Role of ecto-5'-nucleotidase in bladder function.

Purinergic signaling plays an important role in regulating bladder contractility and voiding. Abnormal purinergic signaling is associated with lower urinary tract symptoms (LUTS). Ecto-5'-nucleotidase (NT5E) catalyzes dephosphorylation of extracellular AMP to adenosine, which in turn promotes adenosine-A2b receptor signaling to relax bladder smooth muscle (BSM). The functional importance of this mechanism was investigated using Nt5e knockout (Nt5eKO) mice. Increased voiding frequency of small voids revealed by voiding spot assay was corroborated by urodynamic studies showing shortened voiding intervals and decreased bladder compliance. Myography indicated reduced contractility of Nt5eKO BSM. These data support a role for NT5E in regulating bladder function through modulation of BSM contraction and relaxation. However, the abnormal bladder phenotype of Nt5eKO mice is much milder than we previously reported in A2b receptor knockout (A2bKO) mice, suggesting compensatory response(s) in Nt5eKO mouse bladder. To better understand this compensatory mechanism, we analyzed changes in purinergic and other receptors controlling BSM contraction and relaxation in the Nt5eKO bladder. We found that the relative abundance of muscarinic CHRM3 (cholinergic receptor muscarinic 3), purinergic P2X1, and A2b receptors was unchanged, whereas P2Y12 receptor was significantly downregulated, suggesting a negative feedback response to elevated ADP signaling. Further studies of additional ecto-nucleotidases indicated significant upregulation of the nonspecific urothelial alkaline phosphatase ALPL, which might mitigate the degree of voiding dysfunction by compensating for Nt5e deletion. These data suggest a mechanistic complexity of the purinergic signaling network in bladder and imply a paracrine mechanism in which urothelium-released ATP and its rapidly produced metabolites coordinately regulate BSM contraction and relaxation.

Smooth Muscle Insulin Receptor Deletion Causes Voiding Dysfunction: A Mechanism for Diabetic Bladder Dysfunction.

Diabetic bladder dysfunction (DBD) is the most common complication in diabetes. Myogenic abnormalities are common in DBD; however, the underlying mechanisms leading to these remain unclear. To understand the importance of smooth muscle insulin receptor (IR)-mediated signaling in the pathogenesis of DBD, we conditionally deleted it to achieve either heterozygous (SMIR+/-) or homozygous (SMIR-/-) deletion in smooth muscle cells. Despite impaired glucose and insulin tolerance seen with SMIR-/- mice, both SMIR+/- and SMIR-/- mice exhibited normal blood glucose and plasma insulin levels. Interestingly, these mice had abnormal voiding phenotypes, that included urinary frequency and small voids, and bladder smooth muscle (BSM) had significantly diminished contraction force. Morphology revealed a dilated bladder with thinner BSM layer, and BSM bundles were disorganized with penetrating interstitial tissue. Deletion of IR elevated FoxO and decreased mTOR protein expression, which further decreased the expression of Chrm3, P2x1, Sm22, and Cav1.2, crucial functional proteins for BSM contraction. Furthermore, we determined the expression of adiponectin in BSM, and deletion of IR in BSM inhibited adiponectin-mediated signaling. In summary, disruption of IR-mediated signaling in BSM caused abnormalities in proliferation and differentiation, leading to diminished BSM contractility and a voiding dysfunction phenotype that recapitulates human DBD.

Analysis of World-Scale Mitochondrial DNA Reveals the Origin and Migration Route of East Asia Goats.

Despite much attention on the history of goat evolution, information on origin, demographic history, and expansion route remains controversial. To address these questions, we collected 4,189 published goat DNA sequences including 1,228 sequences from 57 breeds in China and 2,961 sequences including 193 goat breeds from 71 other countries and carried out an integrated analysis. We found goat breeds from South China had the highest genetic diversity of lineage B, and subclades B2 only were found in Southwest China, suggesting that lineage B (particularly, subclade B2) probably originated from Southwest China and its surrounding areas. In addition, in this study, we found that lineage A from South China also presented higher genetic diversity and earlier expansion time (10, 606 years ago), even earlier than breeds from the Middle East. Hence, we speculated that South China and surrounding areas were the origin of lineage B and also the transportation hub for lineage A spreading to North China and Southwest Asia. Furthermore, according to the analysis of correlation between genetic differentiation value λ1 and λ2 and geographical distance, we further confirmed two phases of migration in goat breeds of North China. These results will contribute to a better understanding of the origin and migration history of domestic goat.

Involvement of the lncRNA AFAP1-AS1/microRNA-195/E2F3 axis in proliferation and migration of enteric neural crest stem cells of Hirschsprung's disease.

NEW FINDINGS: What is the central question of this study? Long non-coding RNAs (lncRNAs) are widely involved in the progression of Hirschsprung's disease (HSCR), but the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1), an lncRNA, in HSCR has not been explored before. What is the main finding and its importance? Downregulation of AFAP1-AS1 blocks enteric neural crest stem cell proliferation, differentiation, migration and invasion and promotes the occurrence of HSCR via the miR-195/E2F3 axis, indicating thatAFAP1-AS might be a potential biomarker for HSCR patients. ABSTRACT: Long non-coding RNAs (lncRNAs) are involved in several human disorders. Nevertheless, it remains unclear whether they are implicated in the phenotypes of enteric neural crest stem cells (ENCSCs) in Hirschsprung's disease (HSCR). Therefore, we designed this study to explore the pathogenicity of AFAP1-AS1 for HSCR. Microarray analysis and bioinformatic tools were used to screen out the differentially lncRNAs and microRNAs (miRNAs) in patients with HSCR. Small interference RNA transfection was applied to carry out functional experiments in ENCSCs. Cellular activities were detected by cell counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell assays and flow cytometry. Finally, rescue experiments were performed to examine the cofunction of AFAP1-AS1 and miR-195 and of miR-195 and E2F transcription factor 3 (E2F3). AFAP1-AS1 was reduced in HSCR patients. Meanwhile, knockdown of AFAP1-AS1 reduced the cell migratory and proliferative capacities and facilitated cell apoptosis along with G0/G1 phase arrest. E2F3 was diminished when miR-195 was upregulated, and AFAP1-AS1 inhibition reduced its ability to bind to miR-195. Altogether, AFAP1-AS1 silencing acts as an endogenous RNA by interacting with miR-195 to alter E2F3 expression, thus conferring repressive effects on ENCSC activity and promoting HSCR progression.

NEAT1 Negatively Regulates Cell Proliferation and Migration of Neuroblastoma Cells by miR-183-5p/FOXP1 Via the ERK/AKT Pathway.

Neuroblastoma, a malignant tumor of the sympathetic nervous system, is an aggressive extracranial tumor in childhood. Long noncoding RNAs (lncRNAs) have been discovered to play a key role in the eukaryotic regulatory gene network and be involved in a wide variety of biological processes. We observed that the expression of lncRNA nuclear-enriched abundant transcript-1 (NEAT1) was significantly decreased in human neuroblastoma tissues and cell lines, compared with the normal. We observed cell proliferation, migration, and invasion with Cell Counting Kit-8 assay, colony formation assay, and Transwell assay to investigate the effects of NEAT1, miR-183-5p, or FOXP1 on neuroblastoma cells. And we also used StarBase and luciferase reporter gene assay to predict and confirm the interaction of NEAT1, miR-183-5p, and FOXP1 in neuroblastoma cells. First, overexpression of NEAT1 suppressed cell proliferation and played a key role in cell migration and invasion. In addition, NEAT1 was demonstrated to directly interact with miR-183-5p and exerted its antioncogenic role in neuroblastoma by negatively regulating miR-183-5p expression. miR-183-5p suppressed the expression of FOXP1 and regulated cell proliferation and migration by directly targeting FOXP1 mRNA 3'-untranslated region. Moreover, FOXP1 antagonized the effect of miR-183-5p on the phosphorylation of extracellular-regulated kinase/protein kinase B (ERK/AKT), while FOXP1 siRNA increased the reduced phosphorylation of ERK/AKT caused by miR-183-5p inhibitor in neuroblastoma cells. Taken together, these data showed that NEAT1 negatively regulated cell proliferation and migration of neuroblastoma by the miR-183-5p/FOXP1 axis via suppression of the ERK/AKT pathway. Our findings may provide a new target for the study of pathogenesis and treatment of neuroblastoma.

Expression of TLR4 and its effect on Treg cells in early pregnancy decidual stromal cells after lipopolysaccharide treating.

OBJECTIVE: To investigate the expression of TLR4 in human early pregnancy decidual stromal cells (DSCs) induced by lipopolysaccharide (LPS) and its effect on the peripheral blood regulatory T (Treg) cells subgroup in women of childbearing age. METHOD: Isolating and cultivating normal human early pregnancy DSCs followed by treatment with 0, 25, 50, 100 and 200 ng/ml LPS, and the expression level of TLR4 mRNA was detected by RT-PCR. After 3 or 4 generation we divide the DSCs into 5 groups: ①Control group: Cultivation of peripheral blood lymphocyte (PBLC); ②Co-cultivation group: Co-cultivation of PBLC and DSCs; ③LPS stimulation group: PBLC + DSCs + LPS; ④PDTC blocking-up group: PBLC + DSCs + LPS + PDTC; ⑤TLR4 blocking-up group: PBLC + DSCs + LPS + TLR4mAb. In ①-④ groups, western blot was used to detect the expression of inhibitory factor-κB (IκB-α) protein and RT-PCR was used to detect the expression of FoxP3 mRNA. In ①-⑤ groups, flow cytometry was applied to detect the percentage of Treg cells subgroup. RESULTS: The purity of primary cultured DSCs was more than 95%. RT-PCR results showed that the expression level of TLR4 mRNA increased gradually with the augment of LPS concentration. Western blot and RT-PCR showed that the expression of IκBα protein and FoxP3 mRNA in the other 3 groups was significantly higher than that in the control group (P < 0.05), and the expression of IκBα protein and FoxP3 mRNA in LPS stimulation group was lower than that in the co-cultivation group (P < 0.05). Compared with the LPS stimulation group, the expression of IκBα protein and FoxP3 mRNA in PDTC blocking-up group was higher than that in the LPS stimulation group (P < 0.05), but still lower than the co-cultivation group (P < 0.05). The proportion of Treg cells in the other 4 groups detected by flow cytometry was significantly higher than that in the control group (P < 0.05). Compared with the co-cultivation group, the Treg cells ratio of the LPS stimulation group was significantly decreased (P < 0.05). The proportions of Treg cells in PDTC blocking-up group and TLR4 blocking-up group were higher than that in the LPS stimulation group, but still lower than that in the co-cultivation group (P < 0.05). There was no significant difference between the PDTC blocking-up group and the TLR4 blocking-up group (P > 0.05). CONCLUSION: Human early pregnancy DSCs can promote the differentiation of Treg cells. LPS can stimulate the expression of TLR4 in early pregnancy DSCs and decrease the proportion of Treg cells in PBLC, with NF-κB signaling pathway being the potential underlying mechanisms.

miR-204 Regulates Cell Proliferation and Invasion by Targeting EphB2 in Human Cervical Cancer.

MicroRNAs (miRNAs) are small noncoding RNAs that are involved in human carcinogenesis and progression. miR-204 has been reported to be a tumor suppressor in several cancer types. However, the function and underlying molecular mechanism of miR-204 in cervical cancer (CC) are still unclear. In the present study, the expression level of miR-204 was measured using the qRT-PCR method in 30 paired CC clinical samples and in 6 CC cell lines. We found that the expression of miR-204 was significantly downregulated in CC tissues and cell lines compared to normal cervical tissues and cell line. miR-204 was overexpressed by transfection with the miR-204 mimic in HeLa and C33A cell lines in the following experiments. The results showed that overexpression of miR-204 dramatically suppressed cell proliferation, migration, and invasion, caused cell cycle arrest at the G0/G1 phase, promoted cell apoptosis in vitro, and inhibited tumor growth in vivo. Western blot results indicated that overexpressing miR-204 decreased the expressions of CDK2, cyclin E, MMP2, MMP9, Bcl2, whereas it enhanced Bax expression and suppressed the activation of the PI3K/AKT signaling pathways in CC cells. Ephrin type B receptor 2 (EphB2) was identified as a direct target of miR-204 in CC cells according to bioinformatics analysis and luciferase reporter assay. Furthermore, knockdown of EphB2 mimicked the inhibitory effect of miR-204 on the proliferation, invasion, and migration of CC cells. These findings suggested that miR-204 might serve as a tumor suppressor in the development of CC by directly targeting EphB2.

Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1-13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both P<0.01) throughout 7 days of culture. The addition of bFGF drove a significantly greater proportion of enteric neurosphere cells to differentiate into neuronal cells than that of EGF (P<0.01), whereas addition of EGF resulted in significantly more glial differentiation compared with addition of bFGF (P<0.01). Combining bFGF and EGF drove enteric neurosphere cells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro.

Merging Fargesia dracocephala into Fargesia decurvata (Bambusoideae, Poaceae): implications from morphological and ITS sequence analyses.

AIMS: Fargesia decurvata is closely allied with F. dracocephala and differs in 5 major characters (i.e. the culm sheath blade base shape, the width of the culm sheath blade base, the auricle shape, and the lower surface of leaf blade) in Fargesia. It is difficult to distinguish these two species because of existing of transitional statements of characters. The aims of this paper are to (i) investigate whether the variation of the characters is continuous or not; (ii) reveal whether the publishment of F. dracocephala was the result of discontinuous sampling of F. decurvata or not. METHODS: Ten populations of F. decurvata and F. dracocephala were investigated in their entire distribution (including type localities). The statements of 5 major characters were measured from 693 annual and 693 perennial culms of 231 individuals in 10 populations, and analyzed at population, individual and culm levels. UPGMA cluster analysis was carried out based on 29 characters from 10 populations of F. decurvata and F. dracocephala and 2 populations of F. qinlingensis as outgroup. The ITS sequences were also sequenced and analyzed. IMPORTANT FINDINGS: Five major characters exhibited great variation not only at population level, but at individual level within a population, even the culm level within an individual and in different parts of the same culm. Cluster analyses showed that 10 populations of F. decurvata and F. dracocephala were not divided into two species, but they were well separated with outgroup. There was no difference in floral organ between F. decurvata and F. dracocephala. MP and NJ trees based on ITS sequences showed the same results with the cluster analysis on morphological characters. All the facts indicated that the publishment of F. dracocephala was the result of discontinuous sampling of F. decurvata, and F. dracocephala should be treated as the synonym of F. decurvata.