Exosomal miR-543 derived from umbilical cord mesenchymal stem cells ameliorates endometrial fibrosis in intrauterine adhesion via downregulating N-cadherin.

INTRODUCTION: Human umbilical cord mesenchymal stem cells (UCMSCs) play an important role in repairing the damaged endometrium of intrauterine adhesion (IUA). Meanwhile, exosomes released by UCMSCs can mediate intercellular communication by delivering miRNAs. It has been shown that miR-543 level was reduced in IUA tissues. However, the role of miR-543 in the progression of IUA remains largely unknown. Therefore, we investigated the role of UCMSCs-derived exosomal miR-543 in IUA. METHODS: In this study, human endometrial epithelial cells (hEECs) were treated with TGF-ß1 for mimicking endometrial fibrosis in vitro. In addition, the IUA-like mouse model in vivo was established by a dual damage method of curettage and LPS infection. RESULTS: The level of miR-543 was markedly reduced in hEECs exposed to TGF-ß1 and in endometrium tissues of IUA mice. Additionally, miR-543 could be transferred from UCMSCs to hEECs via exosomes. Meanwhile, exosomal miR-543-derived from UCMSCs significantly reduced the expressions of N-cadherin, α-SMA, fibronectin 1 and elevated the expression of E-cadherin in TGF-ß1-treated hEECs. Furthermore, UCMSCs-derived exosomal miR-543 attenuated IUA-induced endometrial fibrosis in vivo, as shown by the decreased N-cadherin, α-SMA and fibronectin 1 protein expressions. DISCUSSION: Collectively, UCMSCs-derived exosomal miR-543 was able to prevent endometrial fibrosis both in vitro and in vivo via downregulating N-cadherin. These results may provide an insight into the clinical treatment for IUA.

Identification of a novel TP63 mutation causing nonsyndromic cleft lip with or without cleft palate.

BACKGROUND: Cleft lip with or without cleft palate (CL/P) is the most common craniofacial anomaly with a high incidence of live births. The specific pathogenesis of CL/P is still unclear, although plenty of studies have been conducted. Variations of tumor protein 63 (TP63) was reported to be related to the phenotype of CL/P. The case discussed in this report involves a pedigree with mutation at TP63 gene, and the variation was not reported before. CASE PRESENTATION: A Chinese pedigree with CL/P was collected in this study. The proband is a 3-year-old boy with the phenotype of CL/P, while his global development and intelligence are normal. After two CL/P repair operations, he looks almost normal. The proband's uncle and grandmother both have the phenotype of CL/P. Cytogenetic analysis and chromosomal microarray analysis (CMA) were performed, followed by whole exome sequencing (WES) and sanger validation. Analysis of WES revealed a variant of C>T at nucleotide position 1324 (1324C>T) of TP63 gene, possibly producing a truncated protein with a premature stop codon at amino acid position 442 (p.Q442*). This mutation was localized at the oligomerization domain (OD) of TP63 and might impair the capacity of p63 oligomerization. CONCLUSION: The mutation in TP63 was recognized to be the possible cause of the phenotype of CL/P in this pedigree. This report provides some evidence for the clinical diagnosis of CL/P. And our study also provides clinical evidence for the molecular mechanism of TP63 gene causing nonsyndromic cleft lip with or without cleft palate (NSCL/P).

LncRNA-ATB Promotes the Tumorigenesis of Ovarian Cancer via Targeting miR-204-3p.

BACKGROUND: Ovarian cancer ranks fifth among the most prevalent cancer type in females all over the world. It is the second most frequent malignant tumor which accounts for 3% of cancer in females. Therefore, to explore the mechanism of carcinogenesis in ovarian cancer is important to develop new treatment methods. It has been previously found that lncRNA-ATB could promote the tumorigenesis of malignant tumors. However, the role of lncRNA-ATB during the progression of ovarian cancer remains unclear. METHODS: Gene expressions in tissues or cells were detected by using qRT-PCR. Western blot was performed to investigate the protein expressions in ovarian cancer cells. Cell apoptosis was tested by flow cytometry. Moreover, the correction between lncRNA-ATB and miR-204-3p was examined by Dual-luciferase reporter assay and RNA pulldown. Cell proliferation and invasion were detected by CCK-8, Ki-67 staining and transwell assay, respectively. Finally, xenograft mice model was established to confirm the result of in vitro experiments. RESULTS: LncRNA-ATB silencing significantly inhibited the proliferation and induced apoptosis of ovarian cancer cells. In addition, luciferase activity suggested that lncRNA-ATB negatively regulated miR-204-3p in ovarian cancer. Besides, Nidogen 1 (NID1) was the direct target of miR-204-3p. Overexpression of NID1 could notably reverse the inhibitory effect of lncRNA-ATB knockdown on the progression of ovarian cancer. Finally, lncRNA-ATB silencing notably attenuated the severity of ovarian cancer in vivo. CONCLUSION: Downregulation of lncRNA-ATB significantly inhibited the tumorigenesis of ovarian cancer in vitro and in vivo, which may serve as a potential novel target for the treatment of ovarian cancer.

EspR promotes mycobacteria survival in macrophages by inhibiting MyD88 mediated inflammation and apoptosis.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb), leading to about a million deaths each year. EspR is a DNA binding protein of Mtb which regulates expression of multiple genes and the activity of ESX-1 secretion system of the bacteria, with itself being secreted out as a substrate of ESX-1. We explored the function of secreted EspR in host cells by overexpressing the protein in murine macrophage cell line RAW264.7, infecting the cells with BCG which does not secrete EspR, and evaluating the antimicrobial responses of the cells. We found that EspR resulted in an increased intracellular bacteria load in macrophages. This is due to its inhibition on BCG induced expression of inflammatory cytokines and inducible nitric oxide synthase (iNOS), as well as host cell apoptosis. Mechanism study showed that EspR directly interacted with adaptor protein myeloid differentiation factor 88 (MyD88), suppressed MyD88 dependent Toll-like receptor (TLR) and IL-1R signal activation, thus reduced inflammatory responses and apoptosis in macrophages and promoted mycobacteria survival.

UCP2 Mitigates the Loss of Human Spermatozoa Motility by Promoting mROS Elimination.

BACKGROUND/AIMS: To demonstrate the function of uncoupling protein 2 (UCP2) in the regulation of human spermatozoa motility. METHODS: Semen samples were collected from donors with either normal spermatozoa motility (normospermia [NS]) or poor spermatozoa motility (asthenospermia [AS]). UCP2 protein in spermatozoawas quantified by Western blotting. The level of mitochondrial reactive oxygen species (mROS) was evaluated by MitoSOX Red. The activity of mitochondrial membrane potential (MMP) in spermatozoa was evaluated by a JC-1 assay and the ATP level was monitored by a luciferin-luciferase assay. RESULTS: UCP2 was expressed in both NS and AS groups, with the former exhibiting a higher level than the latter. Immunofluorescence analysis shows that UCP2 is mainly located at the mid-region of human spermatozoa. The inhibition of UCP2 by a highly selective inhibitor, Genipin, results in not only impaired spermatozoa mobility (P<.05) but also an elevated level of mROS (P<.05), suggesting that UCP2 is involved in the maintenance of the spermatozoa mobility, which probably is achieved by promoting mROS elimination. Furthermore, H2O2 treatment of spermatozoa increases the mROS level coupled with the loss of spermatozoa mobility. Unexpectedly, this treatment also has a positive impact on the expression of UCP2 within a certain range of supplemental H2O2, indicating the moderate mROS level possibly serves as a feedback signal to stimulate the expression of UCP2. Finally, the treatment of spermatozoa by an ROS scavenger, N-acetyl-l-cysteine (NAC),decreases the level of mROS and increases the curvilinear velocity (VCL) of spermatozoa, but the UCP2 level is not affected. CONCLUSION: These results suggest an UCP2-mROS-motility regulatory system exists for maintaining spermatozoa mobility in humans. In such a system, UCP2 fulfills its function by promoting mROS elimination, and slightly over-produced mROS in turn serves as a signal to stimulates the expression of UCP2. This regulatory system represents a new potential target for the discovery of novel pharmaceuticals for the treatment of patients with low spermatozoa motility.

LpqT improves mycobacteria survival in macrophages by inhibiting TLR2 mediated inflammatory cytokine expression and cell apoptosis.

Tuberculosis is a severe infectious disease caused by Mycobacterium tuberculosis (Mtb). LpqT is a lipoprotein of Mtb identified as a candidate virulence factor by a high-throughput screen searching for genes important for mycobacteria intracellular survival. To investigate its function, we constructed M. smegmatis strains deficient of LpqT or overexpressing LpqT. Wildtype or LpqT modified M. smegmatis strains were used to infect macrophages and mice, and intracellular survival of mycobacteria was measured. We found that LpqT can improve M. smegmatis survival in macrophage cell line, bone marrow derived macrophages (BMDMs), and murine lungs. This survival promoting effect is dependent on TLR2 and Myd88. Western blot analysis of M. smegmatis infected macrophages showed that LpqT suppressed M. smegmatis induced NF-κB and MAPK phosphorylation, indicating that LpqT hampered TLR2 signal activation. In consistent with this, LpqT inhibited M. smegmatis induced inflammatory cytokine expression and cell apoptosis in macrophages, thus supported mycobacteria intracellular survival.

Effects of Folpet, Captan, and Captafol on Human Aromatase in JEG-3 Cells.

BACKGROUND: Estradiol, produced by aromatase (CYP19A1), is very important for reproduction. Folpet, captan, and captafol belong to the phthalimide class of fungicides. They are used to protect the leaves of plants or fruits. They could be endocrine disruptors and may disrupt CYP19A1 activity. METHODS: In the present study, we investigated the effects of folpet, captan, and captafol on estradiol production and human CYP19A1 activity in JEG-3 cells. RESULTS: Folpet, captan, and captafol decreased estradiol production in JEG-3 cells in a concentration-dependent manner. Folpet, captan, and captafol inhibited human CYP19A1 with inhibitory concentration (IC50) values of 3.55, 10.68, and 1.14 µmol/L respectively. These chemicals competitively inhibited human CYP19A1. Molecular docking simulation analysis showed that they tended to bind to the steroid-binding pocket of the CYP19A1. However, the required concentrations may not be relevant to the negligible systemic exposures in humans to these chemicals. CONCLUSION: Folpet, captan, and captafol are potential inhibitors of human CYP19A1.

MptpB Promotes Mycobacteria Survival by Inhibiting the Expression of Inflammatory Mediators and Cell Apoptosis in Macrophages.

Tuberculosis is a severe contagious disease caused by Mycobacterium tuberculosis (Mtb). To develop new vaccines and medicine against TB, there is an urgent need to provide insights into the mechanisms by which Mtb induces tuberculosis. In this study, we found that secreted Mtb virulence factor MptpB significantly enhanced the survival of H37Rv in macrophages. MptpB suppressed the production of iNOS, the expression of inflammatory factors IL-1ß and IL-6, as well as the apoptosis of the macrophage in Mtb infected RAW264.7 cells. Mechanism investigation showed that MptpB simultaneously hampered the NF-κB and MAPK signal pathways, evidenced by its blocking of p65, IKKα, Erk1/2, and p38 phosphorylation induced by Mtb infection. MptpB also inhibited host cell p53 expression. The results demonstrated that MptpB contributed to the survival of H37Rv by inhibiting host inflammatory responses and apoptosis through impeding the NF-κB and MAPK signal pathways and p53 expression in the macrophage.

Geniposide, the component of the Chinese herbal formula Tongluojiunao, protects amyloid-ß peptide (1-42-mediated death of hippocampal neurons via the non-classical estrogen signaling pathway.

Tongluojiunao (TLJN) is an herbal medicine consisting of two main components, geniposide and ginsenoside Rg1. TLJN has been shown to protect primary cultured hippocampal neurons. However, its mechanism of action remains unclear. In the present study, primary cultured hippocampal neurons treated with Aß1-42 (10 µmol/L) significantly increased the release of lactate dehydrogenase, which was markedly reduced by TLJN (2 µL/mL), specifically by the component geniposide (26 µmol/L), but not ginsenoside Rg1 (2.5 µmol/L). The estrogen receptor inhibitor, ICI182780 (1 µmol/L), did not block TLJN- or geniposide-mediated decrease of lactate dehydrogenase under Aß1-42-exposed conditions. However, the phosphatidyl inositol 3-kinase or mitogen-activated protein kinase pathway inhibitor, LY294002 (50 µmol/L) or U0126 (10 µmol/L), respectively blocked the decrease of lactate dehydrogenase mediated by TLJN or geniposide. Therefore, these results suggest that the non-classical estrogen pathway (i.e., phosphatidyl inositol 3-kinase or mitogen-activated protein kinase) is involved in the neuroprotective effect of TLJN, specifically its component, geniposide, against Aß1-42-mediated cell death in primary cultured hippocampal neurons.