Exosomal miR-202 derived from leukorrhea as a potential biomarker for endometriosis.

OBJECTIVE: Endometriosis (EMS) is a chronic gynecological disorder with an urgent need of a reliable non-invasive diagnostic strategy. Recently, there has been increasing interest in using the contents of exosomes, especially exosomal microRNAs (miRNAs), as potential biomarkers for various types of diseases. In this study, we assessed the differentially expressed miRNAs in exosomes derived from primary normal and ectopic endometrial cells. METHODS: We used miRNA microarray analysis to identify differentially expressed exosomal miRNAs. Among the selected exosomal miRNAs, we focused on hsa-miR-202-3p and hsa-miR-202-5p and validated their expression levels using quantitative reverse transcription polymerase chain reaction analysis. We then further examined their expression in exosomes derived from vaginal discharge (leukorrhea) from patients with EMS and the negative control group. RESULTS: The data show that hsa-miR-202-3p and hsa-miR-202-5p were expressed significantly higher in leukorrhea-derived exosomes from EMS patients compared with the negative controls. CONCLUSION: Taken together, our results suggest that leukorrhea-derived exosomal hsa-miR-202 could serve as a potential useful biomarker for diagnosing EMS.

Sunitinib Reduced the Migration of Ectopic Endometrial Cells via p-VEGFR-PI3K-AKT-YBX1-Snail Signaling Pathway.

Endometriosis (EMs) is one of the most common gynecological diseases, lacking effective treatment. EMs are currently being treated with small molecule targeted therapy, which has resulted in a significant reduction in patient suffering. Our previous studies have shown that sunitinib plays an obvious role in migration. Consequently, the purpose of this study is to explore the molecular mechanism by which sunitinib suppressed the ectopic endometrial migration. The ectopic endometrial cells from patients were divided into two groups: the control group and the sunitinib group. Co-IP and protein spectrum assay were employed to filtrate differential proteins between two groups, and then, our study discovered a signaling pathway, p-VEGFR-PI3K-AKT-YBX1-Snail, in the cell of EMs. To confirm this signaling pathway, VEGF165 was added to the sunitinib group to upregulate the expression of VEGFR. Next, the expression of p-VEGFR, PI3K, AKT, YBX1, and snail was measured in the control group and sunitinib group (compared with the control group: p-VEGFR, PI3K, AKT, YBX1, and snail, ∗∗∗∗P < 0.0001) and the VEGFR+sunitinib group (compared with the sunitinib group: p-VEGFR, PI3K, AKT, and snail, ∗∗∗∗P < 0.0001; YBX1, ∗∗∗P < 0.001); finally, the outcome was as expected. In addition to in vitro experiments, we also conducted in vivo experiments in mice. In the EMs mouse model, we found sunitinib reduced the number of heterotopic foci (t = 11.16, ∗∗∗∗P < 0.0001) and inhibited the expression of p-VEGFR, YBX1, and snail by immunofluorescence. To sum up, sunitinib exactly reduced the migration of ectopic endometrial cells with the involvement of the p-VEGFR-PI3K-AKT-YBX1-Snail signaling pathway in both in vitro and in vivo experiments. This study suggests that sunitinib presents a potential targeted drug for EMs therapy.

Role of CTGF gene promoter methylation in the development of hepatic fibrosis.

Connective tissue growth factor (CTGF) plays a critical role in the hepatic stellate cells (HSCs)-mediated development of hepatic fibrosis. Nevertheless, the effects of CTGF gene promoter methylation in the pathogenesis of hepatic fibrosis remain largely unknown. In the current study, we isolated and overexpressed CTGF in primary HSCs. We analyzed the CTGF gene promoter methylation inHSCs that undergo a phenotypic change into myofibroblast-like cellsthat express α-smooth muscle actin (α-SMA) in vitro and in vivo in a CCl4-induced rat hepatic fibrosis model. We found that CTGF promoted the phenotypic changes of HSCs into myofibroblasts in vitro, while inhibition of CTGF promoter methylation augmented the process, suggesting that CTGF gene promoter methylation may negatively regulate hepatic fibrosis. In vivo, CCl4 induced hepatic fibrosis in rats, and the severity of hepatic fibrosis inversely correlated with the levels of CTGF gene promoter methylation in HSCs. Together, our data demonstrate that CTGF gene promoter methylation may prevent the development of hepatic fibrosis, and low level of CTGF gene promoter methylation in HSCs may be a predisposing factor for developing liver fibrotic disease.

[Correlation of the inner diameter parameters of the spermatic vein in different positions and states of the varicocele patient with the results of seminal examination].

OBJECTIVE: To study the correlation of the inner diameter parameters of the spermatic vein in different positions and states of the varicocele (VC) patient with the results of seminal examination. METHODS: A total of 149 VC patients underwent ultrasonography, routine semen examination, and sperm morphological analysis. The parameters obtained from ultrasonography included the bilateral testis volume in a supine position, the largest spermatic vein diameter in a supine position at rest (DSR), the largest spermatic vein diameter in a supine position following Valsalva manoeuvre (DSV), the largest spermatic vein diameter in an upright position at rest (DUR), and the largest spermatic vein diameter in an upright position following Valsalva manoeuvre (DUV). Then we calculated the parameters △DS=DSV－DSR, △DU=DUV－DUR, △DR=DUR－DSR, and △DV=DUV－DSV and analyzed the correlation of the above parameters with the results of semen examination using the ROC curve. RESULTS: Based on the results of semen examination, 119 (79.87%) of the patients were allocated to the abnormal group and the other 30 (20.13%) to the normal group. Statistically significant differences were observed between the two groups in △DU (P=0.007), △DR (P=0.0001), and △DV (P=0.04), but not in DSR (P=0.35), DSV (P=0.34), DUR (P=0.06), DUV (P=0.12), and △DS (P=0.64), nor in the volume of the testis affected (P=0.323). The area under the ROC curve was 0.55 for DSR, 0.57 for DSV, 0.64 for DUR, 0.62 for DUV, 0.49 for △DS, 0.28 for △DU, 0.86 for △DR, and 0.69 for △DV. The corresponding cutoff values were 2.25, 2.51, 2.48, 2.63, 0.30, 0.23, 0.25, and 0.20, the corresponding sensitivities of semen detection were 50.42%, 65.55%, 60.50%, 60.50%, 49.90%, 29.41%, 79.83%, and 65.55%, and the corresponding specificities were 56.67%, 63.33%, 63.33%, 63.33%, 56.67%, 33.33%, 80%, and 63.33%, respectively. CONCLUSIONS: The difference between the largest spermatic vein diameters in supine and upright positions at rest provides a high diagnostic accuracy for semen detection and helps to predict abnormality in seminal examination for VC patients.

[Construction and immunological responses of recombinant adenovirus containing Epstein-Barr nuclear antigen 1 in mice].

This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1 (EBNA1) against nasopharyngeal carcinoma (NPC). The C-terminal region fragment of the ebna1 gene of Epstein-Barr virus was amplified from the standard strain B95-8 by polymerase chain reaction (PCR). The gene fragment was inserted into the pDC316 shuttle plasmid using the EcoRI and BgIII restriction enzyme sites. The pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells after sequencing. The soluble protein was extracted from HEK293 cells, which caused apparent cytopathic effects. The transcription and expression of the ebna1 gene were confirmed using flow cytometry and Western blotting. rAd-ebna1 titers were measured by the TCID50. rAd-ebna1 was injected into BALB/c mice at a dose of 2 x 10(8) VP per mouse, EBNA1 epitope-specific responses were measured at 1st, 2nd, 4th and 8th weeks post-immunization. The target fragment of ebna1 (939 bp) was obtained by PCR, and was in consensus with the sequence from the standard strain B95-8. Cytopathic effects were observed after the pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells. rAd-ebna1 was successfully recombined in HEK293 cells. EBNA1 protein was detected in HEK293 cells, rAd-ebna1 titers reached 10(8) TCID50/mL. Specific responses to CD4+ epitopes of EBNA1 were detected in the immunized mice. In conclusion, rAd-ebna1 was successfully constructed and induced specific responses to CD4+ epitopes of EBNA1 in immunized mice.

[Screening of chlorobenzene-degrading bacterium and purification of its degradation enzyme].

OBJECTIVE: We screened a bacterial strain capable of degrading chlorobenzene, and purified the corresponding degradation enzyme. METHODS: The strain was screened by gradient enrichment culture and sterile filter paper plate method, and identified by morphology and 16S rRNA gene sequence. Chlorobenzene concentration in the liquid culture was determined by gas chromatography. Degradation capability was assayed by the proportion of chlorobenzene degraded per cells. The purity quotient and molecular weight of the purified degradation enzyme were determined by gel per cell. electrophoresis. RESULTS: The isolated bacterium, LW13, used chlorobenzene in activated sludge as sole carbon and energy source. Cells were 2.3 microm long and 0.8 microm wide, with several terminal flagella. Strain LW13 was 95.5% similar to Lysinibacillus fusiformis, and its degradation enzyme was a positively-charged exoenzyme (molecular weight about 57 kDa). The optimal temperature and pH of the purified enzyme were approximately 40 degrees C and 8.0, respectively. CONCLUSION: Strain LW13 belongs to genus Lysinibacillus, and can degrade chlorobenzene (500-2000 mg/L).