N-acetylcysteine and Hydroxychloroquine Ameliorate ADMA-Induced Fetal Growth Restriction in Mice via Regulating Oxidative Stress and Autophagy.

Fetal growth restriction (FGR) seriously threatens perinatal health. The main cause of FGR is placental malperfusion, but the specific mechanism is still unclear, and there is no effective treatment for FGR. We constructed a FGR mouse model by adding exogenous asymmetric dimethylarginine (ADMA) through in vivo experiments and found that ADMA could cause placental dysplasia and induce the occurrence of FGR. Compared with the control group, reactive oxygen species (ROS) production in the placenta was increased in mice with FGR, and the expression of autophagy-related proteins p-AKT/AKT, p-mTOR/mTOR, and P62 was significantly decreased, while the expression of Beclin-1 and LC3-II was significantly increased in the FGR group. Furthermore, ADMA had a favorable effect in promoting the formation of autophagosomes. Hydroxychloroquine (HCQ) and N-acetylcysteine (NAC) improved ADMA-induced disorders of placental development and alleviated ADMA-induced FGR. This study found that ADMA could cause excessive autophagy of trophoblasts by increasing the level of oxidative stress, ultimately leading to the occurrence of FGR, and HCQ and NAC had therapeutic effects on ADMA-induced FGR.

The lncRNA HULC functions as an oncogene by targeting ATG7 and ITGB1 in epithelial ovarian carcinoma.

Highly upregulated in liver cancer (HULC) is a long noncoding RNA (lncRNA), which has recently been identified as a key regulator in the progression of hepatocellular carcinoma, gliomas and gastric cancer. However, its role in epithelial ovarian carcinoma (EOC) remains unknown. In this study, HULC expression was examined in EOC, borderline and benign ovarian tumors, and normal ovarian tissues by RT-PCR. Ovarian cancer cell phenotypes, as well as autophagy-associated proteins were examined after HULC overexpression or downregulation by plasmid or small interfering RNA (siRNA) transfection, respectively. LncRNA-protein interactions were examined by ribonucleoprotein immunoprecipitation (RIP) assays. We found that HULC expression levels were higher in EOC tissues than normal samples. HULC overexpression induced cell proliferation, migration, invasion, whereas reduced cell apoptosis in vitro and induced tumor growth in vivo. In contrast, downregulation of HULC by siRNA transfection reduced cell proliferation, migration and invasion, and induced cell apoptosis and autophagy. Our results showed that HULC overexpression reduced ATG7, LC3-II and LAMP1 expression, while inducing SQSTM1 (P62) and ITGB1 expression. HULC downregulation had the opposite effects. Furthermore, RIP indicated that ATG7 interacted with HULC; ATG7 downregulation also induced cell proliferation, reduced apoptosis and inhibited autophagy in vitro by reducing LC3-II and LAMP1 expression, while inducing SQSTM1 expression. Furthermore, ATG7 co-transfection with HULC reversed the oncogenic effects of HULC both in vitro and in vivo; however, downregulating ATG7 did not affect cell migration and invasive ability. We found that ITGB1 siRNA co-transfection with HULC reversed the function of HULC in inducing ovarian cancer cell migration and invasive ability. Taken together, our results show that HULC may promote ovarian carcinoma tumorigenesis by inhibiting ATG7 and inducing progression by regulating ITGB1.

Fascaplysin inhibit ovarian cancer cell proliferation and metastasis through inhibiting CDK4.

OBJECTIVE: Cyclin-dependent kinases (CDKs) are important regulators of the cell cycle; previous studies have shown that misregulation of CDK4 (cyclin-dependent kinase 4) activity can lead to cancer. The present study investigated the anti-tumor effects of a highly selective CDK4 inhibitor fascaplysin in ovarian carcinoma cell lines. MATERIALS AND METHODS: In our study, cell proliferation, cell cycle, cell apoptosis, cell invasion, and cell migration relative assays were performed in ovarian cancer cell lines A2780 and OVCAR3 in the presence of different concentrations of fascaplysin. The protein expression levels of CDK4, cyclin D1, Bcl-2 (B-cell lymphoma-2), and VEGFA (vascular endothelial growth factor A) were determined by western blot. RESULTS: Our results showed that fascaplysin inhibited ovarian cancer cell proliferation, invasion and migration, as well as inducing S arrest and cell apoptosis. Treatment with fascaplysin also suppressed CDK4, cyclin D1, Bcl-2, and VEGFA expression at protein levels. CONCLUSIONS: Above all, our results showed that fascaplysin has anti-tumor activity against ovarian cancer cell lines through inhibiting CDK4, and may be a therapeutic target for the treatment of ovarian carcinomas.

DLEU1 contributes to ovarian carcinoma tumourigenesis and development by interacting with miR-490-3p and altering CDK1 expression.

Recently, a large number of studies have focused on the important role of long non-coding RNAs (lncRNAs) in metabolism and development and have found that abnormal lncRNA expression is associated with the pathogenesis and development of many diseases. The lncRNA DLEU1 is involved in many solid tumours and haematological malignancies. However, its role in epithelial ovarian carcinoma (EOC) and the associated molecular mechanisms has not been reported. In this study, quantitative reverse transcription-PCR (qRT-PCR) demonstrated higher lncRNADLEU1 expression in EOC tissues than in normal tissues. Plasmid transfection of DLEU1 to up-regulate its expression in the ovarian cancer cell lines A2780 and OVCAR3 increased cell proliferation, migration, and invasion, while inhibited apoptosis. Nude mouse xenograft assay demonstrated that DLEU1 overexpression promoted tumour growth in vivo. QRT-PCR showed decreased miR-490-3p expression, while Western blotting demonstrated increased its target genes CDK1, cyclinD1 and SMARCD1, as well as matrix metalloproteinase-2 (MMP2), Bcl-xL and P70S6K protein expression, respectively. Short interfering RNA silencing of DLEU1 produced opposite results, where qRT-PCR showed increased miR-490-3p expression. The dual-luciferase reporter assay revealed a direct interaction between DLEU1 and miR-490-3p. MiR-490-3p plays a tumour suppressor role in epithelial ovarian cancer by targeting CDK1 regulation and influencing SMARCD1 and cyclin D1 (CCND1) expressions. Therefore, we suggest that through interaction with miR-490-3p, DLEU1 may influence the expression of CDK1, CCND1 and SMARCD1 protein, subsequently promoting the development and progression of EOC.

The role of miR-372 in ovarian carcinoma cell proliferation.

OBJECTIVES: MicroRNA-372 has been shown to be associated with multiple tumors' development and progression, by regulating the expression of proteins involved in cell cycle and apoptosis. However, the specific mechanism and function of miR-372 in ovarian carcinoma are not clear. Our study explored the role of miR-372 in ovarian carcinoma cell cycle and proliferation. MATERIALS AND METHODS: MiR-372 expression was quantified in normal ovarian tissue, benign tumors, primary ovarian carcinomas and metastatic omentum by qRT-PCR. MTT assay and plate clone formation assay were performed to evaluate the cell viability and proliferation. EDU assay and cell apoptosis assay were also used to determine cell growth. We used Western Blot to analysis expression of the known miR-372 targets. RESULTS: We found that miR-372 expression was significantly lower in ovarian carcinoma than normal ovarian tissues and benign tumors. Moreover, miR-372 overexpression showed significant inhibition of cell proliferation and promoted cell apoptosis. Western Blot revealed that miR-372 downregulated the expression of ATAD2, LATS2, P62, DKK1 and cyclinA1 to inhibit the proliferation of cells. CONCLUSIONS: Our findings indicate that miR-372 has a prominent role in inhibiting tumor growth and it is a valuable target for ovarian cancer therapy.

E2F-1 targets miR-519d to regulate the expression of the ras homolog gene family member C.

E2F1 (E2F transcription factor 1) can act as a tumor suppressor or oncogene. We report the molecular mechanism of E2F1 in ovarian carcinoma tumorigenesis and progression. E2F1 expression levels in ovarian carcinoma tissue were examined by immunohistochemistry. After E2F1 plasmid transfection and E2F1-microRNA-519d (miR-519d)/si-RhoC (Ras homolog gene family member C) co-transfection, ovarian cancer cell phenotypes and the related molecules were examined in vitro and in vivo. E2F1 was overexpressed in type I and type II ovarian carcinoma as compared to normal ovary tissues and normal fallopian tube tissues, respectively. E2F1 overexpression promoted cell proliferation, G1-S progression, survival, migration, and invasion in vitro; miR-519d or siRhoC co-transfection reversed E2F1 oncogenic effects. E2F1 overexpression promoted tumor growth in vivo; miR-519d overexpression inhibited it. E2F1 overexpression increased RhoC, Bcl-2, cyclin D1, survivin, MMP2 (matrix metalloproteinase 2), MMP9, STAT3 (signal transducer and activator of transcription 3), and HuR (ELAV-like RNA-binding protein 1) expression; miR-519d overexpression decreased their expression. E2F1 downregulated miR-519d directly and miR-519d downregulated RhoC directly. Conversely, miR-519d directly downregulated E2F1, There is a direct repressive regulatory loop between E2F1 and miR-519d. We provide evidence that E2F1/miR-519d/RhoC is a promising signaling pathway for diagnosing and treating ovarian carcinoma.

MicroRNA-93 Promotes Epithelial-Mesenchymal Transition of Endometrial Carcinoma Cells.

MicroRNA-93, derived from a paralog (miR-106b-25) of the miR-17-92 cluster, is involved in the tumorigenesis and progression of many cancers such as breast, colorectal, hepatocellular, lung, ovarian, and pancreatic cancer. However, the role of miR-93 in endometrial carcinoma and the potential molecular mechanisms involved remain unknown. Our results showed that miR-93 was overexpressed in endometrial carcinoma tissues than normal endometrial tissues. The endometrial carcinoma cell lines HEC-1B and Ishikawa were transfected with miR-93-5P, after which cell migration and invasion ability and the expression of relevant molecules were detected. MiR-93 overexpression promoted cell migration and invasion, and downregulated E-cadherin expression while increasing N-cadherin expression. Dual-luciferase reporter assay showed that miR-93 may directly bind to the 3' untranslated region of forkhead box A1 (FOXA1); furthermore, miR-93 overexpression downregulated FOXA1 expression while miR-93 inhibitor transfection upregulated FOXA1 expression at both mRNA and protein level. In addition, transfection with the most effective FOXA1 small interfering RNA promoted both endometrial cancer cell migration and invasion, and downregulated E-cadherin expression while upregulating N-cadherin expression. Therefore, we suggest that miR-93 may promote the process of epithelial-mesenchymal transition in endometrial carcinoma cells by targeting FOXA1.

Effects and mechanism of RhoC downregulation in suppressing ovarian cancer stem cell proliferation, drug resistance, invasion and metastasis.

Cancer stem cells are considered to be the root cause of tumor initiation, metastasis, recurrence and therapeutic resistance. Recent studies have reported that RhoC plays a critical role in regulating cancer stem cells; however, its function in ovarian cancer stem cells (OCSCs) remains unknown. The ovarian cancer cell line A2780, and the paclitaxel-resistant A2780 cell line (A2780-PTX) were obtained. A2780 cells were used to isolate and identify the highly invasive A2780-PM cells, and A2780-PTX cells were used to isolate and identify the highly drug-resistant and highly invasive A2780-PTX-PM cells by Transwell assay. MTT, Transwell and wound healing assays were used to compare the differences in cell proliferation, invasion and migration ability among the four cell lines. Immunofluorescence was used to detect the expression of stem cell markers CD117 and CD133. OCSCs were sorted by flow cytometry. Following si-RhoC transfection of the OCSCs, cell proliferation, drug resistance, invasion and migration ability and RhoC, CD117 and CD133 expression levels were assayed. RT-PCR was used to assess RhoC, CD117, CD133 and matrix metalloproteinase 9 (MMP9) mRNA expression levels. A2780-PM and A2780­PTX-PM cells exhibited higher cell proliferation, drug resistance, and invasion and migration ability than the A2780 and A2780-PTX cell lines. Furthermore, CD133 and CD117 expression levels were higher in the A2780-PM and A2780­PTX-PM cells than levels in the A2780 and A2780-PTX cells. Transfection of si-RhoC in OCSCs suppressed the proliferation, drug resistance, invasion, migration and CD117 and CD133 expression levels. Furthermore, the expression levels of RhoC, CD117, CD133, MDR1, and MMP9 mRNA were downregulated in the transfected population. Taken together, our results demonstrated that RhoC downregulation may inhibit the proliferation, drug resistance, invasion and migration of OCSCs, and RhoC may play an important role in the formation of OCSCs.

Role of glycogen synthase kinase-3ß inhibitor AZD1080 in ovarian cancer.

BACKGROUND: Glycogen synthase kinase-3ß (GSK-3ß) is a multifunctional serine/threonine kinase that plays an important role in cancer tumorigenesis and progression. We investigated the role of the GSK-3ß inhibitor AZD1080 in ovarian cancer cell lines. METHODS: A2780 and OVCAR3 ovarian cancer cell lines were exposed to AZD1080, after which cell proliferation, cell cycle, invasion, and migration assays were performed. Phalloidin staining was used to observe lamellipodia formation. Reverse transcription polymerase chain reaction and Western blot were used to assess the respective mRNA and protein expression levels of GSK-3ß, CDK2, CDK1, cyclin D1, matrix metalloproteinase-9 (MMP9), and Bcl-xL. RESULTS: AZD1080 exposure suppressed ovarian cancer cell proliferation, invasion, migration, and lamellipodia formation, and induced G1 arrest, which was concentration dependent. AZD1080 also significantly downregulated GSK-3ß, CDK2, CDK1, cyclin D1, MMP9, and Bcl-xL expression at both mRNA and protein levels. CONCLUSION: Taken together, our results demonstrate that the GSK-3ß inhibitor AZD1080 suppresses ovarian cancer development and therefore may indicate a new direction for ovarian cancer treatment.

Immobility responses between mouse strains correlate with distinct hippocampal serotonin transporter protein expression and function.

Mouse strain differences in immobility and in sensitivity to antidepressants have been observed in the forced swimming test (FST) and the tail suspension test (TST). However, the neurotransmitter systems and neural substrates that contribute to these differences remain unknown. To investigate the role of the hippocampal serotonin transporter (5-HTT), we measured baseline immobility and the immobility responses to fluoxetine (FLX) in the FST and the TST in male CD-1, C57BL/6, DBA and BALB/c mice. We observed strain differences in baseline immobility time, with CD-1 mice showing the longest and DBA mice showing the shortest. In contrast, DBA and BALB/c mice showed the highest sensitivity to FLX, whereas CD-1 and C57BL/6 mice showed the lowest sensitivity. Also we found strain differences in both the total 5-HTT protein level and the membrane-bound 5-HTT level (estimated by V max) as follows: DBA>BALB/c>CD-1=C57BL/6. The uptake efficiency of the membrane-bound 5-HTT (estimated by 1/K m) was highest in DBA and BALB/c mice and lowest in CD-1 and C57BL/6 mice. A correlation analysis of subregions within the hippocampus revealed that immobility time was negatively correlated with V max and positively correlated with K m in the hippocampus. Therefore a higher uptake capacity of the membrane-bound 5-HTT in the hippocampus was associated with lower baseline immobility and greater sensitivity to FLX. These results suggest that alterations in hippocampal 5-HTT activity may contribute to mouse strain differences in the FST and the TST.

Subregion-specific decreases in hippocampal serotonin transporter protein expression and function associated with endophenotypes of depression.

Stress influences the development of depression, and depression is associated with structural and functional changes in the hippocampus. The current study sought to determine whether chronic corticosteroid (CORT) treatment influences serotonin transporter (5-HTT) protein expression and function in the CA1, CA3, and dentate gyrus (DG) subregions of the hippocampus. Male CD-1 mice were subcutaneously injected with CORT at a dose of 20 mg/kg once daily for 3 weeks. Behavioral state was assessed using sucrose preference, physical state of the coat, forced swimming test, and tail suspension test. We then determine 5-HTT protein expression and synaptosomal 5-HT uptake in the CA1, CA3 and DG subregions. CORT treatment induced anhedonia and behavioral despair, two core endophenotypes of clinical depression; 5-HTT protein expression levels and synaptosomal 5-HT uptake were both decreased in a subregion-specific manner, with the greatest decrease observed in the DG, a moderate decrease in the CA3, and the CA1 showed no apparent change. In addition, a reduction in tissue mass was detected in the DG following the CORT treatment. These data indicate that subregion-specific decreases in hippocampal 5-HTT protein expression and function are associated with endophenotypes of depression.