IGF2BP3 enhances lipid metabolism in cervical cancer by upregulating the expression of SCD.

Cervical cancer (CC) is the most common gynecologic malignancy, which seriously threatens the health of women. Lipid metabolism is necessary for tumor proliferation and metastasis. However, the molecular mechanism of the relationship between CC and lipid metabolism remains poorly defined. We revealed the expression of IGF2BP3 in CC exceeded adjacent tissues, and was positively associated with tumor stage using human CC tissue microarrays. The Cell Counting Kit-8, colony formation assay, 5-ethynyl-2'-deoxyuridine assay, transwell assays, wound-healing assays, and flow cytometry assessed the role of IGF2BP3 in proliferation and metastasis of CC cells. Besides, exploring the molecular mechanism participating in IGF2BP3-driven lipid metabolism used RNA-seq, which determined SCD as the target of IGF2BP3. Further, lipid droplets, cellular triglyceride (TG) contents, and fatty acids were accessed to discover that IGF2BP3 can enhance lipid metabolism in CC. Moreover, RIP assay and methylated RNA immunoprecipitation experiments seeked the aimed-gene-binding specificity. Lastly, the IGF2BP3 knockdown restrained CC growth and lipid metabolism, after which SCD overexpression rescued the influence in vitro and in vivo using nude mouse tumor-bearing model. Mechanistically, IGF2BP3 regulated SCD mRNA m6A modifications via IGF2BP3-METTL14 complex, thereby enhanced CC proliferation, metastasis, and lipid metabolism. Our study highlights IGF2BP3 plays a crucial role in CC progression and represents a therapeutic latent strategy. It is a potential tactic that blocks the metabolic pathway relevant to IGF2BP3 with the purpose of treating CC.

The traditional uses, phytochemistry, pharmacology and toxicology of Bergenia purparescens: A review comments and suggestions.

Bergenia purpurascens (B. purpurascens, Saxifragaceae) has been used to treat several diseases in different countries, such as lung diseases, stomach problems, rheumatic pains, boosting immunity etc. However, the information on phytochemistry, pharmacology and toxicology of this plant has rarely been comprehensively and critically reported. This paper aims to study and evaluate its therapeutic potential, including the traditional uses and all the latest information of phytochemistry, pharmacology and toxicology. The main components of this plant are phenols compounds and the characteristic substance is bergenin.The results about modern pharmacology have shown that its pharmacological effects include antibacterial, antiviral, cough relieving, anti-inflammatory and so on. In addition, it could inhibit diabetic neuropathy, restore insulin secretion, treat cancer, protect liver and prevent Alzheimer's disease (AD). Thus, its therapeutic fields may be cancer, diabetic and AD in the future. The information will help to further update and study pharmacologic effect and action mechanism of this herb, which is more widely, effectively, and safely used in clinic.

Lactoferrin/Calcium Phosphate-Modified Porous Ti by Biomimetic Mineralization: Effective Infection Prevention and Excellent Osteoinduction.

The surface modification of titanium (Ti) can enhance the osseointegration and antibacterial properties of implants. In this study, we modified porous Ti discs with calcium phosphate (CaP) and different concentrations of Lactoferrin (LF) by biomimetic mineralization and examined their antibacterial effects and osteogenic bioactivity. Firstly, scanning electron microscopy (SEM), the fluorescent tracing method, X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and the releasing kinetics of LF were utilized to characterize the modified Ti surface. Then, the antibacterial properties against S. sanguis and S. aureus were investigated. Finally, in vitro cytological examination was performed, including evaluations of cell adhesion, cell differentiation, extracellular matrix mineralization, and cytotoxicity. The results showed that the porous Ti discs were successfully modified with CaP and LF, and that the LF-M group (200 µg/mL LF in simulated body fluid) could mildly release LF under control. Further, the LF-M group could effectively inhibit the adhesion and proliferation of S. sanguis and S. aureus and enhance the osteogenic differentiation in vitro with a good biocompatibility. Consequently, LF-M-modified Ti may have potential applications in the field of dental implants to promote osseointegration and prevent the occurrence of peri-implantitis.

Analysis of endometrial microbiota in intrauterine adhesion by high-throughput sequencing.

BACKGROUND: Intrauterine adhesions (IUA) arise from scar tissue formation between the endometrial surfaces in response to mechanical or infectious injuries. However, the potential role of endometrial microbiota in IUA remains unclear. We aimed to explore the composition of endometrial microbiota and its potential role in IUA. METHODS: We retrospectively enrolled 46 patients diagnosed with IUA and 21 infertility patients without intrauterine lesions, as control subjects. All cases were diagnosed with hysteroscopy and endometrial tissues were taken from the intrauterine cavity using a hysteroscopic cutting ring without electricity study. After endometrial samples were collected, DNA was extracted and amplified for barcoded Illumina high-throughput next-generation sequencing targeted to the 16S rRNA V4 region for microbiota. Microbiota data were compared between two groups using α-diversity, ß-diversity and Nonmetric Multidimensional Scaling based on Weighted Unifrac distance. RESULTS: At the phyla level, the dominant bacteria included Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. Proteobacteria accounted for more than 64.48%. At the genus level, the proportions of Klebsiella, Shewanella, and Lactobacillus were higher in patients with IUA than in non- IUA participants (20.67% and 8.77%, P=0.006, 13.37% and 4.53%, P=0.175, 12.74% and 6.95%, P=0.882; respectively). The proportion of Acinetobacter was significantly lower in patients with IUA than in non- IUA participants (P=0.005). CONCLUSIONS: Endometrial microbiota differ between patients with IUA and infertility patients without intrauterine lesions, and the potential variation of endometrial microbiota might cause IUA.

si-SNHG5-FOXF2 inhibits TGF-ß1-induced fibrosis in human primary endometrial stromal cells by the Wnt/ß-catenin signalling pathway.

BACKGROUND: Intrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo. METHODS: FOXF2, TGF-ß1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-ß1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/ß-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/ß-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation. RESULTS: FOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10 ng/ml TGF-ß1 for 72 h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-ß1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/ß-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/ß-catenin pathway, thereby altering TGF-ß1-mediated ECM aggregation in endometrial stromal cells ex vivo. CONCLUSIONS: Regulation of the Wnt/ß-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-ß1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-ß1-mediated fibrosis in primary HESCs.

Circular RNA hsa_circ_0003204 promotes cervical cancer cell proliferation, migration, and invasion by regulating MAPK pathway.

Cervical cancer (CC) is the second most common malignancy in women worldwide. The mechanism underlying CC development remains unclear. Recently, Circular RNAs (circRNAs)have attracted attention because of its role in tumorigenesis. To investigate circRNAsin CC, RNA sequencing was employed to characterize circRNA expression profile between CC tissues and matched adjacent normal tissues. The expression of hsa_circ_0003204 was examined in CC tissues and cell lines by real-time PCR. Migration assay and invasion assay were used to verify the effect of hsa_circ_0003204 on migration and invasion ability in CC cell lines. Tumor formation assay in nude mice was used to analyze the effect of hsa_circ_0003204 on the tumorigenicity of CC cell lines in vitro. Western blotting analyzes were performed to investigate the role of hsa_circ_0003204 in the regulation of MAPK signaling activation. We found that circRNA hsa_circ_0003204 was significantly upregulated in CC tissues. The function and potential molecular mechanisms of hsa_circ_0003204 were also investigated in vitro and in vivo. Hsa_circ_0003204 knockdown reduced cell growth, migration, and invasion but promoted cells apoptosis. However, the over-expression of hsa_circ_0003204 had the opposite effect. The MAPK pathway was different in hsa_circ_0003204 over-expression or down-expression cells, compared to parental cells. In addition, over-expression of hsa_circ_0003204 significantly increased tumor volume and tumor weight in vivo.Taken together, results indicated hsa_circ_0003204 may serve as a potential therapeutic target for patients with CC.

Foxf2 and Smad6 co-regulation of collagen 5A2 transcription is involved in the pathogenesis of intrauterine adhesion.

The replacement of normal endometrial epithelium by fibrotic tissue is the pathological feature of intrauterine adhesion (IUA), which is caused by trauma to the basal layer of the endometrium. COL5A2 is a molecular subtype of collagen V that regulates collagen production in fibrotic tissue. Here, we investigated the roles of Foxf2 and Smad6 in regulating the transcription of COL5A2 and their involvement in the pathogenesis of IUA. Small interference-mediated Foxf2 (si-Foxf2) silencing and pcDNA3.1-mediated Smad6 (pcDNA3.1-Smad6) up-regulation were performed in a TGF-ß1-induced human endometrial stromal cell line (HESC) fibrosis model. Assessment of collagen expression by Western blotting, immunofluorescence and qRT-PCR showed that COL5A2, COL1A1 and FN were significantly down-regulated in response to si-Foxf2 and pcDNA3.1-Smad6. Transfection of lentivirus vector-Foxf2 (LV-Foxf2) and pcDNA3.1-Smad6 into HESCs and qRT-PCR showed that Foxf2 promoted COL5A2 expression and Smad6 inhibited Foxf2-induced COL5A2 expression. Co-immunoprecipitation, chromatin immunoprecipitation and dual-luciferase reporter assays to detect the interaction between Foxf2 and Smad6 and their role in COL5A2 transcription showed that Foxf2 interacted with Smad6 and bond the same promoter region of COL5A2. In a rat IUA model, injection of ADV2-Foxf2-1810 and ADV4-Smad6 into the uterine wall showed that Foxf2 down-regulation and Smad6 up-regulation decreased fibrosis and the expression of COL5A2 and COL1A1, as detected by haematoxylin/eosin, Masson trichrome staining and immunohistochemistry. Taken together, these results suggested that Foxf2 interacted with Smad6 and co-regulated COL5A2 transcription in the pathogenesis of IUA, whereas they played opposite roles in fibrosis.

[Analysis of Human Herpes Viruses-Activated Infection Spectra in Patients with Various Immunodeficiencies].

OBJECTIVE: To study the epidemiologic characteristics of human herpes virus (HHV) activated infection in the diseases of blood system and patients received allo-HSCT by statistically analyzing the screening results of 8 human herpes viruses (HHVs) of 4164 patients in Hebei Yanda LU Dao-Pei Hospital from 2012 to 2017. METHODS: PCR was used to screen 8 HHVs. RESULTS: Two thousand and fifty-two patients (49.28%) were HHV-positive among 4164 patients screened. Among these patients screened, the infection spectra of 8 human HHVs in hematological diseases as well as patients received allogeneic hematopoietic stem cell transplantation of totally 2994 patients were summarized as follows: the positive rate of EBV (29.49%) was the highest, that of HCMV (23.15%), HHV-6 was 18.77% and HHV-7 was 17.64%, while the remaining 4 HHVs all≤2.1%. The rate of co-infection of various HHVs was significantly higher than that of single infection of HHV among all these disease groups except familial hemophagocytic lymphohistiocytosis, for which single EBV infection was the most common. The differences of positive rates among these 8 human HHVs in hematological diseases as well as patients received allogeneic hematopoietic stem cell transplantation were statistically significant by Chi-square test of R*C tables (χ2=54.99, P＜0.05). For each HHV, the differences of positive rates among the above-mentioned disease groups were also statistically significant except HHV-8 (P＜0.05). CONCLUSION: The patients with various blood diseases have different activated infection spectra of HHVs. EBV, HCMV, HHV-6 and HHV-7 are most common in HHVs infection. Different HHVs infections correlate with different hematologion diseases.

Aberrantly enhanced melanoma-associated antigen (MAGE)-A3 expression facilitates cervical cancer cell proliferation and metastasis via actuating Wnt signaling pathway.

BACKGROUND: The over-expression of melanoma-associated antigen (MAGE)-A3 in cervical cancer (CC) has been observed in our previous study, suggesting that it possibly take a vital role during the development and metastasis of CC. The present study aimed to investigate the biological function of MAGE-A3 in the progression of CC and explore how it executes its roles. METHODS: The mRNA expression of MAGE-A3 in End1/E6E7 and CC cell lines (HeLa, SiHa and C33A) was measured by real-time quantitative reverse transcription PCR (qRT-PCR). Loss- and gain-of-function methods were used to assess the effect of MAGE-A3 on the proliferative, migratory and invasive abilities of HeLa and SiHa cells. Western blot was performed to measure the expression levels of proteins related to epithelial-mesenchymal transition (EMT) and proteins in the Wnt signaling pathway. In vivo tumorigenesis assay was conducted to evaluate the effect of MAGE-A3 on tumor growth. RESULTS: MAGE-A3 expression was significantly up-regulated in CC cell lines (HeLa, SiHa and C33A) compared with that in End1/E6E7 cell line. Knockdown of MAGE-A3 could significantly suppress migration, invasion and proliferation in HeLa cells; whereas, overexpression of MAGE-A3 in SiHa cells presented the opposite results. Moreover, knockdown of MAGE-A3 presented a suppressive effect on the activation of EMT and Wnt signaling pathway in HeLa cells, whilst up-regulation of MAGE-A3 exhibited the opponent outcomes in SiHa cells. Through in vivo tumorigenesis assay, we further verified that MAGE-A3 acted as a facilitator in tumor growth. CONCLUSIONS: MAGE-A3 is overexpressed in CC cells and possibly facilitates the viability and motility of CC cells via modulating EMT and Wnt signaling. This study implied that MAGE-A3 might be a potential therapeutic target as well as a prognosis predictor for patients with CC.

Sortase A-mediated modification of the Streptococcus mutans transcriptome and virulence traits.

Sortase A contributes to adhesion and biofilm formation of Streptococcus mutans by anchoring surface proteins like P1 onto the cell wall, and few other functional characterization has been annotated to this protein and its coding gene srtA. In this study we investigated that whether srtA deletion would affect S. mutans virulence determinants in addition to adhesion and further explored whether these effects were caused due to changes in S. mutans genomic transcription. We used acid-killing assays, glycolytic rate assessments, and exopolysaccharide (EPS) formation tests to detect whether srtA deletion influenced S. mutans acid tolerance/production and glucan formation. Comparisons between RNA-sequencing data from both the exponential and stationary phases of UA159 and the srtA-deleted strain were made to determine the impact of srtA knockout on S. mutans genomic transcription. Results of our assays indicated that S. mutans aciduricity was enhanced in the srtA deleted strain when bacterial cells were directly subjected to pH 2.8, but the enhancement was repressed when the acid tolerance response was induced in advance. The srtA mutation strain exhibited reduced EPS formation in mature biofilms. SrtA deletion led to pleiotropic changes in the S. mutans transcriptome with a growth phase-dependent pattern. The affected genes mainly included those involved in aciduricity, carbohydrate transport, and EPS formation. It was concluded that S. mutans srtA exhibited multiple effects on the virulence traits of this pathogen, including acid tolerance and glucan formation, and that these alterations could be partially due to transcriptional changes upon loss of srtA.

Exosomal transfer of miR-124 inhibits normal fibroblasts to cancer-associated fibroblasts transition by targeting sphingosine kinase 1 in ovarian cancer.

OBJECTIVE: The interaction between tumor microenvironment and tumor cells plays a key role in tumor progression. However, the mechanisms by which this interaction promotes the transdifferentiation of normal fibroblasts (NFs) to cancer-associated fibroblasts (CAFs) are still unclear. The aim of this study was to investigate whether ovarian cancer (OvCa) cells-derived microRNAs were involved in the transition of resident fibroblasts to CAFs, and in promoting tumorigenesis. METHODS: CAFs and NFs were isolated from the same ovarian site in OvCa and noncancerous prophylactic oophorectomy specimens. The effect of exosomes on the motility of CAFs or NFs was analyzed by wound healing and Transwell assays. The expression of CAFs marker α-smooth muscle actin (α-SMA) and fibroblast activated protein (FAP) were determined by quantitative real-time PCR and Western blotting. A luciferase reporter assay was used to test the interaction between miR-124 and sphingosine kinase 1 (SPHK1). RESULTS: NFs with downregulated miR-124 displayed the characteristics of CAFs, including overexpression of α-SMA and FAP and increased migratory and invasive ability. Overexpression of miR-124 in CAFs reversed some traits of NFs. Human ovarian surface epithelial cells-secreted miR-124 could be transferred via exosomes to CAFs and resulted in decreased α-SMA and FAP expression and attenuated cell motility. Moreover, our finding showed that the expression of SPHK1, a potential target of miR-124, was significantly elevated in CAFs. CONCLUSIONS: The present study provides important and novel perspective into OvCa CAF differentiation and extracellular matrix remodeling, which trigger the tumor progression.

Cross-kingdom interaction of Candida albicans and Actinomyces viscosus elevated cariogenic virulence.

OBJECTIVE: How the interactions between Candida albicans and Actinomyces viscosus contributed to the root caries was not clear. This study aimed to investigate their cross-kingdom interactions on the biomass and the cariogenic virulence in dual-species biofilms. DESIGN: Suspensions of C. albicans and A. viscosus were formed the mono and polymicrobial biofilms in vitro. Crystal violet assay, viable plate count, scanning electron microscopy and fluorescence in situ hybridization were used to analyze the biomass and biofilm structure. Glycolytic pH drop and the spectrophotometric method were used to evaluate the acid production and hydroxyapatite dissolution, respectively. The exopolysaccharide production was measured by the anthrone-sulfuric acid method, while the adhesion force was measured by atomic force microscopy. RESULTS: The biomass and colony-forming units of mixed-species were significantly increased compared to that of the mono-species at 24 h, 48 h, 72 h. The structure of dual-species biofilm had more microcolonies and was much denser. The dual-species biofilms significantly decreased the pH value and damaged the hydroxyapatite compared with the mono-species biofilms at various time points, indicating the strong cariogenic virulence. Moreover, the dual-species biofilms significantly enhanced the exopolysaccharide production and adhesion force suggesting the increase of biofilm adhesion. CONCLUSIONS: Cross-kingdom interactions of C. albicans and A. viscosus significantly elevated the biomass and cariogenic virulence of dual-species biofilm.

[Link between sortase A function and cariogenicity of Streptococcus mutans: a preliminary metabolomics analysis].

OBJECTIVE: This study intends to explore the mechanism underlying the support of sortase A (SrtA) of the cariogenicity of Streptococcus mutans (S. mutans). METHODS: We performed a metabonomics study based on ¹H nuclear magnetic resonance spectroscopy (NMR), in which we compared the extracellular metabolites of wild-type S. mutans UA159 with those of its SrtA-deficient strain. Metabolite differences among strains were identified using a combination of principal component analysis and orthogonality partial least square discriminant analysis. RESULTS: Several differences corresponding mostly to unknown metabolites were identified. Some amino acids such as leucine and valine (δ 0.92×10⁻6-1.20×10⁻6), lactic acid ( δ1.28×10⁻6), oxoglutaric acid (δ 3.00×10⁻6), and glycine (δ 3.60×10⁻6) differed among strains. CONCLUSIONS: This work establishes the feasibility of using ¹H NMR-based metabonomics to provide leads for research into molecular factors that promote caries. The database of microbial metabolites should be also improved in further studies.

Establishment of an miR-137-knockout cell model using CRISPR/Cas9 genome editing.

MicroRNA-137 (miR-137) has been reported to be abnormally expressed in a variety of types of cancer, including ovarian cancer. However, the roles served by miR-137 in cancer are not fully understood. In the present study, 3 single guide RNAs (sgRNAs) were designed, synthesized and inserted into pXPR001 plasmids. The pXPR001-sgRNA plasmids were verified using sequencing and integrated into the genome of the ovarian cancer cell line, A2780, through lentiviral transduction, puromycin selection and single-cell culture. PCR products amplified from single-cell cultures using primers covering miR-137 targeting sites were sequenced to identify clones with miR-137 gene disruption. Genome editing was detected in 72% of the clones derived from sgRNA2, 4% from sgRNA3 and 0% from sgRNA1. Of the clones from sgRNA2, 32% contained 1 edited miR-137 allele and 40% contained 2 edited miR-137 alleles. The expression of miR-137 in clones #2 and #3 could not be detected by reverse transcription-quantitative polymerase chain reaction. In addition, an MTT assay demonstrated that clones #2 and #3 exhibited enhanced proliferation. In conclusion, an miR-137-knockout cell model was successfully established in A2780 cells using CRISPR/Cas9 technology.

Effects of Endodontic Sealers and Irrigation Systems on Smear Layer Removal after Post Space Preparation.

INTRODUCTION: The aim of this study was to investigate the effects of endodontic sealer types and ultrasound on smear layer removal after post space preparation. METHODS: Thirty-six bovine incisors were chemomechanically instrumented and randomly divided into 3 groups (n = 12) according to the endodontic sealer (AH Plus [Dentsply DeTrey, Konstanz, Germany], Apexit Plus [Ivoclar Vivadent AG, Schaan, Fürstentum Liechtenstein], or iRoot SP [Innovative Bioceramix, Vancouver, BC, Canada]) used during root canal obturation, and the groups were further subdivided randomly into 3 subgroups (n = 4) based on the post dowel irrigation systems (ultrasound, regular rinse, or control) used. The samples were examined under a scanning electron microscope and were scored for debris and tubule openings using a 3-scale grading system. The Friedman test, Wilcoxon signed rank test, Kruskal-Wallis analysis, and Mann-Whitney U test were used to statistically analyze the results (α = 0.05). RESULTS: Samples in the AH Plus group were more easily debrided than those in the iRoot SP group (P < .05). The best tubule opening condition was presented in samples in the AH Plus group, whereas those in the iRoot SP group presented the worst (P < .05). The regular rinse and ultrasonic groups were similarly good at smear layer removal and tubule opening (P > .05) compared with the control group (P < .05). The samples using AH Plus in combination with ultrasound or syringe rinsing showed the best cleaning result among all of the subgroups (P < .05). CONCLUSIONS: AH Plus presented the easiest removal from the post space, whereas iRoot SP presented the most difficult removal. Ultrasound improved the cleaning efficacy of post dowels as did the regular rinse.

MicroRNA-16 Promotes Ovarian Granulosa Cell Proliferation and Suppresses Apoptosis Through Targeting PDCD4 in Polycystic Ovarian Syndrome.

BACKGROUND/AIMS: Several miRNAs have been reported to be involved in the pathogenesis of polycystic ovarian syndrome (PCOS). However, the biological roles of miR-16 and its molecular mechanisms in PCOS development remain to be elucidated. METHODS: qRT-PCR was performed to detect the expression levels of miR-16 and programmed cell death protein 4 (PDCD4). GCs proliferation, cell cycle distribution and apoptosis were examined by MTT assay and flow cytometry analysis. Luciferase reporter assay and RIP assay were applied to confirm the regulatory relationship between miR-16 and PDCD4. Western blot was applied to measure the protein levels of PDCD4, PCNA and caspase-3. ELISA kits were used to determine the serum levels of steroids. RESULTS: miR-16 expression was down-regulated in ovarian cortex tissues and serums of PCOS patients. PDCD4 expression was up-regulated in ovarian cortex tissues of PCOS patients. miR-16 overexpression facilitated cell proliferation, induced cell cycle progression, and inhibited apoptosis in GCs. Moreover, PDCD4 was a direct target of miR-16. Also, enforced expression of PDCD4 abated the effects of miR-16 on GCs growth and apoptosis. Additionally, testosterone resulted in a decrease of miR-16 expression and an increase of PDCD4 expression, thus blocking cell growth and enhanced apoptosis in GCs. Furthermore, miR-16 overexpression alleviated PCOS in vivo by regulating PDCD4. CONCLUSIONS: miR-16 promoted ovarian GCs proliferation and inhibited apoptosis through directly targeting PDCD4 in PCOS, contributing to a better understanding of the molecular mechanism of GCs dysregulation and providing a promising target in PCOS.

Effects of human adipose-derived mesenchymal stem cells combined with estrogen on regulatory T cells in patients with premature ovarian insufficiency.

OBJECTIVE: To investigate the effects of human adipose-derived mesenchymal stem cells (hADSCs) combined with estrogen on regulatory T cells (Tregs) in patients with premature ovarian insufficiency (POI). METHODS: hADSCs were isolated by enzymatic digestion and identified by flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated from POI patients and healthy controls. PBMCs were cultured in the following experimental groups: the control group, hADSC group, estrogen group and combined group. The PBMCs in the hADSC group were co-cultured with hADSCs at concentrations of 1×104, 2×104, or 1×105 cells/well, and the estrogen group was co-cultured with 10-9, 10-8, or 10-7mol/L 17ß-estradiol. Cell proliferation was measured with the CCK-8 assay. The percentage of CD4+ CD25+ Foxp3+ Tregs was measured by flow cytometry. The expression levels of Foxp3, TGF-ß1 and IFN-γ were measured by real-time PCR. RESULTS: Treatment with hADSCs, estrogen and their combination promoted Tregs differentiation of PBMCs from POI patients and healthy controls. An increase in the percentage of CD4+ CD25+ Foxp3+ Tregs was observed when PBMCs were co-cultured with hADSCs, 17ß-estradiol and their combination. Foxp3 and TGF-ß1 mRNA expression was higher and IFN-γ mRNA expression was lower in the hADSCs, estrogen and combined groups than in the control group. CONCLUSION: Combined treatment with hADSCs and estrogen played an immunomodulatory role by promoting Tregs proliferation, thereby potentially improving impaired ovarian function.

MicroRNA-17 downregulates expression of the PTEN gene to promote the occurrence and development of adenomyosis.

The aim of the current study was to evaluate the expression of microRNA (miR)-17 in the endometrial tissues of patients with adenomyosis (AM) and determine its biological function in the occurrence and development of the disease. A total of 45 fresh endometrial tissues of AM patients and 32 normal endometrial tissues were collected from healthy controls. The expression of miR-17 was evaluated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The miR-17-targeting gene phosphatase and tensin homolog (PTEN) was predicted using bioinformatics and its expression was evaluated with RT-qPCR and western blot analysis. Endometrial cells were isolated from patients with AM and healthy controls. They were cultured in vitro and transfected with antagomiR-17 to downregulate miR-17 expression, subsequently cell viability and apoptosis were measured using MTT and flow cytometry. The expression of PTEN and cell cycle- and apoptosis-related proteins were evaluated using western blot analysis. Endometrial cells that stably overexpressed PTEN were screened in vitro by co-culture with G418. A dual-luciferase reporter assay was conducted to verify whether miR-17 was directly bound to PTEN mRNA. The results demonstrated that expression of miR-17 was significantly increased in the endometrial tissues of patients with AM compared with control patients (P<0.05). PTEN mRNA and protein expression were significantly lower in the AM group compared with the control group (P<0.05). When the expression of miR-17 in the cells was downregulated, the expression of PTEN was significantly increased (P<0.05). In addition, expression of Bcl-2 protein was significantly decreased and that of Bax protein significantly increased compared with the negative control (both P<0.05). The expression of cyclins E1 and D1 were also significantly downregulated (P<0.05). When PTEN was overexpressed or miR-17 was downregulated, the viability of endometrial cells significantly decreased and cell apoptosis significantly increased (all P<0.05). A dual-luciferase reporter assay indicated that miR-17 could directly bind to the PTEN mRNA 3'-untranslated region to regulate its expression. Thus the current study indicates that expression of miR-17 was increased in the endometrial tissues of patients with AM and may influence cell apoptosis and cyclin expression through the targeted regulation of PTEN. These results suggest that miR-17 promotes the occurrence and development of AM.

Overexpression of miR-21 in stem cells improves ovarian structure and function in rats with chemotherapy-induced ovarian damage by targeting PDCD4 and PTEN to inhibit granulosa cell apoptosis.

BACKGROUND: Chemotherapy-induced premature ovarian failure (POF) is a severe complication affecting tumor patients at a childbearing age. Mesenchymal stem cells (MSCs) can partially restore the ovarian structure and function damaged by chemotherapy. miR-21 is a microRNA that can regulate cell apoptosis. This study discusses the repair effect and mechanism of MSCs overexpressing miR-21 on chemotherapy-induced POF. METHODS: Rat MSCs and granulosa cells (GCs) were isolated in vitro. MSCs were transfected with miR-21 lentiviral vector (LV-miR-21) to obtain MSCs stably expressing miR-21 (miR-21-MSCs). The microenvironment of an ovary receiving chemotherapy was mimicked by adding phosphamide mustard (PM) into the cellular culture medium. The apoptosis rate and the mRNA and protein expression of target genes PTEN and PDCD4 were detected in MSCs. Apoptosis was induced by adding PM into the culture medium for GCs, which were cocultured with miR-21-MSCs. The apoptosis rate and the mRNA and protein expression of PTEN and PDCD4 were detected. The chemotherapy-induced POF model was built into rats by intraperitoneal cyclophosphamide injection. miR-21-MSCs were transplanted into the bilateral ovary. The rats were sacrificed at 15, 30, 45, and 60 days after the last injection. The ovarian weights, follicle count, estrous cycle, and sex hormone levels (estradiol (E2) and follicle-stimulating hormone (FSH)) were detected. Apoptosis of GCs was determined by TUNEL assay. The miR-21 and mRNA and protein expression of PTEN and PDCD4 were determined. RESULTS: The apoptosis decreased in MSCs transfected with miR-21. The mRNA and protein expression of target genes PTEN and PDCD4 was downregulated. GCs cocultured with miR-21-MSCs showed a decreased apoptosis, an upregulation of miR-21, and a downregulation of PTEN and PDCD4. Following the injection of miR-21-MSCs, the ovarian weight and follicle counts increased; E2 levels increased while FSH levels decreased, with less severe apoptosis of GCs. The miR-21 expression in the ovaries was upregulated, while the mRNA expression and protein expression of PTEN and PDCD4 were downregulated. CONCLUSIONS: Overexpression of miR-21 in MSCs promoted efficacy against chemotherapy-induced POF and its improvement of the repair effect was related to the inhibition of GC apoptosis by targeting PTEN and PDCD4.

CPAF selectively degrades chlamydial T cell antigens for inhibiting antigen presentation.

INTRODUCTION: Chlamydia trachomatis is the leading cause of sexually transmitted bacterial disease, which may cause significant threats, such as pelvic inflammatory disease and tubal factor infertility, to women if untreated. The pathological mechanisms of chlamydia-induced disease remain largely unknown, but it has been proposed that CPAF, a chlamydia-secreted serine protease, may play major roles in aiding chlamydial infection and contribute to chlamydia pathogenesis during in vivo infection. According to previous results, CPAF targets host immunity by degrading antimicrobial peptides and neutralizing complement activity; however, whether CPAF is involved in chlamydial antigen presentation has never been reported. METHODOLOGY: Antigen presentation assay was used to monitor the effects of CPAF on OT1-, OT2-, and chlamydia T cell antigen-mediated antigen presentation. In vitro cell-free degradation assay was used to detect CPAF processing of chlamydia T cell antigens. RESULTS: We found that CPAF preferably inhibits OT2- but not OT1-mediated antigen presentation. CPAF inhibits OT2 antigen presentation by direct proteolytic cleavage in the wild type CPAF, but not enzymatic mutants. Importantly, several previously identified chlamydial T cell antigens were selectively degraded by CPAF when co-incubated in vitro. In addition, specific inhibition T cell antigen presentation by CPAF was correlated with T cell antigen cleavage by CPAF in vitro assay. CONCLUSIONS: Our experiments demonstrated that CPAF selectively and specifically degrades chlamydial T cell antigens, which chlamydia may utilize as a novel mechanism for evading host immune responses to promote chlamydia survival.