CONCLUSIONS: The most common pathogenic variant of the BRCA1 and BRCA2 in breast cancer patients was c.2635G > T and c.5164_5165del, respectively. Additionally, a family history of cancer, bilateral cancer, HER2-, and Ki67 ≥ 15% were identified as independent predictors of BRCA1/2 pathogenic variants.

Proteomics of Serum Samples for the Exploration of the Pathological Mechanism of Obstetric Antiphospholipid Syndrome.

Obstetric antiphospholipid syndrome (OAPS) is a multisystem disorder characterized by thrombosis or recurrent fetal loss. In this study, we aim to explore the pathological mechanism of OAPS. Herein, we carried out data-independent acquisition (DIA) mass spectrometry quantitative proteomic analysis of serum samples from OAPS patients and healthy controls. A set of 93 differentially expressed proteins was identified, including 75 upregulated and 18 downregulated proteins compared with the levels in controls. Those proteins are enriched in KEGG pathways related to autoimmune diseases, allergic diseases, and pathogen infection. Interestingly, metabolic pathways such as fatty acid degradation and type I diabetes were enriched, indicating that OAPS is metabolic disease related. The significantly increased triglyceride also supported this idea. The differentially expressed proteins insulin-like growth factor-binding protein-1 (IGFBP-1), C-reactive protein (CRP), and ferritin light chain (FTL) were validated by ELISA. Our study presented a deep serum proteomics of OAPS and advanced our understanding of OAPS pathogenesis.

Association of Apolipoprotein E Gene Polymorphism with Type 2 Diabetic Nephropathy in the Southern Chinese Population.

Background: Common polymorphisms within the apolipoprotein E (APOE) gene are rs429358 and rs7412, which result in three major alleles (ɛ2, ɛ3, and ɛ4) and six genotypes (E2/E2, E2/E3, E3/E3, E3/E4, E4/E4, and E2/E4). Although APOE gene polymorphisms have been suggested to be associated with the development of diabetic nephropathy (DN), their potential association remains unclear in different regions. This study aims to unveil the genetic effects of APOE gene polymorphisms on DN susceptibility and serum lipid profiles in southern Chinese population. Methods: A total of 306 DN patients and 483 type 2 diabetic patients as controls were included in the study. The APOE gene polymorphisms were analyzed by polymerase chain reaction (PCR) microarray gene chip. Relevant medical records and information of these participants were collected. Results: There were statistically significant differences (p < 0.05) in gender, SBP, hypertension, hyperuricemia, UTP, TG and HDL-C between DN patients and controls. DN patients exhibited a higher frequency of the ε2 allele and E2/E3 genotype than controls (p < 0.001). Logistic regression analysis indicated that the ε2 allele and E2/E3 genotype were independent risk factors (adjusted OR: 3.237, 95% CI: 1.789-5.854, p < 0.001; adjusted OR: 3.453, 95% CI: 1.873-6.368, p < 0.001), while the ε3 allele or E3/E3 genotype might serve as protective role (adjusted OR: 0.395, 95% CI: 0.255-0.612, p < 0.001) for development of DN. Conclusion: Our study indicates a correlation between APOE polymorphisms and DN in the southern Chinese Hakka population. Specifically, individuals carrying the APOE ε2 allele and E2/E3 genotype are at a higher risk of developing DN. Conversely, those with the APOE ε3 allele and E3/E3 genotype have a lower risk of DN in southern Chinese population.

Effects of APOE gene ε4 allele on serum lipid profiles and risk of cardiovascular disease and tumorigenesis in southern Chinese population.

BACKGROUND: Human apolipoprotein E (APOE) polymorphisms are attributable to the presence of three common alleles, namely, ε2, ε3, and ε4, which generate six genotypes, viz, E2/E2, E2/E3, E3/E3, E3/E4, E4/E4, and E2/E4. APOE polymorphisms are associated with all types of tumors and cardiovascular diseases (CVD). However, the relationship between the type of APOE polymorphisms and tumorigenesis remains debatable. Therefore, we aimed to investigate the role of APOE polymorphisms on the tumor with or without CVD in southern China. METHODS: A total of 1438 participants were categorized into 4 groups: 409 patients with tumor, 369 patients with CVD, 338 patients with both tumor and CVD, and 322 controls. APOE polymorphisms were determined by genotyping assay. The factors influencing tumor patients with or without CVD were also analyzed by logistic regression analysis. RESULTS: The present study involved different types of solid tumors. Lung cancer was the most common cancer (20.2%, 151/747), followed by colorectal (17%, 127/747), esophageal (9.8%, 73/747), and liver (8.7%, 65/747) cancers. E3/E3 was the most frequent genotype, and ɛ3 was the greatest allele frequency in our study population. The frequencies of the E3/E3, E3/E4, E2/E3, E2/E4, E4/E4, and E2/E2 genotypes in tumor patients were 76.97% (575/747), 14.19% (106/747), 6.83% (51/747), 1.2% (9/747), 0.4% (3/747), and 0.4% (3/747), respectively. Tumor patients carrying ε3 with or without CVD showed higher levels of TG, TC, and LDL-C and lower levels of HDL-C compared to the controls carrying ε3. On the other hand, the tumor patients carrying ε4 with or without CVD showed higher levels of TG and LDL-C and lower levels of HDL-C (all P < 0.05). The frequency of APOE ε4 allele and the E3/E4 genotype was relatively greater in tumor or CVD patients (P < 0.001). In addition, ε4 allele acted as an independent risk factor for tumor patients group (P = 0.037, adjusted OR = 1.92, 95% CI 1.04-3.55) and tumor + CVD patients group (P = 0.012, adjusted OR = 2.53, 95% CI 1.22-5.23). CONCLUSIONS: Individuals carrying ε4 are at a higher risk of tumor with or without CVD, and APOE polymorphisms affect the serum lipid profiles.

Increased Peripheral NKG2A-NKG2D+CD3-CD16+CD56dim NK Cell Subset Was Positively Correlated with Antiphospholipid Antibodies in Patients of Obstetric Antiphospholipid Syndrome.

Obstetric antiphospholipid syndrome (OAPS) is an autoimmune disorder with severe life-threatening complications shown during pregnancy. It has been reported that the increase in CD16+CD56dim natural killer (NK) cells in peripheral blood are risk factors for recurrent miscarriages, but this expression of CD16+CD56dim NK cells in OAPS patients has not been reported, and the mechanism is not clearly illustrated. In this study, we compared the distributional profiles of different NK cell subsets and the expressions of NK cell-activating receptors in peripheral blood of patients with OAPS and healthy women. Our results showed significantly increased NKG2A-NKG2D+ subset and decreased NKG2A+NKG2D- subset in CD3- CD16+CD56dim NK cells, CD3-CD16-CD56bright NK cells and CD56+T cells in OAPS patients compared with those in healthy control women. The CD27-CD11b+ subset significantly increased in CD3-CD16+CD56dim NK cells in OAPS patients compared with those in healthy control women. In addition, the NKG2A-NKG2D+ subset in CD3-CD16+CD56dim NK subset in triple positivity was higher than single positivity OAPS patients. At the optimal diagnostic threshold established by ROC analysis, using the cut-off of NKG2A-NKG2D+ and CD27-CD11b+ subset in CD3-CD16+CD56dim NK cells is 10.10% and 92.75%, the sensitivity of NKG2A-NKG2D+ and CD27-CD11b+ to detect patients with OAPS compared with healthy control results was 94.1% and 60.8%, and specificity was 84.2% and 89.5%, respectively, with an area under the curve (AUC) of 0.903 and 0.829, respectively. The NKG2A-NKG2D+ subset in CD3-CD16+CD56dim NK cells was positively correlated with the antiphospholipid antibodies lg anti-aCL IgG, lg anti-aCL IgM, lg anti-aCL IgA, lg anti-ß2GP1 IgM and Complement 4(C4), while the CD27+CD11b+ subset in CD3-CD16+CD56dim NK cells was correlated with lg anti-ß2GP1 IgG and lg anti-ß2GP1 IgA. These results suggested that the NK cytotoxic function enhanced in OAPS patients and unbalanced of NK activating receptors and inhibiting receptors may contribute to the immune pathogenesis of OAPS.

Imbalance of circulating CTLA4+ follicular helper and follicular regulatory T cells in obstetric antiphospholipid syndrome.

Obstetric antiphospholipid syndrome (OAPS) is a systemic autoimmune disease that is characterized clinically by a variety of obstetric manifestations (fetal death and recurrent abortions) and serologically by the presence of antiphospholipid antibodies (aPLs). Whether dysregulation of Follicular helper T (Tfh) and Follicular regulatory T (Tfr) cells contribute to the immunopathogenesis in OAPS is still unknown. We analyzed phenotypic characterizations of circulating Tfh cells and Tfr cells in OAPS patients and healthy individuals. CTLA4(Cytotoxic T lymphocyte antigen 4)+ Tfh cells and CTLA4+ Tfr cells were declined and CTLA4+ Tfr/Tfh ratio and IL-21 were increased in OAPS patients compared with healthy controls. Percentages of CTLA4+ Tfh cells and CTLA4+ Tfr cells were the lowest in OAPS patients whose antiphospholipid antibodies (aPL) were triple positive. Increased CTLA4+ Tfr/Tfh ratio was positively correlated with anti-ß2 glycoprotein I (anti-ß2GPI) IgM, Complement 4(C4) or IL-21 in OAPS. Increased Th17 subtype and decreased Th1, Th2 subtypes in Tfh cells and Tfr cells, increased effector memory subtype and decreased central memory subtype of Tfh cells and Tfr cells were also observed in OAPS compared with healthy individuals. Our data demonstrated that an imbalance of circulating CTLA4+ Tfh cells, and Tfr cells correlates with the immunopathogenesis of OAPS.

Trimester-specific reference intervals of TAT, TM, tPAI-C and PIC for healthy Chinese pregnant women.

AIM: Pregnancy is a hypercoagulability state, the aim of this study was to observe the changes of thrombin-antithrombin complex (TAT), thrombomodulin (TM), tissue plasminogen activator-inhibitor complex (tPAI-C) and plasmin-α2-antiplasmin complex (PIC) during pregnancy and establish trimester-specific reference intervals for Chinese healthy pregnant women. METHODS: In total 190 Chinese healthy pregnant women (first trimester 59 cases, second trimester 60 cases and third trimester 71 cases) were recruited in North China. TAT, TM, tPAI-C and PIC were processed on Sysmex HISCL 5000 automated chemiluminescence immune detection system. Trimester-specific reference intervals were established with the 2.5th and 97.5th percentile of the distribution. RESULTS: The reference intervals for TAT, TM, tPAI-C, PIC at trimester 1 were 0.40-3.65 ng/mL, 4.85-8.80 TU/mL, 1.75-6.40 ng/mL, 0.25-1.05 µg/mL, respectively. At trimester 2, the reference intervals were 1.65-8.61 ng/mL, 5.70-9.93 TU/mL, 2.91-7.71 ng/mL, 0.33-2.02 µg/mL, respectively. At trimester 3, the reference intervals were 3.16-12.68 ng/mL, 5.50-14.24 TU/mL, 2.70-10.69 ng/mL, 0.24-1.54 µg/mL, respectively. CONCLUSIONS: The changes of TAT, TM, tPAI-C, PIC during pregnancy are presented, and trimester-specific reference intervals for healthy pregnant women are described. The levels of TAT, TM, tPAI-C were increased gradually from trimester 1 to trimester 3, while the PIC level remains stable during all trimesters.

Knockout of EGFL6 by CRISPR/Cas9 Mediated Inhibition of Tumor Angiogenesis in Ovarian Cancer.

Tumor angiogenesis plays an important role in the progression and metastasis of ovarian cancer. EGFL6 protein is highly expressed in ovarian cancer and has been proposed to play an important role in promoting tumor angiogenesis. Here, a CRISPR/Cas9 system was used to knockout the EGFL6 gene in the ovarian cancer cell line SKOV3, using specific guide RNA targeting the exons of EGFL6. The knockout of EGFL6 markedly inhibited the proliferation, migration, and invasion of SKOV3 cells, as well as promoted apoptosis of tumor cells. In the nude mouse model of ovarian cancer, knockout of EGFL6 remarkably inhibited tumor growth and angiogenesis. The transcript profile assays detected 4,220 differentially expressed genes in the knockout cells, including 87 genes that were correlated to proliferation, migration, invasion, and angiogenesis. Moreover, Western blotting confirmed that EGFL6 knockout downregulated the FGF-2/PDGFB signaling pathway. Thus, the results of this study indicated that EGFL6 could regulate cell proliferation, migration, and angiogenesis in ovarian cancer cells by regulating the FGF-2/PDGFB signaling pathway.

CRISPR/Cas9-Mediated OC-2 Editing Inhibits the Tumor Growth and Angiogenesis of Ovarian Cancer.

Ovarian cancer is the leading cancer-related cause of death in women worldwide. It is of great relevance to understand the mechanism responsible for tumor progression and identify unique oncogenesis markers for a higher chance of preventing this malignant disease. The high-expression OC-2 gene has been shown to be a potential candidate for regulating oncogenesis and angiogenesis in ovarian cancer. Hence, we wished to investigate the impact of OC-2 gene on ovarian cancer aggressiveness. CRISPR/Cas9, a gene editing tool, allows for direct ablation of OC-2 at the genomic level, and we successfully generated OC-2 KO cell lines from SKOV3 and CAOV3 cells. In an apoptosis assay, OC-2 KO induced the apoptosis activation of tumor cells, with the up-regulation of Bax/Caspase-8 and the down-regulation of Bcl-2. Consequently, the proliferation, migration, and invasion of OC-2 KO cell lines were significantly inhibited. Assays of qRT-PCR and Western blotting showed that the expression levels of pro-angiogenic growth factors VEGFA, FGF2, HGF, and HIF-1α and the activation of Akt/ERK pathways were significantly down-regulated at the loss of OC-2. In the xenograft model, OC-2 KO potently suppressed the subcutaneous tumor growth, with the inhibition exceeding 56%. The down-regulation of CD31 and relevant pro-angiogenic growth factors were observed in OC-2 KO tumor tissues. Taken together, OC-2 depletion negatively regulated the ovarian cancer progression possibly by apoptosis activation and angiogenesis inhibition. This work revealed a pivotal regulator of apoptosis and angiogenesis networks in ovarian cancer, and we applied the CRISPR/Cas9 system to the transcription factor pathway for developing a broad-acting anti-tumor gene therapy.

The Design, Characterizations, and Tumor Angiogenesis Inhibition of a Multi-Epitope Peptibody With bFGF/VEGFA.

Tumor angiogenesis is dependent on growth factors, and inhibition of their pathways is one of the promising strategies in cancer therapy. However, resistance to single pathway has been a great concern in clinical trials so that it necessitates multiple targetable factors for developing tumor angiogenesis inhibitors. Moreover, the strategy of Fc fusion protein is an attractive platform for novel peptide agents, which gains increasing importance with FDA approval because of better immunogenicity and stability. Here, we applied the Fc fusion protein concept to bFGF/VEGFA pathways and designed a multi-epitope Peptibody with immunogenic peptides derived from human bFGF and VEGFA sequences. Immunization with Peptibody could elicit high-titer anti-bFGF and anti-VEGFA antibodies, activate T cells, and induce Th1/Th2-type cytokines. In in vitro experiments, the isolated anti-Peptibody antibody inhibited the proliferation and migration of A549 cells and human umbilical vein endothelial cells (HUVECs) by decreasing the MAPK/Akt/mTOR signal pathways. In the murine tumor model, pre-immunization with Peptibody suppressed the tumor growth and neovascularization of lung cancer by decreasing the production of bFGF/VEGFA/PDGF, the MAPK/Akt/mTOR signal pathways, and the activation of suppressive cells in tumor sites. Further, the biological characterizations of the recombinant Peptibody were investigated systematically, including protein primary structure, secondary structure, stability, and toxicity. Collectively, the results highlighted the strategy of bFGF/VEGFA pathways and Fc fusion protein in suppressing tumor progression and angiogenesis, which emphasized the potential of multiple targetable factors for producing enduring clinical responses in tumor patients.

miR-6086 inhibits ovarian cancer angiogenesis by downregulating the OC2/VEGFA/EGFL6 axis.

miRNAs have emerged as a pivotal component of gene regulatory networks, mediating cytokines secretion, cell cycle, and differentiation regulation. However, how miRNAs collaborate with transcription factors and downstream effector proteins that determine the fate of ovarian cancer cells remains to be understood, especially regarding to mechanism of tumor angiogenesis regulation. Based on the qRT-PCR and IHC analysis, we found that miR-6086 was maintained a very low level both in ovarian cancer cell lines and tissues. Further, we identified OC2 and EGFL6 as the direct targets of miR-6086 by luciferase assay and we observed an inverse relationship between the expression of miR-6086 and the OC2/VEGFA/EGFL6 axis. The Western blotting analysis suggested that OC2 could directly upregulate VEGFA and indirectly up-regulate EGFL6 through VEGFA. Moreover, miR-6086 could indirectly downregulate VEGFA through OC2. In addition, miR-6086, siOC2 and siEGFL6 could negatively regulate the tumor growth and angiogenesis of ovarian cancer (Skov3) in the animal studies, with the inhibition rates of 77.07%, 69.89%, and 73.62%, respectively (**p < 0.01). Moreover, the tumor cell proliferation, migration, and invasion of ovarian cancer cell lines (Caov3 and Skov3) and vascular formation (HUVECs) were significantly suppressed in vitro, by decreasing the AKT/MAPK pathways (*p < 0.05). Taken together, our results reveal that miR-6086 can suppress the angiogenesis networks in ovarian cancer by down-regulating the OC2/VEGFA/EGFL6 axis, directly or indirectly, which may provide potential targets for tumor therapeutics.

Transfer of MicroRNA-216a-5p From Exosomes Secreted by Human Urine-Derived Stem Cells Reduces Renal Ischemia/Reperfusion Injury.

Human urine-derived stem cells (USCs) protect rats against kidney ischemia/reperfusion (I/R) injury. Here we investigated the role of USCs exosomes (USCs-Exos) in protecting tubular endothelial cells and miRNA transfer in the kidney. Human USCs and USCs-Exos were isolated and verified by morphology and specific biomarkers. USC-Exos played a protective role in human proximal tubular epithelial cells (HK-2) exposed to hypoxia/reoxygenation (H/R). USCs-Exos were rich in miR-216a-5p, which targeted phosphatase and tensin homolog (PTEN) and regulated cell apoptosis through the Akt pathway. In HK-2 cells exposed to H/R, incubation with USC-Exos increased miR-216-5p, decreased PTEN levels, and stimulated Akt phosphorylation. Exposure of hypoxic HK-2 cells to USCs-Exos pretreated with anti-miR-216a-5p can prevent the increase of miR-216-5p and Akt phosphorylation levels, restore PTEN expression, and promote apoptosis. The dual-luciferase reported gene assay in HK-2 cells confirmed that miR-216a-5p targeted PTEN. In rats with I/R injury, intravenous infusion of USCs-Exos can effectively induce apoptosis suppression and functional protection, which is associated with decreased PTEN. Infusion of exosomes from anti-miR-216a-5p-transfected USCs weakened the protective effect in the I/R model. Therefore, USCs-Exos can reduce renal I/R injury by transferring miR-216a-5p targeting PTEN. Potentially, USCs-Exos rich in miR-216a-5p can serve as a promising therapeutic option for AKI.

Anti-domain 1 of beta2-glycoprotein I aids risk stratification in lupus anticoagulant-positive patients.

Lupus anticoagulant (LA) is considered a risk factor for thromboembolism (TE) and adverse pregnancy outcomes (APOs). However, quite a few patients diagnosed with LA positivity do not suffer these adverse events. Further testing of anticardiolipin (aCL), anti-beta2-glycoprotein I (anti-ß2GPI) or anti-domain 1 of ß2GPI (anti-D1) may help to assess the occurrence risk of TE and APOs. Therefore, we aimed to study how to stratify LA-positive patients. In our study, 167 LA-positive patients were consecutively enrolled from January 2015 to December 2016. Serum aCL and anti-ß2GPI (IgG, IgM and IgA) and anti-D1 IgG were simultaneously measured. Among these patients, 114 (68.3%) were followed for an average of 36.5 months for TE and APOs. The outcomes showed that 105 patients experienced TE and/or APOs, and 62 patients were LA carriers. Anti-D1 had good consistency with triple positivity (LA+, aCL+, anti-ß2GPI+) (kappa = 0.742). Elevated anti-D1 was related to increased risks for TE [odds ratio (OR) 29.87, 95% confidence interval (CI) 8.05-110.74] and APOs (OR 8.73, 95% CI 3.41-22.31). Area under curve showed that the diagnostic power of anti-D1 for TE and APOs was 0.856 (95% CI 0.743-0.970) and 0.682 (95% CI 0.599-0.765), respectively. Survival analysis revealed that patients with high anti-D1 titres had a high cumulative incidence of APOs (hazard ratio 4.66, 95% CI 1.46-14.87). In conclusion, anti-D1, based on good consistency with triple positivity in LA-positive patients, has a stronger association with TE and APOs and, to some degree, could predict pregnancy outcomes. Therefore, anti-D1 may aid risk stratification in LA-positive patients.

Blockade of ONECUT2 expression in ovarian cancer inhibited tumor cell proliferation, migration, invasion and angiogenesis.

One cut homeobox 2 (ONECUT2 or OC-2) is a newly discovered transcription factor. Aberrant expression of OC-2 is closely related to cell proliferation, migration, invasion, and angiogenesis. In this study, we found that OC-2 expression was upregulated in ovarian adenocarcinoma cells, by Western blot analysis. The results of immunohistochemistry showed that the expression of OC-2 was also increased in malignant ovarian cancer tissue. In order to explore the role of OC-2 in the development of ovarian cancer, siRNAs that specifically targets OC-2 were designed. The siRNA targeting OC-2 could effectively inhibit the vascular endothelial growth factor A (VEGFA) expression, but silence and overexpression of VEGFA did not affect OC-2 expression. In addition, OC2-siRNA could block the proliferation, migration, and invasion, and inhibit epithelial-mesenchymal transition and the AKT/ERK signaling pathway, of human ovarian cancer cells in vitro. In a mouse model of ovarian cancer xenograft tumors, OC2-siRNA could significantly inhibit tumor cell growth and the tumor inhibition rate reached approximately 73%. The results of immunohistochemistry showed that the densities of microvessels stained with CD31, the expression of OC-2 and VEGFA were significantly decreased in tumors. These data indicated that OC-2 was an upstream regulator of VEGFA and silencing OC-2 could inhibit ovarian cancer angiogenesis and tumor growth.

Protective effects of apigenin against acrylonitrile-induced subchronic sperm injury in rats.

This study aimed to investigate the protective effects of apigenin (AP), a flavonoid found in plants, against acrylonitrile (ACN)-induced subchronic sperm and testes injury in rats. Male Sprague-Dawley rats were randomly divided into four groups: a control group (corn oil), an ACN group (ACN 50 mg kg-1), an ACN + AP1 group (ACN + AP 234 mg kg-1), and an ACN + AP2 group (ACN + AP 468 mg kg-1). The ACN + AP group received AP by gavage after treatment with 50 mg kg-1 ACN for 30 min, whereas the rats in the control group were given an equivalent volume of corn oil. The gavage was conducted 6 days per week for 12 weeks. The results showed that AP increased the sperm concentration, motility, and mitochondrial membrane potential (MMP) (P < 0.05), which were reduced by ACN. Conversely, reactive oxygen species (ROS) and malondialdehyde (MDA) were significantly decreased by AP (P < 0.05). AP improved the damage of the ultrastructure of sperm caused by ACN. AP reduced the pathological injuries and spermatogenic cell apoptosis caused by ACN in rat testes. AP also increased glutathione peroxidase activity and decreased MDA content. In conclusion, AP reduces ACN-induced decreasing sperm quality by inhibition of inflammation and oxidative stress.

[Protective effect and mechanism of neutrophil gelatinase-associated lipocalin against hypoxia/reoxygenation injury of HK-2 renal tubular epithelial cells].

Objective To study the role of neutrophil gelatinase-associated lipocalin (NGAL) in hypoxia/reoxygenation injury and its regulatory effect on autophagy in HK-2 renal tubular epithelial cells. Methods Three sets of designed NGAL short-hairpin RNA (shRNA) sequences were synthesized and transfected into HK-2 cells. The expression levels of NGAL mRNA and protein were analyzed by quantitative real-time PCR and Western blotting, respectively. Then, the HK-2 cells with the best NGAL mRNA interference were selected to establish the hypoxia/reoxygenation model. The levels of microtubule-associated protein 1 light chain 3II (LC-3II) and beclin-1 were detected by Western blotting. Besides, the viability of cells was tested by Cell Titer-Blue and CCK-8 assay. Results Three sets of shRNA plasmids carrying silenced NGAL gene were successfully constructed and transfected into HK-2 cells. The expressions of NGAL mRNA and protein in these cells were significantly lower than those in the controls with blank vector. After NGAL-silenced HK-2 cells were subjected to hypoxia/reoxygenation, the levels of LC-3 II and beclin-1 were lower than those in the controls with blank vector, whereas the levels of LC-3 II and beclin-1 in were higher than those in the ones in which 400 ng/mL recombinant NGAL was added. Cell Titer-Blue and CCK-8 assays showed that the viability of NGAL-knockdown HK-2 cells was significantly lower than the controls. Conclusion NGAL may plays protective role towards HK-2 cells in the process of hypoxia/reoxygenation by enhancing autophagy.

Last intron of the chemokine-like factor gene contains a putative promoter for the downstream CKLF super family member 1 gene.

The genes for chemokine-like factor (CKLF) and four chemokine-like factor super family members (CKLFSF1-4) are tightly linked on chromosome 16, with only 325 bp separating CKLF and CKLFSF1. We used Northern blotting and RT-PCR to show that these two genes are expressed independently of one another. We then used a novel computational promoter prediction method based on the interaction among transcription factor binding sites (TFBSs) to identify a putative promoter region for the CKLFSF1 gene. Our method predicted a promoter region in the last intron of the upstream gene, CKLF. We PCR amplified the predicted promoter region and used a luciferase assay to show that the region was able to drive the luciferase gene. DNA decoy experiments indicated that 214 bp fragment neighboring the TATA box markedly inhibited CKLFSF1 gene expression. Sequence analysis of the region revealed a typical TATA box (TATATAA) and multiple potential transcription factor binding sites, providing further evidence for this being a functional promoter for CKLFSF1. This work provides the first evidence of a promoter from one gene located in an intron of another.