Knockdown of ARHGAP30 inhibits ovarian cancer cell proliferation, migration, and invasiveness by suppressing the PI3K/AKT/mTOR signaling pathway.

The mortality and morbidity rates of ovarian cancer (OC) are high, but the underlying mechanisms of OC have not been characterized. In this study, we determined the role of Rho GTPase Activating Protein 30 (ARHGAP30) in OC progression. We measured ARHGAP30 abundance in OC tissue samples and cells using immunohistochemistry (IHC) and RT-qPCR. EdU, transwell, and annexin V/PI apoptosis assays were used to evaluate proliferation, invasiveness, and apoptosis of OC cells, respectively. The results showed that ARHGAP30 was overexpressed in OC tissue samples and cells. Inhibition of ARHGAP30 suppressed growth and metastasis of OC cells, and enhanced apoptosis. Knockdown of ARHGAP30 in OC cells significantly inhibited the PI3K/AKT/mTOR pathway. Treatment with the PI3K/AKT/mTOR pathway inhibitor buparlisib simulated the effects of ARHGAP30 knockdown on growth, invasiveness, and apoptosis of OC cells. Following buparlisib treatment, the expression levels of p-PI3K, p-AKT, and p-mTOR were significantly decreased. Furthermore, buparlisib inhibited the effects of ARHGAP30 upregulation on OC cell growth and invasiveness. In conclusion, ARHGAP30 regulated the PI3K/AKT/mTOR pathway to promote progression of OC.

miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

BACKGROUND: Our previous two-dimensional electrophoresis experiment showed that the expression of LASP1 in patients with endometriosis was significantly higher than that of control endometrium. However, the molecular mechanism by which LASP1 is regulated in endometriosis/adenomyosis is unknown. METHODS: Herein, qPCR was performed to analyze the expression levels of LASP1 and miR-218-5p between endometriosis (Ems) cells and control cells. Fluorescence in situ hybridization was carried out to measure the expression level of miR-218-5p in ectopic endometrium versus normal endometrium. After miR-218-5p mimic or inhibitor were transfected, the transwell experiment was carried out to see the effect of miR-218-5p on the migration of endometrial stromal cells (ESCs). EdU was used to measure cell proliferation rate. Dual-luciferase reporter assay was used to verify the binding of hsa-miR-218-5p to the 3'UTR of LASP1. Western blot and immunofluorescence analysis were carried out to identify the protein expression pattern of LASP1 and EMT markers in endometrial tissue. RESULTS: The miR-218-5p is mainly secreted from blood vessels and expressed in the muscle layer around the endometrium, which inhibits the expression level of LASP1 by binding the 3'UTR region of LASP1 in normal ESCs. Overexpression of miR-218-5p impedes the epithelial-to-mesenchymal transition (EMT) and prevents the migration of ESCs and the expression of Vimentin in Ems. CONCLUSIONS: Our findings revealed that miR-218-5p in endometrial microenvironment prevents the migration of ectopic endometrial stromal cells by inhibiting LASP1.

Aberrant activation of the Hedgehog signaling pathway in granulosa cells from patients with polycystic ovary syndrome.

The molecular mechanism that triggers polycystic ovary syndrome (PCOS) is mysterious. Abnormal development of ovarian granulosa cells (GCs) is one of the causes of PCOS. Herein, our study was carried out using RNA-seq to detect the different gene expression levels in ovarian GCs between three patients with PCOS and four normal controls. To verify the RNA-seq data, GCs from 22 patients with PCOS and 21 controls with normal ovulation were collected to perform the RT-PCR analysis. Hedgehog signaling pathway (Hh) members, Ihh and Ptch2 were abnormally highly expressed in the PCOS tissue (PT). The qPCR also indicated that the expression levels of Hh signaling pathway downstream members, Ptch1, Gli1, and Gli2 in the PT were significantly higher than those in the normal tissue (NT). Besides, the expression of TNF-α mRNA in PCOS patients was higher than that in the control group. Through the chromatin immunoprecipitation assay (ChIP), we found that the Gli1-IP-DNA enriched from the granular cells of PCOS patients was higher than that of the control group. Finally, the Hh signaling pathway inhibitor, cyclopamine, can decrease the apoptosis of PCOS ovarian granulosa cells. These results suggest that abnormal activation of Hh signaling pathway, especially Ihh signal, may have a profound influence on PCOS.

Endometrial stromal cell proteomic analysis reveals LIM and SH3 protein 1 (LASP1) plays important roles in the progression of adenomyosis.

Adenomyosis is one of the most common gynecological disorders that the molecular events underlying its pathogenesis remain not fully understood. Prior studies have shown that endometrial stromal cells (ESCs) played crucial roles in the pathogenesis of adenomyosis. In this study, we utilized two-dimensional gel electrophoresis combined with protein identification by mass spectrometry (2D/MS) proteomics analysis to compare the differential protein expression profile between the paired eutopic and ectopic ESCs (EuESCs and EcESCs) in adenomyosis, and a total of 32 significantly altered protein spots were identified. Among which, the expression of LIM and SH3 protein 1 (LASP1) was increased significantly in EcESCs compared to EuESCs. Immunohistochemical assay showed that LASP1 was overexpressed in the stromal cells of ectopic endometriums compared to eutopic endometriums; further functional analyses revealed that LASP1 overexpression could enhance cell proliferation, migration and invasion of EcESCs. Furthermore, we also showed that the dysregulated expression of LASP1 in EcESCs was associated with DNA hypermethylation in the promoter region of the LASP1 gene. However, the detailed molecular mechanisms of enhancing cell proliferation, invasion and migration caused by upregulated LASP1 in adenomyosis needs further study. For the first time, our data suggested that LASP1 plays important roles in the pathogenesis of adenomyosis, and could serve as a prognostic biomarker of adenomyosis.

Androgen receptor gene mutations in 258 Han Chinese patients with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) affects 8-13% of reproductive-age females worldwide and mutations or aberrant expression of androgen receptor (AR) may cause the onset of this disease. In the present study, 258 samples from Han Chinese patients with PCOS were analyzed for the presence of AR mutations via sequencing of all coding exons of the AR gene. A total of five heterozygous missense mutations, namely p.V3M, p.Q72R, p.S158L, p.S176R and p.G396R, were identified in five of the patients. Among these, p.S158L was a novel mutation that, to the best of our knowledge, has not been reported previously. Although the remaining four mutations have been reported previously, they existed at low frequencies or were absent in the control subjects and in the Exome Aggregation Consortium database. The results of evolutionary conservation and in silico analysis revealed that the p.V3M, p.S158L and p.S176R mutations were pathogenic, whereas the p.Q72R and p.G396R mutations were benign. Compared with the patients with PCOS without AR mutations or with benign AR mutations, markedly lower estrogen levels on the day of human chorionic gonadotropin injection were observed in the three patients with PCOS with potentially pathogenic mutations. In addition, patients with PCOS with pathogenic mutations had lower numbers of oocytes; however, the difference was not statistically significant. Of note, these observations should be interpreted with caution due to the relatively small sample size in the present study. Therefore, a larger number of samples should be collected to validate the results of the present study in future studies. In summary, the present study identified three potential pathogenic mutations in 258 Han Chinese patients with PCOS and these mutations may have an implication in the pathogenesis of PCOS.

FoxM1 promotes the migration of ovarian cancer cell through KRT5 and KRT7.

Forkhead box M1(FoxM1) played an important role in the pathogenesis of ovarian cancer, but its downstream molecular network is mysterious. Here, we combined ChIP-seq with RNA-seq analysis and identified 687 FoxM1-binding regions and 182 genes regulated by FoxM1. The above data pointed out that KRT5 and KRT7 were downstream target genes of FoxM1. Next, we used qPCR and Western blot to verify that FoxM1 knockdown inhibited the expression levels of KRT5 and KRT7. We also demonstrated that FoxM1 regulated KRT5 and KRT7 genes expression through binding a consensus AP-2 cis element, and showed that KRT5 and KRT7 deficiency could prevent the migration but not proliferation of SK-OV-3 cells. Finally, tissue microarray results indicated that KRT5 and KRT7 were highly expressed in ovarian cancer and positively correlated with FoxM1 expression. TCGA database showed that high expression of KRT5 and KRT7 could significantly reduce the survival rate of patients with ovarian cancer. The above results clarify the specific downstream molecular network of FoxM1 to promote the pathogenesis of ovarian cancer, and provide a basis experiment for the judgment of ovarian cancer prognosis and the design of drug targets.

Suppression of PRDX4 inhibits cell proliferation and invasion of ectopic endometrial stromal cells in endometriosis.

Oxidative stress (OS) has been proposed to play a role in the development of EMs. Peroxiredoxins are a family of antioxidant proteins that exhibit peroxidase activity in a thioredoxin-dependent manner, protecting cells against OS. The Western blotting results showed that the relative expression of PRDX4 was significantly increased in ectopic endometria compared with the normal endometria of EMs-free (p < .05). The H2O2 concentration was also significantly higher in the ectopic endometrium. PRDX4 siRNA was transfected into primary ectopic endometrial stromal cells (EESCs). The viability of the transfected EESCs was measured by CCK-8 assay, and the results showed significantly decreased cell viability. Furthermore, the apoptosis rate and ROS generation in flow cytometry assays were significantly increased after the knockdown of PRDX4 expression (p < .05). Scratch assays and transwell assays revealed that decreased expression of PRDX4 mediated by siRNA inhibited EESC migration and invasion. In conclusion, these findings indicate the potential role of PRDX4 in the development of EMs and PRDX4 as a possible therapeutic target for EMs treatment.

Novel MYH8 mutations in 152 Han Chinese samples with ovarian endometriosis.

Endometriosis is a common gynecological disease affecting up to 10% of women at reproductive age. Prior combined studies implied that MYH8 mutations might exist in endometriosis. Here, 152 Han Chinese samples with ovarian endometriosis were analyzed for the presence of MYH8 mutations. Two heterozygous missense mutations in the MYH8 gene, c.1441A > C (p.I481L) and c.4057G > A (p.E1353K), were identified in our samples. These mutations were neither found in public databases nor detected in our 485 Han Chinese control women without endometriosis. The p.I481L-mutated sample belonged to 34-year-old, who had slightly elevated serum CA 125 (42.09 U/mL); while the sample with p.E1353K mutation belonged to 25 years old, who had a markedly increased serum CA125 (89.86 U/mL). The evolutionary conservation analysis results suggested that these MYH8 mutations caused highly conserved amino acid substitutions among vertebrate species. Both the mutations were predicted to be 'disease causing' by MutationTaster and SIFT programs. In addition, no association was observed between MYH8 mutations and the available clinical data. In summary, the present study identified two novel potential pathogenic mutations in the MYH8 gene in samples with ovarian endometriosis for the first time, implying that MYH8 mutations might play a positive role in the pathogenesis of endometriosis.

Regulatory effect of daphnetin on the balance of Th17 and Treg cells in the peripheral blood mononuclear cells from patients with unexplained recurrent pregnancy loss.

INTRODUCTION: There has a close relationship between the balance of T helper 17 (Th17) cells and Foxp3+ regulatory T cells (Treg) and unexplained recurrent pregnancy loss (URPL). The present study is to investigate the regulatory effect of daphnetin, which is extracted from Daphne odora Var, on the balance of Th17 and Treg cells in the peripheral blood mononuclear cells (PBMCs) from patients with URPL. MATERIAL AND METHODS: PBMCs were isolated from 35 pregnant women with URPL and 35 women with normal early pregnancies, respectively and treated with daphnetin for three days. Flow cytometry was performed to measure the proportions of Th17 and Treg cells. The level of expression of IL-6, TGF-ß1 and IL-2 were detected using enzyme-linked immunosorbent assay (ELISA) and the level of expression of FoxP3, RORγt, signal transducers and activators of transcriotion 3 (STAT3) and STAT5 were detected by RT-PCR. RESULTS: The concentrations of Th17-type cytokines IL-2 were significantly decreased in the URPL group after treatment (p < 0.01). Treg-type cytokines such as TGF-ß1 and IL-6 were significantly increased after treatment (p < 0.01). At the same time, daphnetin may induce a decrease in the ratio of RORγt to Foxp3 and a Treg cell bias, which would be beneficial for pregnancy maintenance. Futhermore the expression level of STAT3 were higher in the URPL patients whereas STAT5 were lower than those in the control subjects. CONCLUSIONS: In conclusion, daphnetin may have regulatory effect on the balance of Th17 and Treg cells in the peripheral blood mononuclear cells from patients with URPL.

Metabolomics study of serum and urine samples reveals metabolic pathways and biomarkers associated with pelvic organ prolapse.

Pelvic organ prolapse (POP) is a common medical condition among women and involves complicated diagnostics and controversial surgical management. The exact molecular mechanism underlying POP is poorly understood, especially at the metabolism level. To explore the metabolic mechanism underlying POP and discover potential biomarkers for POP diagnosis, we applied a non-targeted metabolomics approach using ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS). Metabolomics study of serum samples from patients with POP (n = 24) and controls (n = 22) revealed a total of 59 metabolites that are significantly different (VIP ≥ 1 and p ≤ 0.05) between the two groups. Between urine samples from POP patients (n = 45) and controls (n = 59), 33 metabolites differed significantly (VIP ≥ 1 and p ≤ 0.05). Metabolic pathways affected by these differentially expressed metabolites were analyzed. In both serum and urine samples, three pathways including arginine biosynthesis and purine metabolism were found to be significantly related to POP. Six metabolites including GPC, 1-methyladenosine, maleic acid, L-pyroglutamic acid, inosine, and citrate are significantly changed (VIP ≥ 1 and p ≤ 0.05) in both serum and urine samples from patients with POP. Receiver operating characteristics (ROC) curve analysis showed that using these six metabolites as a biomarker could distinguish patients with POP from controls with good accuracy in both serum (AUC = 1) and urine samples (AUC = 0.854). Collectively, these results further extended our understanding of key regulatory metabolic pathways involved in the pathophysiology of POP, as well as provided some promising biomarkers for effective POP diagnosis.

Estrogen receptor 1 mutations in 260 cervical cancer samples from Chinese patients.

Cervical cancer is one of the leading causes of cancer-associated mortality among females; however, the underlying molecular mechanisms of its carcinogenesis remain largely unclear. Previous comprehensive genomic studies have revealed prevalent estrogen receptor 1 (ESR1) mutations in breast cancer, which are rare in certain other types of cancer. To the best of our knowledge, it is unknown whether ESR1 mutations also exist in cervical cancer. Considering the evidence that cervical cancer shares certain genetic aberrations with breast cancer, and that the progression of both breast and cervical cancers can be affected by estrogen, it is possible that cervical cancer may also harbor ESR1 mutations. In the present study, a total of 260 Chinese cervical cancer samples with distinct subtypes were tested for the presence of ESR1 mutations. A total of three heterozygous missense ESR1 mutations, p.K303R (c.908A>G), p.T311M (c.932C>T) and p.Y537C (c.1610A>G), were identified in 3/207 (1.4%) cervical squamous cell carcinoma samples, which were absent in 27 adenosquamous carcinomas and 26 adenocarcinomas samples. Of the three individuals with an ESR1mutation, 1 patient was also diagnosed with ovarian endometriosis and the other 2 patients were diagnosed with a uterine fibroid. A bioinformatics analysis suggested that these ESR1 mutations may be pathogenic by promoting the development of cervical cancer. Furthermore, a previous comprehensive study confirmed that individuals with cervical squamous cell carcinoma possessed ESR1 mutations. These combined studies indicate that ESR1 mutations may participate in the carcinogenesis of cervical squamous cell carcinoma, albeit at a low frequency. In conclusion, the present study identified three potentially pathogenic ESR1 mutations in Chinese cervical squamous cell carcinoma samples, but not in other subtypes.

The Expression of MBD6 Is Associated with Tumor Size in Uterine Leiomyomas.

Background: Uterine leiomyoma (UL) is the most common benign smooth muscle tumor of the uterus in reproductive women. Prior studies indicated that methyl-CpG-binding domain proteins (MBDs) may be involved in the pathogenesis of UL. Materials and Methods: In this study, UL tissues and paired adjacent myometrium were collected from a total of 51 patients. The expression of MBD mRNAs and their cognate proteins were analyzed via quantitative polymerase chain reaction assays and western blotting, respectively. The relationships between the MBD expression levels and the patients' clinicopathologic variables were assessed using Student's t test, nonparametric tests, or Pearson χ2 methods. Results: Our results show that both the mRNA and protein levels of MBD2 were significantly decreased in ULs compared to the adjacent myometrium. In addition, MBD6 protein expression was also decreased significantly in UL samples when compared to the adjacent myometrium. There was, however, no significant difference on the mRNA expression of MBD6 between these two groups. Neither the mRNA nor the protein levels of the other MBD members (MBD1, MBD3, MBD4, MBD5, and MeCP2) showed any significant differences between ULs and the adjacent myometria. The decreased expression of the MBD6 protein was correlated with the tumor size of ULs. Conclusions: These results suggest that the dysregulated expression of MBD2 and MBD6 in ULs may play a role in their development; however, a larger sample size together with cellular functional assays should be carried out to further elucidate the precise role of MBD6 in ULs.

FBXW7 Mutations Promote Cell Proliferation, Migration, and Invasion in Cervical Cancer.

Aim: Cervical cancer is the most common gynecological cancer. Recent studies have revealed that the F-box and WD repeat domain containing 7 (FBXW7) gene, which encodes a subunit of Skp1-Cul1-F-box protein (SCF) ubiquitin ligase, is frequently mutated in cervical squamous cell carcinomas. In this study, we investigated whether Chinese cervical cancer cells also harbor these mutations. Methods: Using PCR and sequencing assays, a total of 190 specimens from Han Chinese patients with cervical cancer were analyzed for FBXW7 mutations. Results: Two FBXW7 mutations (p.R479P and p.L443H), were identified from a study of 145 (1.4%) cervical squamous cell carcinomas. The p.L443H somatic mutation has not been previously reported. Functional assays showed that both of these FBXW7 mutations could promote cell proliferation, migration, and invasion. Conclusion: A low frequency (1.4%) of cervical squamous cell carcinomas were identified with FBXW7 mutations. We did, however, identify a novel FBXW7 mutation. Our results also demonstrated that the identified FBXW7 mutations could promote cell proliferation, migration, and invasion in cervical cancer cells.

Inhibition of formin like 2 promotes the transition of ectopic endometrial stromal cells to epithelial cells in adenomyosis through a MET-like process.

EMT (Epithelial-Mesenchymal Transition) is one of the factors in the pathogenesis of adenomyosis. FMNL2 induced invasion of cancer cell through promoting EMT, but it is unclear the role of FMNL2 in the adenomyosis. By IHC staining, we found the expression level of FMNL2 was significantly higher in the ectopic endometrial stromal cells from women with adenomyosis when compared with normal endometrial stromal cells. Knockdown of FMNL2 inhibited the invasion and migration of ectopic endometrial stromal cells and promoted the protein levels of E-cadherin and Vimentin. Meanwhile, inhibition of FMNL2 could induce the cell membrane localization of E-cadherin. Our findings reveal that the aberrant activation of FMNL2 promotes the pathogenesis of adenomyosis through inducing the EMT process. On the contrary, inhibition of FMNL2 promotes the transition of ectopic endometrial stromal cells to epithelial cells in adenomyosis through a MET-like process.

The mitochondrial DNA 4977-bp deletion and copy number alteration in Han Chinese samples with uterine fibroids.

Uterine fibroids (UFs) are the most common benign neoplasms, but their pathogenesis is not completely understood. Thus far, alterations in the mitochondrial DNA (mtDNA) content and the mtDNA 4977-bp deletion level in UFs, as well as the corresponding nontumorous tissue, have remained elusive. To test whether large mtDNA deletions and mtDNA content are involved in the pathogenesis of UFs, a total of 309 UF tissues and 28 paired adjacent myometrium from 270 UF patients were enrolled for the analysis of large mtDNA deletions and mtDNA content through the use of nested PCR and qPCR techniques, respectively. In our samples, a 4977-bp deletion was identified: 36 out of 309 UF tissues (11.56%) and 15 out of 28 (53.57%) paired adjacent myometrium were detected to harbor the 4977-bp deletion. In addition, a novel 4838-bp mtDNA deletion was identified in three UF tissues, and other different sizes of deleted fragments (4910, 4926, 5135-bp) were also found in UFs for the first time. Furthermore, older age was significantly associated with an mtDNA large deletion in the paired adjacent myometrium. We also found that increased mtDNA content and higher expression of ND1 occurred in solitary fibroids compared to adjacent myometrium. In conclusion, we identified a lower frequency of mtDNA large deletions and some novel large deletion in UFs for the first time. Furthermore, there was a general increase of mtDNA copy number during solitary UF development. Although the definite mechanism by which mtDNA was altered is supposed to be further confirmed, it will be helpful for further studies on the pathological mechanism of UFs.

High-throughput polymer tip-electrospray ionization mass spectrometry for enhanced detection of neopterin and biopterin in clinical urine samples.

Urinary biopterin (Bio) and neopterin (Neo) are important markers for clinical diagnosis of hyperphenylalaninemia. Herein, we developed a high-throughput analysis method based on electrospray ionization mass spectrometry (ESI-MS) with polymer tips for the rapid quantitative detection of Bio and Neo in clinical urine samples. Different polymer tips were investigated. It is found that the best detection sensitivity was achieved with hydrophobic polymer tip, ie, polyethylene tips. The high-throughput polymer tip-ESI-MS method allowed a rapid analysis speed at ~40 seconds per sample. The limits of quantification (LOQ) (S/N ≥ 10) for the detection of Bio and Neo were improved to be 5.0 ng/mL. Acceptable relative standard deviation (RSD) values for Neo and Bio were measured to be 12.2% and 13.4% for direct measurement of Bio and Neo in raw urine samples, respectively. Furthermore, Bio and Neo were directly quantified from 18 clinical urine samples by presented method. The ratios of urinary Bio-to-Neo were analyzed for diagnosis of hyperphenylalaninemia. The results demonstrated that the present polymer tip-ESI-MS method is a promising strategy for the rapid analysis of clinical samples.

Mutation analysis of ZP1, ZP2, ZP3 and ZP4 genes in 152 Han Chinese samples with ovarian endometriosis.

Endometriosis is characterized by the ectopic implant of endometrial tissue outside the uterine cavity and found in ˜35-50% of subfertile women. Previous studies have found that endometriosis had frequent defects in zona pellucida (ZP), and mutations in ZP genes could lead to ZP defects, raising the possibility that mutations in ZP genes might exist in endometriosis. We analyzed a total of 152 Han Chinese samples with ovarian endometriosis for the presence of mutations in the ZP1, ZP2, ZP3 and ZP4 genes. Two novel nonsynonymous ZP4 mutations were identified in three out of 152 (2.0%) samples: a p.M1?/(c.3 G > C) mutation in a 27- and 35-year-old sample, respectively, and a p.A433 V (c.1298C > T) mutation in a 31-year-old patient. No mutations were detected in ZP1, ZP2 or ZP3 genes; furthermore, no mutations in ZP genes were identified in 85 female control samples without endometriosis. The p.M1?/(c.3 G > C) mutation could lead to the usage of a downstream translation initiation site, while the evolutionary conservation and protein structural modeling analyses suggested that the p.A433 V mutation might be functionally important. However, there were strikingly different fertility outcomes among the three samples with ZP4 mutations: the p.A433V-mutated sample had no problem in fertility; while the p.M1?-mutated samples presented with paradoxical effects on fertility: the 35-year-old patient had a child while the 27-year-old patient was infertile, who underwent two spontaneous abortions and an implantation failure after IVF treatment. These results suggested that the potential role of ZP4 mutations on human fertility might be more complex than we thought, and other genetic and environment factors might play a role. In conclusion, we identified two novel mutations in the ZP4 gene in 2.0% of Han Chinese patients with ovarian endometriosis for the first time, our results suggested that mutations in ZP4, but not ZP1, ZP2 and ZP3, might play active roles in the pathogenesis of ovarian endometriosis, despite the mutation-carriers present with complex fertility outcomes.

Analysis of CARD10 and CARD11 somatic mutations in patients with ovarian endometriosis.

Endometriosis is a complex and heterogeneous pre-malignant inflammatory disease harboring multiple gene mutations. Previous studies have suggested that caspase recruitment domain family member (CARD)10 and CARD11 mutations may exist in endometriosis. In the present study, a collection of endometriotic lesions and paired peripheral blood from 101 patients with ovarian endometriosis were obtained, and the entire coding sequences of the CARD10 and CARD11 genes were sequenced. Evolutionary conservation analysis and online prediction programs were applied to analyze the disease-causing potential of the identified mutations. A total of 4 novel somatic mutations were identified in 4 out of the 101 (4.0%) samples: 2 in-frame deletions in CARD10 (c.785_790delAGGAGA, p.K272_E273delKE; c.785_802delAGGAGAAGGAGAAGGAGA, p.K272_V277delKEPDNV) and 2 heterozygous missense mutations in CARD11 (c.49G>T, p.D17Y; c.160G>C, p.E54Q). The sample with CARD10 p.K272_E273delKE deletion was obtained from a 47-year-old patient who was also diagnosed with uterine leiomyoma, while the CARD10 p.K272_V277delKEPDNV-mutated sample was from a 43-year-old patient exhibiting a decreased blood eosinophil granulocyte ratio (0.3%) and an elevated serum creatine kinase level (314 U/l). The patient with the CARD11 p.D17Y mutation was 38 years old and exhibited an increased level of cancer antigen 125 (45.4 U/ml), while the patient with the CARD11 p.E54Q mutation was 46 years old and exhibited no other gynecological conditions. Evolutionary conservation analysis and online prediction programs suggested that these mutations may be disease-causing. In summary, 4 novel somatic mutations in the CARD10 and CARD11 genes were identified from amongst 101 cases of ovarian endometriosis for the first time, these mutations may serve active roles in the development of ovarian endometriosis.

The presence of KRAS, PPP2R1A and ARID1A mutations in 101 Chinese samples with ovarian endometriosis.

Endometriosis is a potential premalignant disorder. The underlying molecular aberrations, however, are not fully understood. A recent exome sequencing study found that 25% (10/39) of deep infiltrating endometriosis harbored cancer driver gene mutations. However, it is unclear whether these mutations also exist in ovarian endometriosis. Here, a total of 101 ovarian endometriosis samples were analyzed for the presence of these gene mutations, including KRAS, PPP2R1A, PIK3CA and ARID1A. In addition, 6 other cancer-associated genes (BRAF, NRAS, HRAS, ERK1, ERK2 and PTEN) were also analyzed. In total, four somatic mutations were identified in three out of 101 ovarian endometriotic lesions (4%, 4/101), including a KRAS p.G12V, a PPP2R1A p.S256F and two ARID1A nonsense mutations (p.Q403* and p.G1926*); while no mutations were identified in the remaining 7 genes (BRAF, NRAS, HRAS, ERK1, ERK2, PTEN and PIK3CA). Note that the KRAS G12V and ARID1A Q403* mutations co-occurred in a 36-year-old sample who had a high serum CA125 (308.4 U/mL) and a late menarche age (18-year-old). Additionally, no mutations in any of the 10 genes were identified in either the healthy eutopic endometrial tissues from 85 control individuals without endometriosis, or in 62 healthy ovarian tissues from ovarian cysts samples (without endometriosis). Our study revealed, for the first time, the presence of classical cancer driver gene mutations in ovarian endometriosis. Furthermore, the co-occurrence of KRAS and ARID1A mutations was identified in a single individual for the first time. The observations of cancer driver gene mutations in our ovarian endometriosis samples, together with several prior observations, further support the notion that endometriosis is a premalignant disorder.

Rapid determination of carbendazim in complex matrices by electrospray ionization mass spectrometry with syringe filter needle.

The determination of pesticide residues is an indispensable task in controlling food safety and environment protection. Carbendazim is one of the extensive uses of pesticides in the agricultural industry. In this study, a simple method utilizing syringe filter has been applied as electrospray ionization emitter for mass spectrometric identification and quantification of carbendazim in complex matrices including soil, natural water, and fruit juice samples, which contain many insoluble materials. With online syringe filter of the complex samples, most of insoluble materials such as soil were excluded in spray ionization process due to the filter effect, and analytes were subsequently sprayed out from syringe needle for mass spectrometric detection. The pore sizes of filters and diameters of syringe needles also were investigated. The analytical performances, including the linear range (1-200 ng·mL-1 ), limit of detection (0.2-0.6 ng·mL-1 , S/N > 3), limit of quantitation (3.5-8.6 ng·mL-1 , S/N > 10), reproducibility (6.4%-12.5%, n = 6), and recoveries (72.1%-91.0%, n = 6) were well acceptable for direct analysis of raw samples. Matrix effect for detection of carbendazim in soil samples also was experimentally investigated. This study demonstrated that syringe filter needle coupled with electrospray ionization mass spectrometry is a simple, efficient, and sensitive method for detection of pesticide residues in water, soil, and fruit juice for risk assessment.