miR-188-3p-targeted regulation of ATG7 affects cell autophagy in patients with nonobstructive azoospermia.

BACKGROUND: Nonobstructive azoospermia (NOA) is one of the most difficult forms of male infertility to treat, and its pathogenesis is still unclear. miRNAs can regulate autophagy by affecting their target gene expression. Our previous study found that miR-188-3p expression in NOA patients was low. There are potential binding sites between the autophagy gene ATG7 and miR-188-3p. This study aimed to verify the binding site between miR-188-3p and ATG7 and whether miR-188-3p affects autophagy and participates in NOA by regulating ATG7 to influence the autophagy marker genes LC3 and Beclin-1. METHODS: Testicular tissue from 16 NOA patients and 16 patients with normal spermatogenesis and 5 cases in each group of pathological sections were collected. High-throughput sequencing was performed to detect mRNA expression differences. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, immunohistochemical staining and immunofluorescence were used to detect protein localization and expression. Autophagosome changes were detected by electron microscopy. The targeting relationship between miR-188-3p and ATG7 was confirmed by a luciferase assay. RESULTS: ATG7 protein was localized in the cytoplasm of spermatogenic cells at all levels, and the ATG7 gene (p = 0.019) and protein (p = 0.000) were more highly expressed in the NOA group. ATG7 expression after overexpression/inhibition of miR-188-3p was significantly lower (p = 0.029)/higher (p = 0.021) than in the control group. After overexpression of miR-188-3p, the ATG7 3'UTR-WT luciferase activity was impeded (p = 0.004), while the ATG7 3'UTR-MUT luciferase activity showed no significant difference (p = 0.46). LC3 (p = 0.023) and Beclin-1 (p = 0.041) expression in the NOA group was significantly higher. LC3 and Beclin-1 gene expression after miR-188-3p overexpression/inhibition was significantly lower (p = 0.010 and 0.024, respectively) and higher (p = 0.024 and 0.049, respectively). LC3 punctate aggregation in the cytoplasm decreased after overexpression of miR-188-3p, while the LC3 punctate aggregation in the miR-188-3p inhibitor group was higher. The number of autophagosomes in the miR-188-3p mimic group was lower than the number of autophagosomes in the mimic NC group. CONCLUSIONS: LC3 and Beclin-1 were more highly expressed in NOA testes and negatively correlated with the expression of miR-188-3p, suggesting that miR-188-3p may be involved in the process of autophagy in NOA. miR-188-3p may regulate its target gene ATG7 to participate in autophagy anDual luciferase experiment d affect the development of NOA.

Promoter methylation of protocadherin8 is an independent prognostic factor for biochemical recurrence of early-stage prostate cancer.

BACKGROUND: Protocadherin8 has been demonstrated to play critical roles in initiation and progression of several human cancers. It is frequently inactivated by promoter methylation in cancers and may be used as a potential biomarker. However, the methylation status of protocadherin8 and its clinical significance in prostate cancer remains largely unknown. The purpose of this study was to evaluate the clinical significance of protocadherin8 methylation in early-stage prostate cancer. MATERIAL AND METHODS: The promoter methylation status of protocadherin8 in 162 prostate cancer tissues and 47 normal prostate tissues was examined using methylation-specific PCR (MSP). Subsequently, the relationships between protocadherin8 methylation and clinicopathological features of prostate cancer patients and biochemical recurrence-free survival of patients were analyzed. RESULTS: We found that protocadherin8 methylation occurred frequently in prostate cancer tissues but not in normal prostate tissues. Moreover, protocadherin8 methylation was significantly associated with advanced pathologic stage, higher level of preoperative prostate specific antigen (PSA), higher Gleason score, positive lymph node metastasis, and biochemical recurrence. In addition, patients with protocadherin8 methylated have shorter biochemical recurrence-free survival time than patients without. Multivariate Cox regression analysis revealed that protocadherin8 methylation was an independent predictor of biochemical recurrence-free survival in prostate cancer patients. CONCLUSIONS: Promoter methylation of protocadherin8 is a frequent event in prostate cancer, and might be used as an independent prognostic factor for biochemical recurrence-free survival in patients with prostate cancer.

Generation of a human haploid neural stem cell line for genome-wide genetic screening.

BACKGROUND: Haploid embryonic stem cells (haESCs) have been established in many species. Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens. AIM: To derive human haploid neural stem cells (haNSCs) to carry out lineage-specific screens. METHODS: Human haNSCs were differentiated from human extended haESCs with the help of Y27632 (ROCK signaling pathway inhibitor) and a series of cytokines to reduce diploidization. Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency. Global gene expression analysis was con-ducted to compare haNSCs with diploid NSCs and haESCs. Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs. Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools. RESULTS: Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies, a higher efficiency in neural differentiation, a higher cell survival ratio and higher stability in haploidy maintenance. These characteristics effectively facilitated the derivation of human haNSCs. These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro. After PiggyBac transfection, there were multiple insertion sites in the human haNSCs' genome, and the insertion sites were evenly spread across all chromosomes. In addition, after the cells were treated with manganese, we were able to generate a list of manganese-induced toxicity genes, demonstrating their utility as genetic screening tools. CONCLUSION: This is the first report of a generated human haploid somatic cell line with a complete genome, proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.

Heterogeneous Structural Color Conductive Photonic Organohydrogel Fibers with Alternating Single and Dual Networks.

Intelligent interactive electronic devices can dynamically respond to and visualize various stimuli, promoting the rapid development of flexible electronics. In this paper, an alternating single- and dual-network design strategy was developed for ingeniously constructing an interactive electronic fiber sensor with heterogeneous structural color (HSCEF sensor). The resulting sensor can rapidly output the synchronous electrical and optical dual signals under strain by adjusting the transport distance of conductive ions and the lattice spacing of the photonic crystal (∼200 ms). Meanwhile, the addition of low-freezing-point glycerol endowed the HSCEF sensor with excellent low-temperature tolerance (-25 °C) and cyclic stability. Notably, benefiting from the alternating single- and dual-network structure, the HSCEF sensor exhibits attractive heterogeneous structural color, which achieves colorimetric changes in the full visible light region with high mechanochromic sensitivity (2.25 nm %-1) and large wavelength shift (Δλ ∼ 225 nm). An intelligent wearable interactive sensor is finally used for real-time dynamic detection of joint movements, realizing precise resolution of different amplitudes. This work provides a general strategy to transform conventional photonic gels into heterogeneous structural color ones, and the developed new interactive sensor with rich optical information could be further used for visual health and exercise monitoring, intelligent soft robotics, wearable sensors, etc.

Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma.

Nucleobindin 2 (NUCB-2) is a multifunctional protein that contains several functional domains and is associated with a wide variety of biological processes, such as food intake and energy homeostasis. NUCB-2 has been demonstrated to be associated with worse malignant outcomes and cell migration in breast and prostate cancer. However, to the best of our knowledge, its clinical and biological significance in renal cell carcinoma remains unknown. In the present study, tissue specimens from 68 patients with renal cell carcinoma and 10 normal controls were collected for NUCB-2 mRNA and protein assays. The NUCB-2 level in the patients with renal cell cancer was significantly increased compared with the normal control patients. NUCB-2-knockout in the renal cancer cell line SK-RC-52 inhibited migration and invasion. In addition, the expression levels of molecules associated with epithelial-mesenchymal transition (EMT), including E-cadherin, ß-catenin, Slug and Twist, were affected by NUCB-2 suppression and the zinc finger E-box binding to homeobox 1 (ZEB1)-dependent pathway. The AMP-dependent protein kinase (AMPK)/target of rapamycin complex (mTORC) 1 signaling pathway participates in the regulation of NUCB-2-mediated metastasis and EMT. Suppression of NUCB-2 also inhibited tumor nodule formation in a murine renal cell carcinoma tumor model. In summary, NUCB-2 increased migration, invasion and EMT in renal cell carcinoma cells through the AMPK/TORC1/ZEB1 pathway in vitro and in vivo.

The role of AMH and its receptor SNP in the pathogenesis of PCOS.

The etiology of polycystic ovaries syndrome (PCOS) is unknown. Studies probing the role of genetic variants of anti-Mullerian hormone (AMH) and its type II receptor (AMHR2) in the pathogenesis of PCOS have yielded inconsistent results. Thus, we performed a systematic review and meta-analysis to determine the role of genetic variants of AMH/AMHR2 in the pathogenesis of PCOS. A systematic search of electronic databases was performed. Statistical analysis was performed using the Comprehensive Meta-Analysis software (Version 3). Pooled Odds Ratios (OR) (95% confidence intervals) were determined to assess the association between genetic variants of AMH/AMHR2 and PCOS. Five studies, involving a total of 2042 PCOS cases and 1071 controls, were included in the meta-analysis. Single nucleotide polymorphisms of AMH and AMHR2 did not appear to confer a heightened risk for PCOS (OR: 0.954, 95% CI: 0.848-1.073; P = 0.435; and OR: 1.074, 95% CI: 0.875-1.318; P = 0.494, respectively). In this study, genetic variants of AMH or AMHR2 were not found to be associated with a higher risk for PCOS.

Role of Wnt5a in the differentiation of human embryonic stem cells into endometrium-like cells.

OBJECTIVE: To explore the effects of Wnt5a and Wint7a on the differentiation of human embryonic stem cells (hESCs) into endometrium-like cells, and provide a basis for establishing endometrium-like cell models and a cell source for carrying out further endometrium-related experiments. METHODS: The hESCs established by our center were differentiated into endometrium-like cells in 4 different media including Wnt5a (Group A), Wnt7a (Group B), secreted frizzled related protein (sFRP, an inhibitor of Wnt signal pathway, Group C) and medium alone (Group D). In the differentiated terminal cells, the expressions of cytokeratin (CK) and vimentin were detected with immunofluorescence, and the mRNA levels of CK18, epithelial cell adhesion molecule (EPCAM), estrogen receptor (ER) and progesterone receptor (PR) were determined with RT-PCR. At the same time, the differentiated terminal cells were incubated in medium containing medroxyprogesterone followed by determination of prolactin (PRL). RESULTS: RT-PCR indicated that mRNA levels of CK18, EPCAM, ER and PR were significantly higher in Group A (Wnt5a) than in other groups (all P<0.05), but were significantly lower in Group C (sFRP2) than in other groups (all P<0.05). The changing trend of PRL mRNA was consistent with that of above genes in the 4 groups. Immunofluorescence displayed that the expression of cytokeratin was the strongest in Group A (Wnt5a), and the weakest in Group C (sFRP2) among the 4 groups. CONCLUSION: Wnt5a has promotive effects on the differentiation of hESCs into endometrium-like cells, but Wnt7a has no marked effects.