Brown Adipose Transplantation Improves Polycystic Ovary Syndrome-Involved Metabolome Remodeling.

Polycystic ovary syndrome (PCOS) is a complex reproductive, endocrine, and metabolic disorder in reproductive-age women. In order to explore the active metabolites of brown adipose tissue (BAT) transplantation in improving the reproductive and metabolic phenotypes in a PCOS rat model, the metabolites in the recipient's BAT were explored using the liquid chromatography-mass spectrometry technique. In total, 9 upregulated and 13 downregulated metabolites were identified. They were roughly categorized into 12 distinct classes, mainly including glycerophosphoinositols, glycerophosphocholines, and sphingolipids. Ingenuity pathway analysis predicted that these differentially metabolites mainly target the PI3K/AKT, MAPK, and Wnt signaling pathways, which are closely associated with PCOS. Furthermore, one of these differential metabolites, sphingosine belonging to sphingolipids, was randomly selected for further experiments on a human granulosa-like tumor cell line (KGN). It significantly accelerated the apoptosis of KGN cells induced by dihydrotestosterone. Based on these findings, we speculated that metabolome changes are an important process for BAT transplantation in improving PCOS. It might be a novel therapeutic target for PCOS treatment.

mir-21-5p inhibits the progression of human chondrosarcoma by regulating CCR7/STAT3/NF-κB pathway.

Purpose: MicroRNAs (miRNAs or miRs) play an important role in the initiation and development of chondrosarcoma (CS). However, the role of miR-21-5p in CS progression and its underlying molecular mechanisms remains unclear.Materials and Methods: miR-21-5p expression was measured by qRT-PCR. Cell proliferation, migration, and invasion were detected by CCK-8 and Transwell assay. Dual-luciferase reporter assay was used to validate the target of miR-21-5p. Western blot was applied to explore the expressions of CCR7 and EMT biomarkers. Then, the xenograft model was established to confirm the effects of miR-21-5p.Results: miR-21-5p was significantly downregulated in CS tissues and cells and negatively correlated with grade, recurrence, and 5-year overall survival. In vitro, miR-21-5p caused G0/G1 cell cycle arrest and induced apoptosis by decreasing cyclin D1 expression and Bcl-2/Bax ratio. miR-21-5p suppressed cell migration and invasion of CS cells by inhibiting epithelial-mesenchymal transition (EMT). In vivo, miR-21-5p inhibited xenograft tumor formation of SW1353 cells. Mechanistically, miR-21-5p targeted the 3'-UTR of C-C chemokine receptor 7 (CCR7) mRNA to inhibit its expression. Overexpression of CCR7 reversed the inhibitory effects of miR-21-5p on CS cell behaviors. However, the silencing of CCR7 enhanced the inhibitory effects of miR-21-5p on CS cells. Besides, the overexpression of miR-21-5p or silencing of CCR7 obviously reduced the expression levels of p-STAT3, p-p56, and p-IκBα.Conclusion: miR-21-5p targeted CCR7 expression to inhibit the STAT3 and NF-κB signaling, thereby suppressing cell proliferation, migration, invasion, and EMT in CS cells, which might be a novel mechanistic study for CS therapy.Abbreviations: 3'-UTR: 3'-untranslated region; CCR7: C-C chemokine receptor type 7; CS: chondrosarcoma; DMEM: dulbecco's modified eagle's medium; EMT: epithelial-mesenchymal transition; HEK-293T: human embryonic kidney-293T; miR-21-5p: microRNA-21-5p; miR-NC: negative control miRNA; SD: standard deviation; si-CCR7: CCR7 siRNAs.

Changes of miRNA Expression Profiles from Cervical-Vaginal Fluid-Derived Exosomes in Response to HPV16 Infection.

As an oncogenic virus, HPV16 can lead to the dysfunction of cervical epithelial cells and contribute to the progression of cervical cancer. Components from the cervical-vaginal fluid (CVF) could be used as the basis for cervical cancer screening. Exosomes are widely present in various body fluids and participate in intercellular communication via its cargos of proteins, mRNAs, and miRNAs. This study was conducted to explore the changes of miRNAs in exosomes isolated form the cervical-vaginal fluid during HPV16 infection and to predict the potential effects of exosomal miRNAs on the development of cervical cancer. CVF was collected from volunteers with or without HPV16 infection. The exosomes in CVF were identified by electron microscopy. Microarray analysis was subjected to find the differentially expressed miRNAs in CVF exosomes. To confirm the results, 16 miRNAs were randomly selected to go through real-time quantitative polymerase chain reaction. In addition, GO and pathway analyses were conducted to reveal potential functions of differentially expressed miRNAs. A total of 2548 conserved miRNAs were identified in the cervical-vaginal fluid-derived exosomes. In response to HPV16 infection, 45 miRNAs are significantly upregulated and 55 miRNAs are significantly downregulated (P < 0.05). The GO and KEGG pathway analyses revealed that these differentially expressed miRNAs are tightly associated with cervical cancer tumorigenesis, through interaction with the Notch signaling pathway, TNF signaling pathway, and TGF-ß signaling pathway. These results suggest that exosomal miRNAs in CVF are differentially expressed in HPV16 infection patients and HPV16-free volunteers. It provided a novel insight to understand the underlying mechanism of HPV16 infection in regulating cervical cancer progression.

Immunomodulatory significance of natural peptides in mammalians: Promising agents for medical application.

Modulation of immune responses by immunoregulatory agents, such as the natural or synthetic immunomodulatory peptides, has been suggested as a potential strategy to modulate immune system against infection and other immune-related diseases. These compositionally simple peptides have attracted much attention for many drug developers, due to their high activity, low toxicity and clear target specificity. Host defence peptides and milk-derived peptides are two kinds of natural immunomodulatory peptides which have been widely studied in mammalians. They could participate at the interface of innate and adaptive immunity by regulating immune effector cells. This review summarizes the recent advances in host defence peptides and milk-derived peptides as well as their general characteristics, immunomodulatory functions and possible applications.

Androgen upregulates NR4A1 via the TFAP2A and ETS signaling networks.

Hyperandrogenism is one of the clinical and biochemical characteristics of polycystic ovary syndrome (PCOS). Our previous studies confirmed that nuclear receptor subfamily 4 group A member 1 (NR4A1), as a differentially expressed gene in the ovaries of PCOS patients, was upregulated by increased androgen. However, the potential mechanism of NR4A1 upregulation remains unknown. To elucidate the molecular mechanisms involved in NR4A1 regulation, we cloned and characterized the promoter regions of the NR4A1 gene using a series of truncated promoter plasmids in luciferase reporter assays. We identified two unique core promoters of NR4A1 located within the +1055/+1251 and +3183/+3233 regions relative to the transcription start site. TFAP2A downregulated NR4A1 expression, while five ETS transcription factors, ETS1, ELK1, ERG, FLI1 and SPI1, could upregulate NR4A1 promoter activity in HeLa cells. Of these transcription factors, ETS1 and ELK1 were the most effective ones. Moreover, all six transcription factors were confirmed to interact directly with the NR4A1 promoter. In conclusion, this study presents the first description that TFAP2A and ETS family signaling networks are involved in the androgen-mediated transcriptional regulation of NR4A1, which contributes to the understanding of the molecular mechanisms involved in the TFAP2A-NR4A1 and ETS-NR4A1 signaling networks in PCOS.

Profiling Analysis Reveals the Potential Contribution of Peptides to Human Adipocyte Differentiation.

PURPOSE: Peptide drugs provide promising regimes in anti-obesity treatment. In order to identify potential bioactive peptides involved in adipogenesis. EXPERIMENTAL DESIGN: The intracellular peptides between preadipocytes and adipocytes are compared by liquid chromatography/mass spectrometry. The underlying biological function of the identified peptides are evaluated by gene ontology (GO) and pathway analysis of their precursors. To find more potential bioactive peptides, 50 peptide sequences are identified located in the functional domains with the use of the SMART and UniProt databases. Finally, the Open Targets Platform database is used to investigate the precursors related to metabolic diseases. RESULTS: A total of 181 downregulated peptides and 89 upregulated peptides after differentiation (fold change > 1.5 and p-value < 0.05) are identified. The GO and pathway analysis indicate that these differentially expressed peptides play a role in adipogenesis. A total of 10 putative peptides 6 to 26 amino acids in length are identified that might have bioactive effects in adipogenesis and metabolic diseases. CONCLUSIONS AND CLINICAL RELEVANCE: On one hand, present preliminary research provides a better understanding of the intracellular peptides during adipocyte differentiation. On the other hand, it lays a foundation for discovering new peptide drugs in anti-obesity treatment.

Development and identification of Set transgenic mice.

As a multifunctional protein involved in numerous biological processes, Set is expressed in several embryonic and adult organs. Furthermore, Set is overexpressed in numerous types of human cancers, including acute myeloid leukemia, breast cancer and pancreatic cancer. The expression of Set in germ cells is involved in gonad development, and the overexpression of Set has been observed in polycystic ovaries. In order to elucidate the physiological and pathological roles of Set, a Set transgenic mouse model was developed, in which the global overexpression of Set in adult tissues could be induced via the Cre/loxP system with the precise deletion of the Stop fragment in double-transgenic hybrids. This result was then confirmed by genotypical and protein analysis using polymerase chain reaction and bioluminescence imaging. In conclusion, the conditional Set transgenic mice carrying a reporter system were successfully generated. The transgenic mice open a new window for the further investigation of the function of Set using tissue-specific Cre mice and inducible Cre systems.

Analysis of secreted peptidome from omental adipose tissue in polycystic ovarian syndrome patients.

Polycystic ovarian syndrome (PCOS) is a common endocrinopathy associated with increased risk of metabolic disorders. Prevalence of adiposity and obesity is greater in women suffering from PCOS. Moreover, adipose tissue dysfunction has been demonstrated in PCOS patients, particularly in abdominal adipose tissue. This dysfunction likely aggravates the metabolic and reproductive abnormalities. We used liquid chromatography-mass spectrometry to compare the peptides secreted from PCOS and non-PCOS abdominal adipose tissue. We detected 298 upregulated peptides and 31 downregulated peptides (absolute fold change ≥ 2 and p < 0.05). Twenty-nine peptides were only detected in the PCOS group, while 18 were only detected in the control group. In addition, we demonstrate that these cleavage products are not degradation products of the proteasome based on previous studies reported. Gene Ontology enrichment and pathway analysis were performed to study differentially secreted peptides through their precursor proteins. We identified 12 peptides from 10 precursor proteins associated with PCOS, and 6 peptide sequences were located in the functional domains of their corresponding precursor proteins. These results provide a deeper understanding of adipose tissue-derived peptides in PCOS for future functional studies.

Oncogenic Role of SET/I2PP2A for Gynecologic Cancers.

SET (SE translocation, SET) is an evolutionarily conserved gene broadly expressed in various human tissues, especially in the gonadal and neural system. As a multitasking protein, SET is involved in essential cell processes such as histone modification, chromatin remodeling, DNA repair, gene transcription, and androgen synthesis. Recent studies showed that SET is overexpressed in breast cancers, ovary cancers and a variety of other malignancies. The strong correlation between SET expression levels and survival of ovarian cancer patients, and SET-mediated activation of androgen synthesis, strongly indicated that this factor may play a significant role in gynecologic cancers. Here, we summarized data pertaining to the pathological implications of SET in tumorigenesis and cancer progression. We analyzed how SET, through the PP2A-dependent and PP2A-independent pathways, may regulate different cell functions. Potential interactions among these pathways and future studies on SET's oncogenic activities are also discussed.

Pathologic significance of SET/I2PP2A-mediated PP2A and non-PP2A pathways in polycystic ovary syndrome (PCOS).

SET (SE translocation, SET), a constitutive inhibitor of protein phosphatase 2A (PP2A), is a multifunctional oncoprotein involved in DNA replication, histone modification, nucleosome assembly, gene transcription and cell proliferation. It is widely expressed in human tissues including the gonadal system and brain. Intensive studies have shown that overexpressed SET plays an important role in the development of Alzheimer's disease (AD), and may also contribute to the malignant transformation of breast and ovarian cancers. Recent studies indicated that through interaction with PP2A, SET may upregulate androgen biosynthesis and contribute to hyperandrogenism in polycystic ovary syndrome (PCOS) patients. This review article summarizes data concerning the SET expression in ovaries from PCOS and normal women, and analyzes the role/regulatory mechanism of SET for androgen biosynthesis in PCOS, as well as the significance of this action in the development of PCOS. The potential value of SET-triggered pathway as a therapeutic target and the application of anti-SET reagents for treating hyperandrogenism in PCOS patients are also discussed.

Zinc Finger and X-Linked Factor (ZFX) Binds to Human SET Transcript 2 Promoter and Transactivates SET Expression.

SET (SE Translocation) protein carries out multiple functions including those for protein phosphatase 2A (PP2A) inhibition, histone modification, DNA repair, and gene regulation. SET overexpression has been detected in brain neurons of patients suffering Alzheimer's disease, follicle theca cells of Polycystic Ovary Syndrome (PCOS) patients, and ovarian cancer cells, indicating that SET may play a pathological role for these disorders. SET transcript 2, produced by a specific promoter, represents a major transcript variant in different cell types. In this study, we characterized the transcriptional activation of human SET transcript 2 promoter in HeLa cells. Promoter deletion experiments and co-transfection assays indicated that ZFX, the Zinc finger and X-linked transcription factor, was able to transactivate the SET promoter. A proximal promoter region containing four ZFX-binding sites was found to be critical for the ZFX-mediated transactivation. Mutagenesis study indicated that the ZFX-binding site located the closest to the transcription start site accounted for most of the ZFX-mediated transactivity. Manipulation of ZFX levels by overexpression or siRNA knockdown confirmed the significance and specificity of the ZFX-mediated SET promoter activation. Chromatin immunoprecipitation results verified the binding of ZFX to its cognate sites in the SET promoter. These findings have led to identification of ZFX as an upstream factor regulating SET gene expression. More studies are required to define the in vivo significance of this mechanism, and specifically, its implication for several benign and malignant diseases related to SET dysregulation.

Slug signaling is up-regulated by CCL21/CCR7 [corrected] to induce EMT in human chondrosarcoma.

In recent decades, the CXC chemokine receptor 7 (CCR7) [corrected] and its ligand CCL21 have been extensively reported to be associated with tumorigenesis. Meanwhile, Slug signaling induces the epithelial-mesenchymal transition (EMT) process in chondrosarcoma development. In the present study, we explored the functions of CCL21/CCR7 [corrected] in Slug-mediated EMT in the chondrosarcoma. We analyzed protein expression of CCR7 [corrected] and Slug in human chondrosarcoma samples. Effects of CCR7 [corrected] on chondrosarcoma cells were assessed by in vitro assays. Additionally, CCR7 [corrected] pathways were further investigated by pharmacological and genetic approaches. We found that the altered CCR7 [corrected] (81.7 %) and Slug (85.0 %) expression in human chondrosarcoma tissues were significantly associated with grade, recurrence, and 5-year overall survival. According to in vitro assays, CCL21 stimulation induced the expression of phosph-ERK, phosph-AKT, Slug and N-cadherin in SW1353 cells, while the expression of E-cadherin was down-regulated. Furthermore, Slug signaling modulated E- to N-cadherin switch, which was influenced by the kinase inhibitor PD98059 and LY294002. In addition, the genetic silencing of Slug inhibited the capacity of migration and invasion of SW1353 cells. In conclusion, CCL21/CCR7 [corrected] pathway activates ERK and PI3K/AKT signallings to up-regulate Slug pathway, leading to the occurrence of EMT process in human chondrosarcoma. This study lays a new foundation for molecule-targeted therapy of human chondrosarcoma.