Aquaporins (AQPs), the rapid transition pores for water molecules, play an important role in maintenance of intracellular water balance. Studies showed that AQPs were also involved in occurrence, development, invasion and metastasis of tumors. In this study, we aimed to explore the distribution and expression differences of aquaporin 6 (AQP6) and aquaporin 8 (AQP8) in epithelial ovarian tumors. The expression of AQP6 and AQP8 in 47 cases of epithelial ovarian tumors were measured by immunochemical technique and Western blotting. AQP6 was strongly expressed in benign ovarian tumors, but weak signal was shown in malignant tumors. The difference was not statistically significant (P > 0.05). Compared with serous adenoma and normal tissues, AQP6 expression in serous carcinoma was obviously decreased (P < 0.05). AQP8 expressions were both identified in benign and malignant tumors, but there was no significantly statistical difference (P > 0.05). For patients with large volume of malignant ascites (>1000 ml), AQP8 expression was increased (P < 0.05). AQP8 expression in malignant tumors was not related to different clinical stages, presence of lymphatic metastasis, and differentiation degrees (P > 0.05). These data showed that AQP6 and AQP8 had different expression degrees in epithelial ovarian tissues, which suggests that AQP6 and AQP8 may play certain roles in epithelial ovarian tumors.
Aquaporins (AQPs) are important mediators of water permeability and are closely associated with tumor cell proliferation, migration, angiogenesis and chemoresistance. Moreover, the chemosensitivity of tumor cells to cisplatin (CDDP) is potentially affected by osmotic pressure. The present study was undertaken to determine whether hyperosmosis regulates ovarian cancer cell sensitivity to CDDP in vitro and to explore whether this is associated with AQP expression. The hyperosmotic stress was induced by D-sorbitol. 3AO ovarian cancer cells were treated with different concentrations of hypertonic medium and/or CDDP for various times, followed by measuring the inhibition rate of cell proliferation using an MTT assay. In addition, AQP expression in response to osmotic pressure and/or CDDP was measured by reverse transcription-quantitative polymerase chain reaction and western blotting. Cell proliferation in response to hypertonic stress was also measured when AQP5 was knocked down by small interfering (si)RNA. 3AO cell proliferation was inhibited by hyperosmotic stress, while the expression of AQP5, but not that of AQP1, AQP3 or AQP9, was increased in a dose- and time-dependent manner in hypertonic sorbitol-containing medium. When AQP5 was silenced by siRNA, cells were susceptible to hypertonic stress. MTT analyses showed that the inhibition of cell proliferation by a low dose of CDDP increased significantly with exposure to a hyperosmotic stimulus, and this effect was reduced when a high dose of CDDP was used. AQP5 expression was induced by a low dose of CDDP, but was reduced by a high dose of CDDP. However, hyperosmosis enhanced AQP5 mRNA expression at every dose of CDDP tested, compared with isotonic medium. With prolonged treatment time, AQP5 expression was reduced by CDDP in hypertonic and isotonic culture medium. Thus, the effects of hyperosmosis on cell sensitivity to CDDP were associated with AQP5 expression. These results suggest that AQP5 expression in ovarian cancer cells is induced by hypertonic medium, and that the sensitivity of ovarian cancer cells to CDDP can be regulated by hyperosmosis associated with AQP5 expression.
Background Hedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC). Methods Protein–protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination. Results Gli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo. Conclusion miR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC. Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.
Background: Hedgehog (Hh) signaling pathway, which is essential for cell proliferation and differentiation, is noted to be aberrantly activated in tumor from increasing studies in recent years. MicroRNAs (miRNAs) as an important non-coding RNA in cells have been proven to possess a regulatory role specific to the Hh signaling pathway. Here, in vitro and in vivo cellular/molecular experiments were adopted to clarify the regulatory mechanism linking miR-636 to the Hh signaling pathway in ovarian cancer (OVC). Methods: Protein-protein interaction analysis was performed to identify the hub gene in the Hh pathway. TargetScan database was used to predict the potential upstream regulators for Gli2. qRT-PCR was performed to test the expression of miR-636, while Western blot was conducted to detect the expression of proteins related to the Hh pathway and epithelial-mesenchymal transition (EMT). For cell functional experiments, HO-8910PM OVC cell line was used. MTT assay and wound healing assay were used to measure the effect of miR-636 on cell proliferation and migration. Flow cytometry was carried out to examine the effect of miR-636 on cell cycle, and Western blot was used to identify the change in expression of Hh and EMT-related proteins. Dual-luciferase reporter gene assay was implemented to detect the targeting relationship between miR-636 and Gli2. Xenotransplantation models were established for in vivo examination.Results: Gli2 was identified as the hub gene of the Hh pathway and it was validated to be regulated by miR-636 based on the data from TargetScan and GEO databases. In vitro experiments discovered that miR-636 was significantly lowly expressed in OVC cell lines, and overexpressing miR-636 significantly inhibited HO-8910PM cell proliferation, migration and induced cell cycle arrest in G0/G1 phase, while the inhibition of miR-636 caused opposite results. Dual-luciferase reporter gene assay revealed that Gli2 was the target gene of miR-636 in OVC. Besides, overexpressed miR-636 decreased protein expression of Gli2, and affected the expression of proteins related to the Hh signaling pathway and EMT. Rescue experiments verified that overexpression of Gli2 reversed the inhibitory effect of miR-636 on HO-8910PM cell proliferation and migration, and attenuated the blocking effect of miR-636 on cell cycle. The xenotransplantation experiment suggested that miR-636 inhibited cell growth of OVC by decreasing Gli2 expression. Besides, overexpressing Gli2 potentiated the EMT process of OVC cells via decreasing E-cadherin protein expression and increasing Vimentin protein expression, and it reversed the inhibitory effect of miR-636 on OVC cell proliferation in vivo.Conclusion: miR-636 mediates the activation of the Hh pathway via binding to Gli2, thus inhibiting EMT, suppressing cell proliferation and migration of OVC.Trial registration: The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine (IR2019001235). Written informed consent was obtained from individual or guardian participants.
Endometrial carcinoma (EC) is a common malignancy of female genital system which exhibits a unique immune profile. It is a promising strategy to quantify immune patterns of EC for predicting prognosis and therapeutic efficiency. Here, we attempted to identify the possible immune microenvironment-related prognostic markers of EC. We obtained the RNA sequencing and corresponding clinical data of EC from TCGA database. Then, 3 immune scores based on the Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) algorithm were computed. Correlation between above ESTIMATE scores and other immune-related scores, molecular subtypes, prognosis, and gene mutation status (including BRCA and TP53) were further analyzed. Afterwards, gene modules associated with the ESTIMATE scores were screened out through hierarchical clustering analysis and weighted gene co-expression network analysis (WGCNA). Differentially expressed analysis was performed and genes shared by the most relevant modules were found out. KEGG pathway enrichment analysis was conducted to explore the biological functions of those genes. Survival analysis was carried out to identify prognostic immune-related genes and GSE17025 database was further used to confirm the correlation between immune-related genes and the ImmuneScore. The immune-related scores based on ESTIMATE algorithm was closely related to the immune microenvironment of EC. 3 gene modules that had the closest correlations with 3 ESTIMATE scores were obtained. 109 immune-related genes were preliminarily found out and 29 pathways were significantly enriched, most of which were associated with immune response. Univariate survival analysis revealed that there were 14 genes positively associated with both OS and PFS. Among which, 11 genes showed marked correlations with ImmuneScore values in GSE17025 database. Our current study profiled the immune status and identified 14 novel immune-related prognostic biomarkers for EC. Our findings may help to investigate the complicated tumor microenvironment and develop novel individualized therapeutic targets for EC.
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