Mechanism of Jinteng Qingbi granules in the treatment of rheumatoid arthritis using metabolomics analysis.

This study investigated the differential metabolites after rheumatoid arthritis (RA) rats were treated with Jinteng Qingbi granules. Collagen-induced arthritis rats were divided into three groups, namely normal group, model group, and Jinteng Qingbi granules group. Serum compounds were identified, annotated, and classified using metabolomics to explain the physicochemical properties and biological functions. The metabolites were screened using univariate and multivariate statistical analyses. There were differences in serum metabolites between RA and normal rats; Jinteng Qingbi granules improved RA and recovered the metabolite levels to normal. Compared to the normal group, 51 differential ions were screened, and 108 ions were changed in the Jinteng Qingbi granules group compared to the RA model. Eight metabolites were upregulated in the RA model group compared to the normal group, whereas 10 metabolites were downregulated. Treatment with Jinteng Qingbi granules increased the levels of 12 metabolites such as cinnamate and decreased the levels of 16 metabolites such as allamandin in the RA model. Differential ion enrichment was mainly related to the histidine metabolic pathway in amino acid metabolism. Jinteng Qingbi granules resulted in improvements in the RA model, which were mainly associated with lipids and lipid-like molecules, organic acids, and derivatives, providing a new possibility and basis for screening biomarkers for the diagnosis and treatment of RA.

Tripterygium glycosides for safely controlling disease activity in systemic lupus erythematosus: a systematic review with meta-analysis and trial sequential analysis.

Background: Tripterygium glycosides have been used to treat systemic lupus erythematosus (SLE) for a long time, showing the effects of immune regulation. We aimed to evaluate the benefits and risks of Tripterygium Glycosides Tablets (TGT) for patients with SLE. Methods: We searched electronic databases and clinical trial registries for relevant randomized controlled trials (RCTs). We identified eligible RCTs and assessed risk of bias. We conducted a meta-analysis to estimate the pooled effects. The Trial Sequential Analysis (TSA) 0.9.5.10 software was used to verify the reliability of the results. Results: Eight RCTs encompassing 538 patients with SLE were included. TGT combined with conventional treatments (CTs) was superior to CTs alone in reducing lupus activity (MD = -1.66, 95% CI = -2.07 to -1.26, p < 0.00001, low-certainty evidence) and improving overall response rate (ORR) (RR = 1.21, 95% CI = 1.11 to 1.32, p < 0.0001, moderate-certainty evidence). The robustness of the results was confirmed by TSA. Regarding safety, there was no statistical difference in the overall incidence of adverse reactions between the two groups. Conclusion: In patients with SLE, TGT might safely reduce disease activity. However, further high-quality studies are needed to firmly establish the clinical efficacy of TGT. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022300474; Identifier: CRD42022300474.

Insights into the Degradation Mechanisms of TCH by Magnetic Fe3S4/Cu2O Composite.

Unique Fe3S4/Cu2O composites were constructed with high Fenton-like photocatalytic activity through the impregnation coprecipitation method. The structure, morphology, optical, magnetic, and photocatalytic properties of the as-prepared composites were explored in detail. The findings suggest that small Cu2O particles were grown on the surface of Fe3S4. The removal efficiency of TCH by Fe3S4/Cu2O was 65.7, 4.75, and 3.67 times higher than that of pure Fe3S4, Cu2O, and the Fe3S4 + Cu2O mixture, respectively, when the mass ratio of Fe3S4 and Cu2O was 1:1 at pH 7.2. The synergistic effect between Cu2O and Fe3S4 was the main factor for TCH degradation. The Cu+ species from Cu2O increased the Fe3+/Fe2+ cycle during the Fenton reaction. •O2- and h+ were the main active radicals; however, •OH and e- played the second role in the photocatalytic degradation reaction. Moreover, the Fe3S4/Cu2O composite retained good recyclability and versatility, and could be conveniently separated by a magnet.

Evaluation of second-line apatinib plus irinotecan as a treatment for advanced gastric adenocarcinoma or gastroesophageal conjunction adenocarcinoma: a prospective, multicenter phase II trial.

Objective: Apatinib and irinotecan are used as systematic therapies for advanced gastric adenocarcinoma (GAC) and gastroesophageal junction adenocarcinoma (GEJA), while the evidence for their combination as second-line therapy in these patients is limited. This study aimed to evaluate the efficacy and safety of second-line apatinib plus irinotecan for the treatment of GAC and GEJA. Methods: In this prospective, multicenter phase II clinical study, 28 patients with advanced GAC or GEJA who received second-line apatinib plus irinotecan were recruited. Results: In total, 1 (3.6%) patient achieved complete response, 7 (25.0%) patients achieved partial response, 13 (46.4%) patients had stable disease, and 4 (14.3%) patients showed progressive disease, while clinical response was not evaluable or not assessed in 3 (10.7%) patients. The objective response rate and disease control rate were 28.6% and 75.0%, respectively. Meanwhile, the median (95% confidence interval (CI)) progression-free survival (PFS) was 4.5 (3.9-5.1) months, and the median (95% CI) overall survival (OS) was 11.3 (7.4-15.1) months. By multivariate Cox regression analysis, male sex, liver metastasis, and peritoneal metastasis were independently associated with worse PFS or OS, while treatment duration ≥5 months was independently associated with better OS. In terms of the safety profile, 89.3% of patients experienced treatment-emergent adverse events of any grade, among which 82.1% of patients had grade 1-2 adverse events and 64.3% of patients had grade 3-4 adverse events. Conclusion: Apatinib plus irinotecan as second-line therapy achieves a good treatment response and satisfactory survival with tolerable safety in patients with advanced GAC or GEJA.

Identification of MKNK1 and TOP3A as ovarian endometriosis risk-associated genes using integrative genomic analyses and functional experiments.

The risk of endometriosis (EM), which is a common complex gynaecological disease, is related to genetic predisposition. However, it is unclear how genetic variants confer the risk of EM. Here, via Sherlock integrative analysis, we combined large-scale genome-wide association studies (GWAS) summary statistics on EM (N = 245,494) with a blood-based eQTL dataset (N = 1490) to identify EM risk-related genes. For validation, we leveraged two independent eQTL datasets (N = 769) for integration with the GWAS data. Thus, we prioritised 14 genes, including GIMAP4, TOP3A, and NMNAT3, which showed significant association with susceptibility to EM. We also utilised two independent methods, Multi-marker Analysis of GenoMic Annotation and S-PrediXcan, to further validate the EM risk-associated genes. Moreover, protein-protein interaction network analysis showed the 14 genes were functionally connected. Functional enrichment analyses further demonstrated that these genes were significantly enriched in metabolic and immune-related pathways. Differential gene expression analysis showed that in peripheral blood samples from patients with ovarian EM, TOP3A, MKNK1, SIPA1L2, and NUCB1 were significantly upregulated, while HOXB2, GIMAP5, and MGMT were significantly downregulated compared with their expression levels in samples from the controls. Immunohistochemistry further confirmed the increased expression levels of MKNK1 and TOP3A in the ectopic and eutopic endometrium compared to normal endometrium, while HOBX2 was downregulated in the endometrium of women with ovarian EM. Finally, in ex vivo functional experiments, MKNK1 knockdown inhibited ectopic endometrial stromal cells (EESCs) migration and invasion. TOP3A knockdown inhibited EESCs proliferation, migration, and invasion, while promoting their apoptosis. Convergent lines of evidence suggested that MKNK1 and TOP3A are novel EM risk-related genes.

Efficacy and Safety of Pyrotinib in Human Epidermal Growth Factor Receptor 2-Positive Advanced Breast Cancer: A Multicenter, Retrospective, Real-World Study.

Purpose: Pyrotinib, a novel human epidermal growth factor receptor 2 (HER2)-targeted tyrosine kinase inhibitor (TKI), has led to remarkable survival outcomes in HER2-positive advanced breast cancer (ABC) in clinical trials and was approved for second-line standards of treatment for HER2+ ABC in China. However, the clinical trials could not fully reflect reality of clinical practice, and predictive factors were still lacking. This study aimed to assess the actual efficacy and safety of pyrotinib in HER2+ ABC in real-world setting. Patients and Methods: In this multicenter, retrospective, observational real-world study, we analyzed 171 patients with HER2+ ABC, who received pyrotinib-based treatment from November 2017 to November 2020. The primary end point was progression-free survival (PFS). Secondary end points included overall survival (OS), objective response rate (ORR), clinical benefit rate (CBR) and safety. Results: Up to November 30, 2021, the median PFS (mPFS) was 12.0 months for all patients. One hundred and sixty-two patients (94.7%) with measurable lesions had been included in efficacy assessment. The ORR and CBR were 45.1% and 81.5%, respectively. A significantly longer PFS was reported in patients who received pyrotinib as first-line treatment, had the ECOG-PS of 0-1, as well as those who were lapatinib-naive. In addition, multivariable analysis indicated that ECOG-PS of 2-4, positive hormone receptor (HR) status, and presence of visceral metastasis were independent negative predictors of PFS. As far as we know, this study first reported the survival outcome of pyrotinib cross-line treatment, with a mPFS of 5.0 months. All grades of adverse events (AEs) occurred in 171 patients (100%), and the most common AE was diarrhea (86.5%). Conclusion: This study further demonstrated the outstanding efficacy and safety of pyrotinib and reported the potential predictors of survival in HER2+ ABC.

PGAM1 Promotes Glycolytic Metabolism and Paclitaxel Resistance via Pyruvic Acid Production in Ovarian Cancer Cells.

BACKGROUND: Enhanced glycolysis occurs in most human cancer cells and is related to chemoresistance. However, detailed mechanisms remain vague. METHODS: Using proteinomics analysis, we found that the glycolytic enzyme Phosphoglycerate mutase 1 (PGAM1) was highly expressed in the paclitaxel-resistant ovarian cancer cell line SKOV3-TR30, as compared to its parental cell line SKOV3. Cell Counting Kit-8 proliferation experiment, plasmids and siRNA transfection, pyruvic acid and lactic acid production detection, immunofluorescence staining of functional mitochondria and oxygen consumption rate and extracellular acidification rate measurement were uesd to assess the glycolytic metabolism and paclitaxel resistance in ovarian cancer cells. The expression and prognostic effect of PGAM1 in 180 ovarian cancer patients were analyzed. RESULTS: SKOV3-TR30 cells display higher glycolytic flux and lower mitochondrial function than SKOV3 cells. Down-regulation of PGAM1 in SKOV3-TR30 cells resulted in decreased paclitaxel resistance. Up-regulation of PGAM1 in SKOV3 cells led to enhanced paclitaxel resistance. Analysis of the glycolytic flux revealed that PGAM1-mediated pyruvic acid or lactic acid production could modulate the capabilities of ovarian cancer cell resistance to paclitaxel. Our data also show high expression of PGAM1 as significantly correlated with reduced overall survival and reduced progression free survival in ovarian cancer patients. CONCLUSIONS: PGAM1 acts to promote paclitaxel resistance via pyruvic acid and/or lactate production in ovarian cancer cells. Inhibiting PGAM1 may provide a new approach to favorably alter paclitaxel resistance in ovarian cancer.

Inhibition of myeloid-derived suppressive cell function with all-trans retinoic acid enhanced anti-PD-L1 efficacy in cervical cancer.

PD-1/PD-L1 inhibitor treatments are relatively inefficacious in advanced cervical cancer patients. The presence of myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment may be one significant barrier to efficacy. It has been shown that all-trans retinoic acid (ATRA) can differentiate MDSCs into mature myeloid cells. However, whether ATRA suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. Here, the frequency of tumor-infiltrating MDSCs in cervical cancer patients was measured. ATRA was used to target MDSCs both in vitro and in tumor-bearing mice. The impact of ATRA on the human cell line HeLa was also investigated. The frequency of MDSCs and T cells was determined by flow cytometry. The expression of immunosuppressive genes was measured with quantitative real time-PCR and infiltration of immune cells was assessed by immunohistochemical examination. We found that tumor-infiltrating PD-L1+ MDSCs were more prevalent in cervical cancer patients. Blockade of PD-L1 expression in MDSCs with anti-PD-L1 antibody cannot relieve the suppressive activity of MDSCs induced by HeLa cells, while ATRA efficiently abrogated the suppressive activity of MDSCs. Furthermore, ATRA had no effect on PD-L1 expression in HeLa cells in vitro. In in vivo treatment, ATRA decreased MDSCs accumulation and increased the frequency of CD8+ T cells in BALB/C mice with U14 cervical tumors. Importantly, a combination treatment of ATRA and anti-PD-L1 antibody further delayed U14 tumor growth and increased the proportion of CD62L-CD8+ T cells, CD62L-CD4+ T cells, CD107a+CD8+ T cells as well as IFN-γ and TNF-α levels in tumors. Our results provide a rationale for the use of ATRA to suppress MDSCs and enhance anti-PD-L1 cancer immunotherapy in cervical cancer.

The Impact of Traditional Chinese Medicine QingreHuoxue Treatment and the Combination of Methotrexate and Hydroxychloroquine on the Radiological Progression of Active Rheumatoid Arthritis: A 52-Week Follow-Up of a Randomized Controlled Clinical Study.

Traditional Chinese medicine (TCM) has been used successfully to treat rheumatoid arthritis (RA). QingreHuoxue treatment (QingreHuoxue decoction [QRHXD]/QingreHuoxue external preparation [QRHXEP]) is a Chinese medicine treatment for RA. To date, very few studies have compared the long-term effects of QRHXD with those of conventional disease-modifying antirheumatic drugs on RA disease activity and radiological progression. QRHXD delayed the radiological progression and showed long-term clinical efficacy of RA. In clinical experiments, the clinical evidence of delaying the radiological progression of RA patients was obtained. A portion of the patients who participated in the "Traditional Chinese Medicine QingreHuoxue Treatment vs. the Combination of Methotrexate and Hydroxychloroquine for Active Rheumatoid Arthritis" study were followed up for 52 weeks, and intention-to-treat (ITT) and compliance protocol (PP) analyses were used to collect and compare the clinical indicators and imaging data between baseline and week 52. Two radiologists who were blind to treatment scored the images independently. Of the 468 subjects, 141 completed the 52-week follow-up. There were no significant differences among the three groups: the traditional Chinese medicine comprehensive treatment group, the Western medicine treatment group, and the integrated traditional Chinese and Western medicine treatment group. There were no differences in the total Sharp score, joint space stenosis score, and joint erosion score at baseline or 52 weeks. In the comparison of the estimated annual radiographic progression (EARP) and the actual annual Sharp total score changes among the three groups, the actual changes were much lower than the EARP at baseline. The radiological progress in all three groups was well controlled. Results of the ITT and PP data sets showed that the disease activity score 28 level of the three groups at 52 weeks was significantly lower than that at baseline. During the 52-week treatment period, the clearance of heat and promotion of blood circulation controlled disease activity and delayed the radiological progress of active RA.

Traditional Chinese Medicine Qingre Huoxue Treatment vs. the Combination of Methotrexate and Hydroxychloroquine for Active Rheumatoid Arthritis: A Multicenter, Double-Blind, Randomized Controlled Trial.

Traditional Chinese medicine (TCM) has been used successfully to treat rheumatoid arthritis (RA). Qingre Huoxue treatment (Qingre Huoxue decoction (QRHXD)/Qingre Huoxue external preparation (QRHXEP)) is a therapeutic scheme of TCM for RA. To date, there have been few studies comparing the efficacy and safety of QRHXD and conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) for the treatment of active RA. This was investigated in a multicenter, double-blind, randomized controlled trial involving 468 Chinese patients with active RA [disease activity score (DAS)-28 > 3.2] treated with QRHXD/QRHXEP (TCM group), methotrexate plus hydroxychloroquine [Western medicine (WM) group], or both [integrative medicine (IM) group]. Patients were followed up for 24 weeks. The primary outcome measure was the change in DAS-28 from baseline to 24 weeks. The secondary outcome measures were treatment response rate according to American College of Rheumatology 20, 50, and 70% improvement criteria (ACR-20/50/70) and the rate of treatment-related adverse events (TRAEs). The trial was registered at ClinicalTrials.gov (NCT02551575). DAS-28 decreased in all three groups after treatment (p < 0.0001); the score was lowest in the TCM group (p < 0.05), while no difference was observed between the WM and IM groups (p > 0.05). At week 24, ACR-20 response was 73.04% with TCM, 80.17% with WM, and 73.95% with IM (based on the full analysis set [FAS], p > 0.05); ACR-50 responses were 40.87, 47.93, and 51.26%, respectively, (FAS, p > 0.05); and ACR-70 responses were 20.87, 22.31, and 25.21%, respectively, (FAS, p > 0.05). Thus, treatment efficacy was similar across groups based on ACR criteria. On the other hand, the rate of TRAEs was significantly lower in the TCM group compared to the other groups (p < 0.05). Thus, QRHXD/QRHXEP was effective in alleviating the symptoms of active RA-albeit to a lesser degree than csDMARDs-with fewer side effects. Importantly, combination with QRHXD enhanced the efficacy of csDMARDs. These results provide evidence that QRHXD can be used as an adjunct to csDMARDs for the management of RA, especially in patients who experience TRAEs with standard drugs. Clinical Trial Registration: ClinicalTrials.gov, identifier NCTNCT025515.

Novel deep intronic and frameshift mutations causing a TRIP11-related disorder.

Mutations of the thyroid hormone receptor interactor 11 gene (TRIP11, OMIM: 604505) at 14q32.12 have been associated with the autosomal recessive achondrogenesis type IA (ACG1A, OMIM: 200600) or osteochondrodysplasia (ODCD, OMIM: 184260). In this clinical report of a Chinese family, the mother had two consecutive pregnancies with similar aberrant phenotypes in the fetuses showing severe limb shortening. Whole exome sequencing (WES) of DNA from the second fetus identified a heterozygous frameshift mutation (NM_004239: c.3852delT) of TRIP11. Although this was consistent with the fetal clinical phenotypes, initial review of the WES results implied another novel mutation. To test this, we used high-precision clinical exome sequencing (HPCES) and found a mutation in Intron 18 of TRIP11 (c.5457+77T>G). Moreover, the sequencing depth of this mutation was only 3× that of WES compared with 161× that by HPCES. To ascertain the pathogenesis of the mutation (c.5457+77T>G), RT-PCR conducted using the parents' blood samples showed a 77-bp intronic sequence in the transcripts, which might have encoded for a shortened protein because of early termination due to code shifting. Our study furthers current understanding of deep intron function and provides a novel diagnostic method of deep intragenic mutations in families having two or more consecutive pregnancies with similar aberrant fetal phenotypes.

High expression of ENPP1 in high-grade serous ovarian carcinoma predicts poor prognosis and as a molecular therapy target.

Recent studies have shown that the expression of ENPP1 is related to differentiation, death, dissemination and chemosensitivity of tumor cells. So far, there is no research in ovarian carcinoma. This study aimed at exploring the role of ENPP1 gene in ovarian carcinoma, the relationship with prognostic indicators and chemotherapy resistance, and investigates the possibility of molecular targeted therapy. The expression of ENPP1 in 41 normal ovarian epithelial tissues, 97 ovarian serous cystadenoma and 103 HGSOC tissues was detected by IHC. In ovarian cancer tissues and ovarian cancer cell lines, mRNA and protein expression of ENPP1 was determined by qRT-PCR and Western blot. The ENPP1 expression was knockdowned by siRNA. Cell proliferation was measured with the BrdU Cell Proliferation ELISA. Cell migration and invasion were detected by Wound-Healing, Transwell migration and Matrigel invasion assay. Caspase 3 activity was determined by the CaspACE. The expression of EMT markers such as E-cadherin, N-cadherin, and Vimentin was measured, and the expression of PCNA and MMP9 was also be detected. The results showed that the expression of ENPP1 was significantly increased in high-grade ovarian serous carcinoma, the number of strong expression was 85.4% (22.3%+63.1%) and only 1.03% (1.03%+0.0%) in serous cystadenoma, but no in normal ovarian epithelium (P< 0.05). And the stronger the expression of ENPP1, the later the FIGO stage and the poorer differentiation of cells (P = 0.001 or <0.001, respectively). However, no correlation was found between the expression of ENPP1 and chemosensitivity. ENPP1 was also highly expressed in ovarian cancer tissues and in epithelial ovarian cancer cell lines (A2780, CaoV3, OVCAR3, SKOV3 and 3ao). After down-regulation of ENPP1 expression by RNA interference, the cell proliferation, migration and invasion of ovarian cancer cell decreased significantly, the expression of apoptosis related gene caspase 3 increased significantly, while the expression of PCNA and MMP9 was significantly down regulated. In addition, EMT biological characteristics of A2780 and SKOV3 cells were also inhibited. In summary, the increased expression of ENPP1 may be related to the occurrence of HGSOC, and indicate that the disease progresses rapidly and the prognosis is poor. ENPP1 may be considered as a potential molecular therapeutic target.

Elevated expressions of SHP2 and GAB2 correlated with VEGF in eutopic and ectopic endometrium of women with ovarian endometriosis.

Aims: Protein tyrosine phosphatase Src-homology-2-domain-containing phosphatase 2 (SHP2) and adaptor protein Grb2-associated binding protein 2 (GAB2) can bind to each other in various signal transduction. However, the expression of SHP2 and GAB2 have not been investigated in endometriosis. The aim of the study was to evaluate the expressions of SHP2 and GAB2, and explore the correlation with Ki67 and VEGF in ovarian endometriosis.Materials and methods: The protein expressions and localizations were assessed immunohistochemically in ectopic, eutopic endometrium and normal endometrium from patients with (n = 30) and without (n = 30) ovarian endometriosis.Results: SHP2 was mainly present in the endometrial glandular epithelium, with increased expression in eutopic endometrium and even higher expression in ectopic endometrium compared to control endometrium (p < .05). GAB2 was immunolocalized in endometrial epithelium and stroma, increasing its expression from control endometrium to eutopic and ectopic endometrium (p < .05). Positive correlation was found between SHP2 and GAB2 in endometrium (p < .01). SHP2 and GAB2 both positively correlated with VEGF (p < .05), but not Ki67 in endometrium.Conclusions: We provide the first evidence that the protein expressions of SHP2 and GAB2 were elevated in ectopic and eutopic endometrium, suggesting GAB2-SHP2 axis regulating VEGF might contribute to the pathomechanism of endometriosis.

Variation of PD-L1 expression in locally advanced cervical cancer following neoadjuvant chemotherapy.

BACKGROUND: High Programmed death ligand 1 (PD-L1) expression are thought to be necessary to PD-1/PD-L1 axis blockades in many tumors. The aim of the study was to explore the variation of PD-L1 expression after neoadjuvant chemotherapy (NAC) in cervical squamous cell carcinoma (SCC) and its clinical implications. METHODS: A total of 142 paired SCC specimens before and after platinum-based NAC were obtained from cervical cancer patients. The expression of PD-L1 and CD3+, CD4+, CD8+ tumor infiltrating lymphocytes (TILs) was detected by immunohistochemistry and the association between TILs, chemotherapy response, clinical outcome and PD-L1 expression was evaluated. RESULTS: The fraction of patients with high PD-L1 expression was significantly increased from 32.4 to 46.5% after NAC (χ2 = 5.897, p = 0.015), while the increase of CD3+, CD4+, CD8+ TILs was not significant. High PD-L1 expression was not associated with CD3+, CD4+, CD8+ TILs before NAC, however CD8+ TILs infiltration was positively associated with high PD-L1 expression after NAC (r = 0.205, p = 0.014). The decreased PD-L1 expression was more observed in patients with clinical response to NAC (χ2 = 6.890, p = 0.009). A longer DFS was seen in patients with decreased PD-L1 expression than those with elevated or stable PD-L1 expression (p = 0.048, 95% CI: 0.091-0.987), while the difference was not significant in multivariate analysis (p = 0.113, 95% CI: 0.108-1.266). CONCLUSIONS: Cisplatin based chemotherapy can increase PD-L1 expression in cervical cancer. The increased PD-L1 expression and a lymphocyte predominant microenvironment after chemotherapy provide a rational for use of PD-1/PD-L1 axis-inhibitor in the neoadjuvant setting.

The effects and mechanisms of GM-CSF on endometrial regeneration.

BACKGROUND: Endometrial injury can result in thin endometrium and subfertility. Granulocyte macrophage colony stimulating factor (GM-CSF) contributes to tissue repair, but its role in endometrial regeneration has not been investigated. METHODS: To determine the effect of GM-CSF on endometrial regeneration, we established a mouse model of thin endometrium by uterine perfusion with 20 µL 90% ethanol. Thin endometrium in mice was featured by lowered endometrial thickness, decreased expression of Ki67 in glandular cells, and a reduced number of implantation sites. To explore the mechanism of GM-CSF on endometrial regeneration, endometrium was obtained from patients undergoing hysterectomy or hysteroscopy and endometrial biopsy. Effects of GM-CSF on primary cultured human endometrial glandular and stromal cells were examined by the 5-bromo-2'-deoxyuridine (BrdU) proliferation assay and transwell migration assay, followed by exploration of the potential signaling pathway. RESULTS: GM-CSF intraperitoneal (i.p.) injection significantly increased endometrial thickness, expression of Ki67 in endometrial glandular cells, and the number of implantation sites. GM-CSF significantly promoted proliferation of primary human endometrial glandular cells and migration of stromal cells. GM-CSF activated p-Akt and increased expressions of p70S6K and c-Jun, which were blocked by LY294002. CONCLUSION: We found that GM-CSF could improve endometrial regeneration, possibly through activating PI3K/Akt signaling pathway.

Inhibition of RNA-Binding Protein Musashi-1 Suppresses Malignant Properties and Reverses Paclitaxel Resistance in Ovarian Carcinoma.

Background and Aims: Ovarian carcinoma (OC) is one of the most lethal malignant tumors with a high reoccurrence and chemoresistance. The key mechanism relationship with chemoresistance in ovarian carcinoma is still unclear. The existence of cancer stem cells involves in chemoresistance and reoccurrence in OC. The objective of this study was to investigate the expression, suppression of malignant properties and reversal of paclitaxel resistance inhibiting RNA-binding protein Musashi-1 with siRNA in ovarian cancer cells. Methods: The expression of MSI-1 was analyzed in 39 normal ovarian epithelia tissues, 92 serous cystadenomas, 68 borderline serous cystadenomas, and 97 serous cystadenocarcinomas by immunohistochemistry. pLKO.1-MSI-1-siRNA expression vector was transfected into ovarian carcinoma cell line A2780 and its paclitaxel-resistant cell subline A2780/Taxol. The roles of MSI-1 in proliferation, apoptosis, migration and invasion were explored by cell proliferation analysis, Caspase 3 activity assay, wound healing assay, migration and matrigel invasion assay, respectively. Western Blotting and Real-time quantitative PCR were conducted to detect the expression of MSI-1 and the ERK signaling pathway. Reversal of paclitaxel resistance assay was used to evaluate the role of MSI-1 in paclitaxel resistance of OC cells. Finally, therapeutic effects of MSI-1 inhibition were investigated the xenogratfs of SCID mice in vivo of the paclitacel-resistant. Results: MSI-1 is overexpressed and associated with an unfavorable prognosis in OC patients. Knockdown of MSI-1 by small interfering RNA (siRNA) inhibits proliferation, promotes apoptosis, and reduces migration and invasion of cancer cells. Moreover, MSI-1 expression inhibition reverses paclitaxel-resistance in OC cells. We further display that MSI-1 effectively protects OC cells from paclitaxel-induced apoptosis by increasing the expression of p-Bcl-2 through ERK signaling pathway activation. In vivo, MSI-1 siRNA clearly showed a strong effect on tumor growth inhibition and paclitaxel-resistance reversal. Conclusions: These findings suggest that MSI-1 overexpression is associated with the prognosis of OC patients, and knockdown of MSI-1 can suppress malignant properties and reverse paclitaxel-resistance in OC cells. MSI-1 maybe act as a potential prognostic indicator and a therapeutic target in OC.

Cervical Adenoid Basal Carcinoma: Clinicopathologic Features of 9 Cases With Reference to CK17 and Ki-67 Expression.

OBJECTIVE: The aims of this study were to clarify the histological features of adenoid basal carcinoma (ABC) and determine whether cytokeratin 17 (CK17) and Ki-67 can facilitate the differential diagnosis of ABC from squamous cell carcinoma (SCC). MATERIALS AND METHODS: Nine cases of pure ABC were collected from the files of the Division of Pathology at the Zhejiang University Hospital Women's School of Medicine. For comparison, 20 cases of moderately to poorly differentiated cervical SCC, including 2 of basaloid SCC, were also retrieved from the same period. Blocks were recut, reread, and immunostained for CK17 and Ki-67. RESULTS: Morphologically, ABCs were mainly composed of small basaloid cell nests and variable squamous differentiation foci. For immunohistochemical staining, 1 of 9 cases showed diffuse CK17 staining, 5 of 9 showed focal positive staining, and 3 of 9 showed negative staining in the basaloid cell area of ABC, whereas no CK17 expression was found in ABC squamous foci. Eighteen of the 20 invasive SCCs showed diffuse CK17 staining, and 2 showed focal staining. The Ki-67 proliferative index varied in different ABC areas, with a relatively high index in squamous differentiation foci and a low index in basaloid cell areas. In contrast, Ki-67 staining was unevenly intense in SCC. CONCLUSIONS: Adenoid basal carcinoma had characteristic morphological features, and the differential diagnosis of ABC from SCC is usually simple, based on morphology. In select cases, when histological findings are equivocal, the loss of CK17 expression in the squamous differentiation area, and a lower Ki-67 index in basal cell foci support ABC diagnosis.

STON2 negatively modulates stem-like properties in ovarian cancer cells via DNMT1/MUC1 pathway.

BACKGROUND: Cancer stem cells (CSCs) possess abilities of self-renewal and differentiation, have oncogenic potential and are regarded to be the source of cancer recurrence. However, the mechanism by which CSCs maintain their stemness remains largely unclear. METHODS: In this study, the cell line-derived ovarian CSCs (OCSCs), 3AO and Caov3, were enriched in serum-free medium (SFM). Differentially expressed proteins were compared between the OCSC subpopulation and parental cells using liquid chromatography (LC)-mass spectrometry (MS)/MS label-free quantitative proteomics. Sphere-forming ability assays, flow cytometry, quantitative real-time polymerase chain reaction (qPCR), western blotting, and in vivo xenograft experiments were performed to evaluate stemness. RNA-sequencing (RNA-seq) and pyrosequencing were used to reveal the mechanism by which STON2 negatively modulates the stem-like properties of ovarian cancer cells. RESULTS: Among the 74 most differentially expressed proteins, stonin 2 (STON2) was confirmed to be down-regulated in the OCSC subpopulation. We show that STON2 negatively modulates the stem-like properties of ovarian cancer cells, which are characterized by sphere formation, a CD44+CD24- ratio, and by CSC- and epithelial mesenchymal transition (EMT)-related markers. STON2 knockdown also accelerated tumorigenesis in NOD/SCID mice. Further investigation revealed a downstream target, mucin 1 (MUC1), as up-regulated upon the down regulation of STON2. A decrease in both DNA methyltransferase 1 (DNMT1) expression and methylation in the promoter region of MUC1 was associated with subsequently elevated MUC1 expression, as detected in STON2 knockdown in 3AO and Caov3 cells. Direct DNMT1 knockdown simultaneously elevated MUC1 expression. The functional significance of this STON2-DNMT1/MUC1 pathway is supported by the observation that STON2 overexpression suppresses MUC1-induced sphere formation of OCSCs. The paired expression of STON2 and MUC1 in ovarian cancer specimens was also detected revealing the prognostic value of STON2 expression to be highly dependent on MUC1 expression. CONCLUSIONS: Our results imply that STON2 may negatively regulate stemness in ovarian cancer cells via DNMT1-MUC1 mediated epigenetic modification. STON2 is therefore involved in OCSC biology and may represent a therapeutic target for innovative treatments aimed at ovarian cancer eradication.

Basonuclin 1 deficiency is a cause of primary ovarian insufficiency.

Primary ovarian insufficiency (POI) leads to infertility and premature menopause in young women. The genetic etiology of this disorder remains unknown in most patients. Using whole exome sequencing of a large Chinese POI pedigree, we identified a heterozygous 5 bp deletion inducing a frameshift in BNC1, which is predicted to result in a non-sense-mediated decay or a truncated BNC1 protein. Sanger sequencing identified another BNC1 missense mutation in 4 of 82 idiopathic patients with POI, and the mutation was absent in 332 healthy controls. Transfection of recombinant plasmids with the frameshift mutant and separately with the missense mutant in HEK293T cells led to abnormal nuclear localization. Knockdown of BNC1 was found to reduce BMP15 and p-AKT levels and to inhibit meiosis in oocytes. A female mouse model of the human Bnc1 frameshift mutation exhibited infertility, significantly increased serum follicle-stimulating hormone, decreased ovary size and reduced follicle numbers, consistent with POI. We report haploinsufficiency of BNC1 as an etiology of human autosomal dominant POI.