Iron-overloaded follicular fluid increases the risk of endometriosis-related infertility by triggering granulosa cell ferroptosis and oocyte dysmaturity.

Endometriosis (EMs) occurs in approximately 50% of women with infertility. The main causes of EMs-related infertility are follicle dysplasia and reduced oocyte quality. Iron overload occurs in ovarian follicular fluid (FF) of patients with EMs, and this condition is associated with oocyte maturation disorder. However, the underlying molecular mechanism remains largely unknown. In the present study, we identified the mechanism underlying ferroptosis in ovarian granulosa cells and oocyte maturation failure in EMs based on a retrospective review of in vitro fertilization/intracytoplasmic sperm injection-frozen embryo transfer outcomes in infertile patients with EMs. Mouse granulosa cells were treated with EMs-related infertile patients' follicular fluid (EMFF) in vitro. Western blot analysis, quantitative polymerase chain reaction, fluorescence staining, and transmission electron microscopy were used to assess granulosa cells ferroptosis. The effects of exosomes were examined by nanoparticle tracking analysis, RNA-seq, and Western blot analysis. Finally, the therapeutic values of vitamin E and iron chelator (deferoxamine mesylate) in vivo were evaluated in an EMs-related infertility model. Patients with ovarian EMs experienced poorer oocyte fertility than patients with non-ovarian EMs. We observed that EMFF with iron overload-induced granulosa cell ferroptosis in vitro and in vivo. Mechanically, nuclear receptor coactivator four-dependent ferritinophagy was involved in this process. Notably, granulosa cells undergoing ferroptosis further suppressed oocyte maturation by releasing exosomes from granulosa cells. In therapeutic studies, vitamin E and iron chelators effectively alleviated EMs-related infertility models. Our study indicates a novel mechanism through which EMFF with iron overload induces ferroptosis of granulosa cells and oocyte dysmaturity in EMs-related infertility, providing a potential therapeutic strategy for EMs-related infertility.

Androgen Excess Induced Mitochondrial Abnormality in Ovarian Granulosa Cells in a Rat Model of Polycystic Ovary Syndrome.

Background: Androgen excess could profoundly lead to follicular dysplasia or atresia, and finally result in polycystic ovary syndrome (PCOS); however, the exact mechanism remains to be fully elucidated. Methods: PCOS model rats were induced by dehydroepiandrosterone, and their fertility was assessed. The ovarian granulosa cells (GCs) from matured follicles of PCOS model rats were collected and identified by immunofluorescence. The mitochondrial ultrastructure was observed by transmission electron microscope and the mitochondrial function was determined by detecting the adenosine triphosphate (ATP) content and mtDNA copy number. Besides, the expressions of respiratory chain complexes and ATP synthases in relation to mitochondrial function were analyzed. Results: The PCOS model rats were successfully induced, and their reproductive outcomes were obviously adverse. The GCs layer of the ovarian was apparently cut down and the mitochondrial ultrastructure of ovarian GCs was distinctly destroyed. The ATP content and mtDNA copy number of ovarian GCs in PCOS model rats were greatly reduced, and the expressions of NDUFB8 and ATP5j were significantly down-regulated without obvious deletion of mtDNA 4834-bp. Conclusions: Androgen excess could damage mitochondrial ultrastructure and function of GCs in rat ovary by down-regulating expression of NDUFB8 and ATP5j in PCOS.

The Mechanism Exploration of Follicular Fluids on Granulose Cell Apoptosis in Endometriosis-Associated Infertility.

OBJECTIVE: To explore the mechanisms of follicular fluids (FFs) on granulose cell (GC) apoptosis in endometriosis-associated infertility. MATERIALS AND METHODS: 60 infertile women were enrolled. The FFs from 30 endometriosis-associated infertility (EI) patients were collected and processed by ELISA hormone assay and proteomic profiling. The ovary GCs collected from 30 tubal-associated infertility (TI) patients were cultured in follicular fluids of endometriosis-associated infertility patients (EI-FFs), and the apoptosis mechanisms were explored by flow cytometry assay, real-time PCR, Western blotting, and protein-protein interaction (PPI) network analysis. RESULTS: Our results showed that the expression of 22 specific proteins was significantly different in the FFs from EI and TI patients, and the level of testosterone and anti-Müllerian hormone was not obviously different between the two groups. EI-FFs could accelerate the apoptosis process of granulose cells of tubal-associated infertility patients (TI-GCs) by regulating the expression of 5 apoptosis-related proteins including BCL2, BAX, CASP3, CASP9, and TP53. The correlation of these 22 specific proteins and 5 apoptosis-related proteins was analyzed by PPI, and 5 protein biomarkers (INS, CXCL10, ICAM1, WIF1, and TNFRSF13C) and 5 signaling pathways (cytokine-cytokine receptor interaction, apoptosis, regulation of actin cytoskeleton, MAPK, and p53 signaling pathway) were predicted. CONCLUSION: This research clarified the effect and explored the mechanisms of EI-FFs on the apoptosis of TI-GCs and indicated the protein biomarkers and signaling pathways for further study.

Aggregative Perivascular Tumor Cell Growth Pattern of Primary Central Nervous System Lymphomas Is Associated with Hypoxia-Related Endoplasmic Reticulum Stress.

Primary central nervous system lymphomas (PCNSLs) often present a unique histopathological feature of aggregative perivascular tumor cells (APVT). Our previous studies showed that patients of PCNSL with APVTs exhibited poor long-term outcomes and increased expression of the endoplasmic reticulum stress (ERS) factor X-box-binding protein (XBP1). However, very little is known about molecular mechanism of the APVT formation in PCNSLs. The aim of this study is to determine if hypoxia-induced ERS is related to the APVT formation in PCNSLs. In this study, cell culture was used to observe the interplay between diffuse large B cell lymphoma (DLBCL) tumor cells and human brain microvascular endothelial cells (HBMECs) in different oxygen conditions. The expression of XBP1, CXCR and CD44 was manipulated by molecular cloning and siRNA technology. Mouse in vivo experiments and clinical studies were conducted to confirm our hypothesis. Our results showed that activated B-cell type-DLBCL cells easily migrated and invaded, and expressed high levels of XBP1 and stromal molecules CXCR4 and CD44 during hypoxia-induced ERS and dithiothreitol unfolded protein response (UPR). The gene upregulation (using overexpression vector) and downregulation (siRNA gene knock-out) in cultured cells and in mouse models further confirmed a close relation of the expression of XBP1, CXCR4, and CD44 with APVT formation, which is coincided with our clinical observation that increased expression of XBP1, CXCR4, and CD44 in the APVT cells in PCNSLs were associated with poor clinical outcomes. The results suggest that hypoxia-induced ERS and UPR might be associated with APVTs formation in PCNSL and its poor clinical outcomes. The results will help us better understand the progression of PCNSL with APVTs feature in daily pathological work and could be valuable for future target treatment of PCNSLs.

Correlation Between Fecal Metabolomics and Gut Microbiota in Obesity and Polycystic Ovary Syndrome.

Objective: This study aimed to explore the relationship between the fecal metabolites and gut microbiota in obese patients with PCOS and provide a new strategy to elucidate the pathological mechanism of obesity and PCOS. Methods: The fecal samples of obese patients with PCOS (n = 18) and obese women without PCOS (n = 15) were analyzed by 16S rRNA gene sequencing and untargeted metabolomics. The peripheral venous blood of all subjects was collected to detect serum sex hormones. The association among fecal metabolites, gut microbiota, and serum sex hormones was analyzed with the R language. Results: A total of 122 named differential fecal metabolites and 18 enrichment KEGG pathways were obtained between the groups. Seven fecal metabolites can be used as characteristic metabolites, including DHEA sulfate. The richness and diversity of gut microbiota in the obese PCOS group were lower than those in the control group. Lachnoclostridium, Fusobacterium, Coprococcus_2, and Tyzzerela 4 were the characteristic genera of the obese patients with PCOS. Serum T level significantly and positively correlated with the abundance of fecal DHEA sulfate (p < 0.05), and serum DHEAS level significantly and negatively correlated with the abundance of fecal teasterone (p < 0.05). Conclusion: Specific fecal metabolites may be used as characteristic metabolites for obese patients with PCOS. The closely relationship among gut microbiota, fecal metabolites, and serum sex hormones may play a role in the related changes caused by hyperandrogenemia.

Transferrin Insufficiency and Iron Overload in Follicular Fluid Contribute to Oocyte Dysmaturity in Infertile Women With Advanced Endometriosis.

Purpose: To screen out specific protein with different concentration in follicular fluid from advanced endometriosis and determine its direct effect on mouse oocytes matured in vitro. Methods: FF samples were obtained from 25 patients (EMS group, n = 15; control group, n = 10) to screen the differential proteins by using iTRAQ Labeling and 2D LC-MS. Transferrin (TRF) in was found significantly decreased in EMS group, which was verified using ELISA in enlarged FF samples (EMS group, n = 31; control group, n = 27). The contents of ferric ion in FFs were detected by ELISA and TRF saturations were calculated in two groups. Germinal vesicle (GV) oocytes of mouse were maturated in vitro interfered with the FFs in five groups, whose concentrations of TRF were modulated, and maturation in vitro rates were compared among groups. Results: The reduced concentration of TRF with three analogs and increased concentration of ferric ion were found in the FF of the EMS group (p < 0.05). The numerical values of TSAT was 54.8% in EMS group, indicating iron overload in the FF. The EMS-FF showed significantly decreased maturation in vitro rate (p < 0.05) of mouse oocytes, which was improved with the supplementation of TRF, compared with the control-FF. The effect was blocked by the TRF antibody (p < 0.05). Conclusions: Being aware of the relatively small sample size, our results possibly suggest that TRF insufficiency and iron overload in FF from advanced EMS contribute to oocytes dysmaturity, which may be a cause of EMS-related infertility.

Characteristic gut microbiota and predicted metabolic functions in women with PCOS.

Polycystic ovary syndrome (PCOS) is a chronic endocrine and metabolic disease. Gut microbiota is closely related to many chronic diseases. In this study, we conducted a cross-sectional study and recruited 30 obese (OG) and 30 non-obese (NG) women with PCOS, 30 healthy women (NC) and 11 healthy but obese women (OC) as controls to investigate the characteristic gut microbiota and its metabolic functions in obese and non-obese patients with PCOS. The blood and non-menstrual faecal samples of all the participants were collected and analysed. As a result, the Hirsutism score, LH/FSH and serum T level in NG and OG both increased significantly compared with their controls (P < 0.05). High-throughput 16S rRNA gene sequencing revealed that the abundance and diversity of the gut microbiota changed in patients with PCOS. The linear discriminant analysis (LDA) indicated that Lactococcus was the characteristic gut microbiota in NG, while Coprococcus_2 in OG. Correlation heatmap analysis revealed that the sex hormones and insulin levels in human serum were closely related to the changes in the gut microbiota of NG and OG. Functional prediction analysis demonstrated that the citrate cycle pathway enriched both in NG and OG, and other 12 gut bacterial metabolic pathways enriched in NG. This study highlighted significant differences in the gut microbiota and predictive functions of obese and non-obese women with PCOS, thereby providing insights into the role and function of the gut microbiota that may contribute to the occurrence and development of PCOS in obese and non-obese women.

Baicalin inhibits recruitment of GATA1 to the HSD3B2 promoter and reverses hyperandrogenism of PCOS.

High androgen levels in patients suffering from polycystic ovary syndrome (PCOS) can be effectively reversed if the herb Scutellaria baicalensis is included in traditional Chinese medicine prescriptions. To characterize the effects of baicalin, extracted from S. baicalensis, on androgen biosynthesis in NCI-H295R cells and on hyperandrogenism in PCOS model rats and to elucidate the underlying mechanisms. The optimum concentration and intervention time for baicalin treatment of NCI-H295R cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and enzyme-linked immunosorbent assays. The functional genes affected by baicalin were studied by gene expression profiling (GEP), and the key genes were identified using a dual luciferase assay, RNA interference technique, and genetic mutations. Besides, hyperandrogenic PCOS model rats were induced and confirmed before and after baicalin intervention. As a result, Baicalin decreased the testosterone concentrations in a dose-and time-dependent manner in NCI-H295R cells. GEP revealed that 3ß-hydroxysteroid dehydrogenase type II (HSD3B2) was the key enzyme of androgen biosynthesis, and baicalin inhibited the expression of HSD3B2 by regulating the binding of transcription factor GATA-binding factor 1 (GATA1) to the HSD3B2 promoter. Hyperandrogenic PCOS model rats treated with baicalin significantly reversed the high androgen levels of serum and the abnormal ovarian status, restored the estrous cyclicity, and decreased the expression of HSD3B2 in ovarian. In summary , our data revealed that GATA1 is an important transcription factor activating the HSD3B2 promoter in steroidogenesis, and baicalin potentially be an effective therapeutic agent for hyperandrogenism in PCOS by inhibiting the recruitment of GATA1 to the HSD3B2 promoter in ovarian tissue.

Targeted therapy and immunotherapy for platinum-refractory advanced ovarian adenosquamous carcinoma: a case report.

BACKGROUND: Ovarian adenosquamous carcinoma is an extremely rare type of ovarian histology. Platinum-refractory disease is also uncommon, but can be fatal because of the lack of available treatment options. To date, there is no study or case report on platinum-refractory ovarian adenosquamous carcinoma or its relevant treatment. CASE PRESENTATION: Herein, we report the case of a 38-year-old Chinese woman with platinum-refractory advanced ovarian adenosquamous carcinoma who received clinical benefit from poly adenosine diphosphate ([ADP] ribose) polymerase and programmed death-1 inhibitors after failure of prior multiline chemotherapies and antiangiogenic agents. The targeted therapy and immunotherapy-controlled disease deterioration and improved performance status. Thus far, the patient has survived longer than 15 months, and she is taking nivolumab as maintenance treatment. CONCLUSION: Targeted therapy and immunotherapy may be options for rare categories of ovarian cancer, but this warrants more clinical evidence of efficacy and toxicity.

Mining and Functional Verification of Calcitonin Genes from the Genome Database.

BACKGROUND: With more countries in the world entering elderly society, osteoporosis is a common disease among the elderly, especially middle-aged and elderly women. Although calcitonin is an effective drug used to treat osteoporosis in clinical practice, it also exists such problems as high cost, short half-life, and high immunogenicity. Therefore, to explore more efficient calcitonin has important clinical significance. OBJECTIVE: Given the emergence of new-generation gene sequencing, numerous genome sequences of marine species have been revealed. This study aimed to identify new, highly active Calcitonins (CTs) from the gene database. METHODS: Candidate CT sequences were obtained by BLAST and analyzed. The evolutionary tree of these sequences was constructed using the Neighbor-Joining method of MEGA 7 software. Secondary structures were analyzed by Circular Dichroism (CD). The biological activities of CTs were estimated using the standard of the rat hypocalcemic activity assay in vivo. The half-life and immunogenicity of CT sequences were determined by ELISA. The physicochemical properties of peptides were analyzed with ProtParam and HeliQuest. RESULTS: A total of 64 candidate CT gene and amino acid sequences from different species were obtained by BLAST using the salmon CT (sCT) sequence as the query sequence. These sequences were clustered to 27 different CT polypeptide sequences, and then the evolutionary tree was constructed. A total of 13 sequences were selected for chemical synthesis and activity assay. Results showed that although their secondary structures were similar, four types of candidate CTs had 30% higher activities than sCT, three other types had similar activities to sCT, and the remaining four types had much lower activities than sCT. Among the three designed CTs, the activities of CT-01 and CT-02 were at least 50% higher than those of sCT. Furthermore, all three CT sequences had a similar half-life to sCT and lower immunogenicity. CONCLUSION: CTs from Monodelphis domestica, Gallus gallus, Ornithorhynchus anatinus, and Carassius auratus had high activities. The exploration and mining of the marine-life genome database can be extremely valuable considering broad application prospect.

Meta-Analysis of Chinese Traditional Medicine Bushen Huoxue Prescription for Endometriosis Treatment.

OBJECTIVES: To evaluate the efficacy and safety of Bushen Huoxue prescription (BSHXP) for endometriosis. METHODS: A meta-analysis was performed, and studies were searched from the seven databases from the date of database establishment to April 30, 2017. Randomized controlled trials (RCTs) that explored the efficacy and safety of BSHXP for patients with endometriosis were included. Two assessors independently reviewed each trial. The Cochrane Risk of Bias assessment tool was used for quality assessment. RESULTS: In the 13 included studies, the total effectiveness rates of BSHXP were higher than those of Western medicine (RR, 1.55; 95% CI, 1.03-2.32; P = 0.04), but the dysmenorrhea alleviation rates of the two treatments did not significantly differ (RR, 1.28; 95% CI, 0.70-2.34; P = 0.42). The pregnancy rates of BSHXP were also higher than those of hormone therapy (RR, 1.99; 95% CI, 1.17-3.39; P = 0.01). However, whether BSHXP is more effective than Western medicine in diminishing endometriotic cyst remains unknown. CONCLUSIONS: Our study provides evidence that BSHXP is effective and safe for endometriosis, but this evidence is inconclusive because of the low methodological quality of the included RCTs. Our findings suggest that BSHXP is an alternative drug for endometriosis, but it should be further examined in future clinical research.

Increased Expression of SETD7 Promotes Cell Proliferation by Regulating Cell Cycle and Indicates Poor Prognosis in Hepatocellular Carcinoma.

PURPOSE: To investigate the role of SET domain containing 7 (SETD7) in hepatocellular carcinoma (HCC) and determine whether SETD7 can be used as a predictor of overall survival in HCC patients. METHODS: mRNAs and proteins of SETD7 and related genes in HCC tumor samples and paired adjacent non-tumorous liver tissues (ANLTs) (n = 20) or culture cells were determined by quantitative real-time PCR and Western blot. Cell proliferation and apoptosis with SETD7 knockdown SMMC-7721 cells or SETD7 overexpressed HepG2 cells were analyzed by CCK8 assay or flow cytometry. Gene expression alterations in SETD7 knockdown of SMMC-7721 cells were determined by digital gene expression (DGE) profiling. Defined data on patients (n = 225) with HCC were retrieved for the further study. Tissue microarrays (TMAs) were performed using paraffin tissues with tumor and ANLTs. SETD7 and related proteins were determined by TMAs immunohistochemistry. Statistical analyses were conducted to associate SETD7 expression with tumor features and patient outcomes, as well as related proteins expression. RESULTS: SETD7 expression was significantly higher in HCC tumor tissues than in ANLTs. SETD7 overexpression in vitro can promote HepG2 cell proliferation, whereas SETD7 knockdown can inhibit SMMC-7721 cell proliferation by regulating the cell cycle. SETD7 expression was significantly correlated with five genes expression. Increased SETD7 is associated with metastasis, recurrence, large tumor size, and poor tumor differentiation, and indicates poor prognosis in HCC patients. CONCLUSIONS: SETD7 plays a critical role in HCC, and its immunohistochemistry signature provides potential clinical significance for personalized prediction of HCC prognosis.

The effects and possible mechanisms of puerarin to treat endometriosis model rats.

Objective. To explore the effects of puerarin to treat endometriosis (EMT) model rats and the possible regulatory mechanisms. Methods. EMT model rats were surgically induced by autotransplantion of endometrial tissues. The appropriate dosage of puerarin to treat EMT model rats was determined by observing the pathologic morphology of ectopic endometrial tissues and by detecting the levels of estradiol (E2) and prostaglandin E2 (PGE2) of both serum and ectopic endometrial tissues. The related genes and proteins of ectopic endometrial tissues were analyzed by Real-time PCR and immunohistochemistry (IHC) to explore the possible mechanisms. Results. Puerarin could reduce the levels of E2 and PGE2 and prevent the growth of ectopic endometrium tissues by inhibiting the expression of aromatase cytochrome P450 (p450arom) and cyclooxygenase-2 (cox-2); puerarin could adjust the anabolism of E2 by upregulating the expression of 17ß-hydroxysteroid-2 (17ß-hsd-2) and downregulating the expression of 17ß-hydroxysteroid-1 (17ß-hsd-1) of the ectopic endometrium tissues; puerarin could increase the expression of ERß and improve the inflammatory microenvironment of EMT model rats. Conclusions. Our data suggest that puerarin has a therapeutic effect on EMT model rats and could be a potential therapeutic agent for the treatment of EMT in clinic.

A decrease in miR-150 regulates the malignancy of pancreatic cancer by targeting c-Myb and MUC4.

OBJECTIVES: Pancreatic cancer is an aggressive cancer with high mortality. Conventional treatments have little impact on its progression. Limited research investigating the role of oncogene miR-150 specifically in pancreatic cancer has been published. The purpose of this study was to determine the tumorigenesis of miR-150 in pancreatic cancer. METHODS: One hundred six pancreatic ductal adenocarcinomas were analyzed together with their adjacent benign pancreatic tissues. The associations of miR-150, c-Myb, and MUC4 expression with survival rates were determined. Functional studies on miR-150 in pancreatic cancer were used to assess its effect on proliferation and malignancy in several pancreatic cell lines. RESULTS: miR-150 expression was significantly down-regulated in pancreatic ductal adenocarcinoma tissues compared with adjacent benign pancreatic tissues. Patients with low miR-150 expression had significantly higher mortality rates than those with high miR-150 expression. The in vitro and in vivo assays of pancreatic cancer cells showed that miR-150 overexpression leads to reduced cell growth, clonogenicity, migration, invasion, modular cell cycles, and induced apoptosis. Moreover, miR-150 expression was inversely correlated with c-Myb and MUC4 activities in pancreatic tissue, cell lines, and nude mouse model. CONCLUSIONS: miR-150 is an important suppressor of pancreatic ductal carcinoma and acts as a regulator of c-Myb and MUC4 in aggressive progress.

Role of JNK Activation and Mitochondrial Bax Translocation in Allicin-Induced Apoptosis in Human Ovarian Cancer SKOV3 Cells.

Background. Allicin, the major component of freshly crushed garlic, is one of the most biologically active compounds of garlic; it has been reported to induce apoptosis in cancer cells; however, the mechanism by which allicin exerts its apoptotic effects is not fully understood. The aim of the present study was to further elucidate the apoptotic pathways induced by allicin in the human ovarian cancer cell line SKOV3. Methods. Cell proliferation and apoptosis were measured by cell-counting assay and flow cytometry analysis. Activation of the signaling pathway was screened by human phospho-kinase array analysis, and the activated pathway and its related proteins were further confirmed by western blot analysis. Results. Allicin induced SKOV3 cell apoptosis and JNK phosphorylation in a time- and dose-dependent manner, but these were significantly blocked by SP600125 (an inhibitor of JNK). The findings suggest that JNK phosphorylation is related to the action of allicin on SKOV3 cells. Furthermore, JNK activation induced Bcl-2 family activation, triggered mitochondria-mediated signaling pathways, and led to the translocation of a considerable amount of Bax and cytochrome c release. Conclusions. JNK activation and mitochondrial Bax translocation are involved in allicin-induced apoptosis in SKOV3 cells. Our data input new insights to the literature of allicin-induced apoptosis.

Cryptotanshinone Reverses Reproductive and Metabolic Disturbances in PCOS Model Rats via Regulating the Expression of CYP17 and AR.

Objective. To explore the effect of Cryptotanshinone on reversing the reproductive and metabolic disturbances in polycystic ovary syndrome (PCOS) model rats and the possible regulatory mechanisms. Methods. PCOS model rats were induced by subcutaneous injection of dehydroepiandrosterone (DHEA) and verified by histological screening of vaginal exfoliated cells. After Cryptotanshinone intervention, the rats' body weight and ovary morphological were observed; the serum biochemical assessments were analyzed by radioimmunoassay (RIA) and key genes and proteins related with anabolism of androgen and insulin were detected by Real-Time PCR and Immunohistochemical (IHC). Results. The estrous cyclicity of PCOS model rats was significantly recovered by Cryptotanshinone. The body weight, ovarian coefficient, and ovarian morphology had been improved and the serum biochemical indicators including testosterone (T), androstenedione (A2), luteinizing hormone (LH), LH/follicle stimulating hormone (FSH), sexual binding globulin (SHBG), low density cholesterol (LDL-C), fasting insulin (FINS) were reversed after Cryptotanshinone intervention. Specifically, the levels of Cytochrome P450, 17-a hydroxylase/17,20 lyase (CYP17), and androgen receptor (AR) were downregulated significantly. Conclusions. Our data suggest that Cryptotanshinone could rebalance reproductive and metabolic disturbances in PCOS model rats and could be a potential therapeutic agent for the treatment of PCOS.

The loss of miR-26a-mediated post-transcriptional regulation of cyclin E2 in pancreatic cancer cell proliferation and decreased patient survival.

BACKGROUND: miR-26a plays a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. However, the function of miR-26a in pancreatic cancer has not been clearly elucidated. The present study was designed to determine the roles of miR-26a in pancreatic cancer and its association with the survival of patients with pancreatic cancer. METHODS: The expression of miR-26a was examined in 15 pairs of pancreatic duct adenocarcinoma (PDAC) and their adjacent benign pancreatic tissues (ABPT), by qRT-PCR. The results were confirmed by in situ hybridization using two panels of 106 PDACs and their ABPT microarray. The association of miR-26a expression with overall survival was determined. The proliferation and cell cycle distribution of Capan-2, SW-1990, and Panc-1 cells, transfected with miR-26a mimics or a miR-26a inhibitor, were assessed using the Cell Counting Kit-8 assay and flow cytometry, respectively. The cell tumorigenicity was evaluated via murine xenograft experiments. Cyclin D2, E2, EZH2, and PCNA levels were analyzed by Western blot and immunohistochemistry. RESULTS: miR-26a was expressed in the cytoplasm of pancreatic ductal epithelial cells, whereas its expression was significantly downregulated in PDAC tissues compared with that of ABPT. Patients with low miR-26a expression had a significantly shorter survival than those with high miR-26a expression. The in vitro and in vivo assays showed that overexpression of miR-26a resulted in cell cycle arrest, inhibited cell proliferation, and decreased tumor growth, which was associated with cyclin E2 downregulation. CONCLUSIONS: miR-26a is an important suppressor of pancreatic ductal carcinoma, and can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.

Puerarin suppresses proliferation of endometriotic stromal cells in part via differential recruitment of nuclear receptor coregulators to estrogen receptor-α.

BACKGROUND AND OBJECTIVES: Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17ß-estradiol and producing an anti-estrogenic effect. In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. This study was to investigate whether puerarin could suppress the proliferation of endometriotic stromal cells (ESCs) and to further elucidate the potential mechanism. METHODS AND RESULTS: The ESCs were successfully established. The effects of puerarin on the proliferation of ESCs, cell cycle and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry. The mRNA and protein levels of cyclin D1 and cdc25A were detected by real-time PCR and Western blot analysis. Coimmunoprecipitation was applied to examine the recruitment of nuclear receptor coregulators to the estrogen receptor-α. We found that puerarin can suppress estrogen-stimulated proliferation partly through down-regulating the transcription of cyclin D1 and cdc25A by promoting the recruitment of corepressors to estrogen receptor-α as well as limiting that of coactivators in ESCs. CONCLUSIONS: Our data suggest that puerarin could suppress the proliferation of ESCs and could be a potential therapeutic agent for the treatment of endometriosis.

Prognostic significance of the aggregative perivascular growth pattern of tumor cells in primary central nervous system diffuse large B-cell lymphoma.

BACKGROUND: Primary central nervous system lymphomas, predominantly diffuse large B-cell lymphomas (PCNS-DLBCL), are aggressive malignancies, and no histopathological variables with independent prognostic value are currently available. The aim of this study is to determine the prognostic value of histopathological variables of PCNS-DLBCL. METHODS: Aggregative perivascular tumor cells (APVTs) and reactive perivascular T cell infiltrates (RPVIs) in tumor samples from 62 immunocompetent patients with PCNS-DLBCL were histopathologically and immunohistochemically studied. A mouse brain DLBCL model was established to confirm the special morphological features of PCNS-DLBCL. The therapy, overall response rate (ORR), and overall survival (OS) among patients were followed up. RESULTS: APVT was present in 54 (87%) of the 62 cases, whereas RPVI was present in 20 (32%). Patients with APVT-positive lesions exhibited significantly worse OS, with intermediate to high International Extranodal Lymphoma Study Group (IELSG) scores, compared with patients with RPVI-positive lesions. Among cases of APVT-positive lymphoma, the semiquantitative score of immunostaining of X-box-binding protein (XBP1) and CD44 demonstrated prognostic significance. Multivariate analysis confirmed independent associations between APVT and XBP1 and between CD44 staining and survival. CONCLUSIONS: The presence of APVT and staining of XBP1 and CD44 are independently associated with survival among patients with PCNS-DLBCL. These features could be routinely assessed in histopathological and immunohistochemical specimens.

Puerarin suppresses proliferation of endometriotic stromal cells partly via the MAPK signaling pathway induced by 17ß-estradiol-BSA.

BACKGROUND: Puerarin is a major isoflavonoid compound extracted from Radix puerariae. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included Radix puerariae. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E(2)-BSA). METHODOLOGY: ESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway. PRINCIPAL FINDINGS: Our data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E(2)). E(2)-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E(2)-BSA. CONCLUSIONS/SIGNIFICANCE: Puerarin suppresses proliferation of ESCs induced by E(2)-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.