Background: Bladder cancer is of compelling morbidity and mortality due to its high recurrence rate. Little development has been made in the last decades in the therapy methods. Thus, the mechanism of its growth and invasiveness involving novel molecular targets are needed. Objective: Our research objective is to confirm the hypothesis that miR-1-3p suppresses the proliferation, invasion and migration of bladder cancer cells. Methods: The expression levels of miR-1-3p and SFRP1 were evaluated using RT-qPCR in bladder cancer tissues and cells as well as in normal tissues and cells. J82 cell lines were selected as experiment subjects due to their low expression levels of miR-1-3p. Plasmids carrying miR-1-3p mimics, miR-1-3p inhibitors and SFRP1 were transfected into the J82 cell lines. Subsequently, the protein expression of SFRP1 was detected using Western Blot analysis, and cell proliferation, apoptosis, invasion and migration ability was measured using MTT, the flow cytometry, the Transwell test and wound healing assays, respectively Results: Bladder cancer tissues and cells exhibited significant decrease in the expression of miR-1-3p and SFRP1 compared to normal tissues and cells, and human bladder cancer cell line J82 exhibited the most significant decrease in these expressions (P < 0.05). MiR-1-3p up-regulates SFRP1 expression in bladder cancer cells, and the over-expression of miR-1-3p can suppress the proliferation, invasion and migration ability of bladder cancer cells. This mechanism is similar to the effect of SFRP1 over-expression on bladder cancer cells. Conclusion: MiR-1-3p suppresses the proliferation, invasion and migration of bladder cancer cells by up-regulating SFRP1 expression.

Autophagy plays important roles in the host immune response against mycobacterial infection. Mycobacterium tuberculosis (M. tuberculosis) can live in macrophages owing to its ability to evade attacks by regulating autophagic response. MicroRNAs (miRNAs) are small noncoding, endogenously encoded RNA which plays critical roles in precise regulation of macrophage functions. Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that BCG infection of macrophages resulted in enhanced expression of miRNA-20a, which inhibits autophagic process by targeting ATG7 and ATG16L1 and promotes BCG survival in macrophages. Forced overexpression of miR-20a decreased the expression levels of LC3-II and the number of LC3 puncta in macrophages, and promoted BCG survival in macrophages, while transfection with miR-20a inhibitor had the opposite effect. Moreover, the inhibitory effect of miR-20a on autophagy was further confirmed by transmission electron microscopy (TEM) analysis. Quantification of autophagosomes per cellular cross-section revealed a significant reduction upon transfection with miR-20a mimic, but transfection with miR-20a inhibitor increased the number of autophagosomes per cellular cross-section. Moreover, silencing of ATG7 significantly inhibited autophagic response, and transfection with ATG7 siRNA plus miR-20a mimic could further decrease autophagic response. Collectively, our data reveal that miR-20a inhibits autophagic response and promotes BCG survival in macrophages by targeting ATG7 and ATG16L1, which may have implications for a better understanding of pathogenesis of M. tuberculosis infection.

Pulmonary tuberculosis caused by a Mycobacterium infection remains a major public health problem in most part of the world, in part owing to the transmission of its pathogens between hosts including human, domestic and wild animals. To date, molecular mechanisms of the pathogenesis of TB are still incompletely understood. In addition to alveolar macrophages, airway epithelial cells have also been recently recognized as main targets for Mycobacteria infections. In an effort to understand the pathogen-host interaction between Mycobacteria and airway epithelial cells in domestic animals, in present study, we investigated the Toll-like receptor (TLR) signaling in bovine and sheep airway epithelial cells in response to an infection of Mycobacterium tuberculosis avirulent H37Ra stain or Mycobacterium bovis BCG vaccine strain, using primary air-liquid interface (ALI) bronchial epithelial culture models. Our results revealed a host and pathogen species-specific TLR-mediated recognition of pathogen-associated molecular patterns (PAMPs), induction and activation of TLR signaling pathways, and substantial induction of inflammatory response in bronchial epithelial cells in response to Mycobacteria infections between these two species. Interestingly, the activation TLR signaling in bovine bronchial epithelial cells induced by Mycobacteria infection was mainly through a myeloid differentiation factor 88 (MyD88)-independent TLR signaling pathway, while both MyD88-dependent and independent TLR signaling cascades could be induced in sheep epithelial cells. Equally noteworthy, a BCG infection was able to induce both MyD88-dependent and independent signaling in sheep and bovine airway epithelial cells, but more robust inflammatory responses were induced in sheep epithelial cells relative to the bovines; whereas an H37Ra infection displayed an ability to mainly trigger a MyD88-independent TLR signaling cascade in these two host species, and induce a more extent expression of inflammatory cytokines in bovine cells in comparison with that in sheep. These data thus provide an evidence for a host and pathogen species-specific response in bovine and sheep airway epithelial cells in response to Mycobacteria infections, which also suggest there is a need to consider in the interpretation of data generated using a species other than the primary host for analysis of a function role or mechanism of ligands or pathogens. (C) 2016 Elsevier Ltd. All rights reserved.

To explore the potential roles of miRNAs in controlling the survival of mycobacteria in macrophages, miR-17-5p in the regulation of Bacillus Calmette-Guérin(BCG)growth in the macrophage RAW264.7 cells was interrogated. Our results reveal that an infection of BCG shows a time-dependent up-regulation of miR-17-5p in RAW264.7 cells in early phase; importantly, excessive expression of miR-17-5p in these cells exhibits an increased propagation of intracellular BCG. Mechanistically, the Unc-51 like autophagy activating kinase 1 (ULK1), an initial molecular of autophagy are identified as novel target of miR-17-5p, the miR-17-5p is capable of targeting down-regulating the expression of ULK1 protein. In addition, an overexpression of miR-17-5p in RAW264.7 cells is correlated with repression of ULK1 and the autophagosome related proteins LC3I/II. These results imply that miR-17-5p may be able to arrest the maturation of mycobacterial phagosomes in part by targeting ULK1, subsequently reduces the ability of host cells to kill intracellular BCG.

MicroRNAs (miRNAs) have been shown to be important regulators of TLR signaling pathway at the post-transcriptional level. In this study, the potential role of miR-149 was explored in murine alveolar macrophage RAW264.7 cells. Our results demonstrated a dynamic change of the expressions of miR-149 expression and MyD88 in macrophage RAW264.7 upon Mycobacterium bovis Bacillus Calmette-Guerlin (BCG) infection or lipopolysaccharide (LPS) stimulation. The presence of BCG or LPS dynamically reduced the miR-149 expression, along with a substantially striking increase of MyD88 expression in these cells. More importantly, overexpression of miR-149 in RAW264.7 cells was associated with a significant decrease of MyD88 protein expression, as well as a reduced production of inflammatory mediator NF-κB 1, TNF-α and IL-6 in response to BCG infection or LPS stimulation. Further studies using immunoblotting assay against anti-MyD88 antibody and microRNA targeting luciferase reporter assay revealed that miR-149 was able to directly target the 3'-UTR of MyD88 mRNA and post-transcriptionally regulated MyD88 protein expression. These data suggested that miR-149 might be a key player of immune modulator for TLR/MyD88 signaling pathway in macrophages, which may through a mechanism of negatively regulating MyD88-dependent Toll-like receptors signaling pathway.

MicroRNAs (miRNAs) have been demonstrated to play a pivotal role in the regulation of target gene expression at the post-transcriptional level. In order to better understand the role of microRNA-203 (miR-203) in the immunological regulation, the function of miR-203 was explored in the macrophage RAW264.7 cells against lipopolysaccharide (LPS) or Bacille Calmette-Guerin (BCG) stimulation. The results evidenced that myeloid differentiation factor 88 (MyD88) was a novel target of miR-203, miR-203 was capable of directly targeting the 3' untranslated region (3'UTR) of MyD88 and post-transcriptionally down-regulating the expression of protein. In addition, an overexpression of miR-203 in RAW264.7 cells was correlated with repressions of MyD88, as well as its downstream signaling of NF-kappa B (NF-kappa B1),TNF-alpha and IL-6. These results suggest that miR-203 may be an important regulator in macrophages against LPS or mycobacteria infection, which may through a mechanism of negatively regulating MyD88-dependent Toll-like receptors signaling pathway. (c) 2013 Elsevier Ltd. All rights reserved.

C-type lysozyme genes (Lyzls) belong to the class of lysozymes and are highly expressed in the testis and epididymis. The members Lyzl4 and Spaca3 have been reported to play a role in sperm-egg binding and fertilisation in mice. However, the function of the remaining two mouse c-type lysozyme genes, Lyzl1 and Lyzl6, is still not clear. In the present study, we analysed the tissue expression and androgen-dependent expression of mouse c-type lysozyme genes and the possible contribution of human recombinant LYZL6 (rLYZL6) to immunity. The expression of Lyzls was detected by RT-PCR, Western blots, immunohistochemistry and immunofluorescence. The bacteriolytic activity of rLYZL6 was analysed by a colony-forming assay. In mice, the expression of Lyzl genes was mainly in the testis and epididymis in a developmentally regulated manner and androgen- or testicular factor-regulated manner. Immunodetection revealed the presence of LYZL6 protein in primary spermatocytes and round spermatids of the testis and on the post-acrosomal area and midpiece of mature epididymal spermatozoa. The rLYZL6 protein exhibited antibacterial activity. From the results, Lyzls may play a role in mitochondrial function of spermatozoa and LYZL6 may contribute to the innate immunity of the male genital tract.

MicroRNAs (miRNAs) have been demonstrated to play a pivotal role in the regulation of target gene expression at the post-transcriptional level. In order to better understand the role of miRNA in the immunological regulation of macrophages against Mycobacterium bovis BCG infection, we explored the alteration of immune-related miRNA profile in macrophage RAW264.7 cells in response to BCG infection in this study. Our results demonstrated that miR-142-3p was a potential to negatively regulate the production of pro-inflammatory mediators NF-κB (NF-κB1), TNF-α and IL-6 in the macrophages in part through a mechanism of targeting IRAK-1 gene and post-transcriptionally down-regulating IRAK-1 protein expression.

AIM:To construct the recombinant adenoviral vector which can efficiently inhibit mature miR-21 expression and explore its effects and the underlying mechanisms on hepatoma cell line HepG2.METHODS:Based on miRNA-sponge technology, we synthesized 8 duplicated fragments, fully complementary with the miR-21 sequence and cloned them into a shuttle vector pAdTrack-CMV. The constructed plasmid was sequenced and linearized for homologous recombination with pAdEasy-1 vector in BJ5183 bacteria, then transfected into 293A cells. The recombinant adenovirus was used to challenge HepG2 cells. Mature miR-21 level was detected by real-time PCR. Apoptosis and proliferation of the HepG2 cells were detected by Hochest 33258 staining, Western blotting and MTT assay.RESULTS:The restriction enzyme digestion, DNA sequencing and detection of GFP expression demonstrated that recombinant adenoviral vector was constructed successfully. The recombinant adenovirus inhibited the expression of miR-21 in HepG2 cells, and also depressed the proliferation of HepG2 cells and promoted the apoptosis.CONCLUSION:The recombinant adenoviral vector we successfully constructed could efficiently reduce the expression of miR-21 in HepG2 cells and depress the proliferation of HepG2 cells.

Objective To learn about the infection status of 5 common sexually transmitted disease(STD) pathogens of neisseria gonorrhoeae(NGH),chlamydia trachomatis(CT),ureaplasma urealyticum(Uu),herpes simplex virus(HSV-Ⅱ),human papilloma virus(HPV6,11) among male outpatients in our hospital and to provide evidence for the prevention and treatment of these STDs.Methods Samples were taken routinely from 1 460 outpatients to detect NGH,CT,Uu,HSV-Ⅱ and HPV6,11 by FQ-PCR.Results Total positive rate was 20.34%.The positive rates of HPV6,11,HSVⅡ,Uu,CT and NGH were 69.57%,30.00%,24.21%,19.15% and 6.32%.The patients aged 21-40 years accounted for 86.20%.Conclusion HPV6,11 pathogen is the main infectious factor for STD,and should be paid attention to by the whole society.