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Diabetic bladder dysfunction (DBD) is the most common complication in diabetes mellitus (DM). Myogenic abnormalities are common in DBD, however, the underlying mechanisms leading to these remains 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.

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.

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.

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.

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

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.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.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 (P0.05).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.