Pretreatment with inflammatory factors altered the secretome of human amniotic epithelial cells.

Human amniotic epithelial cells (hAECs) are novel and promising therapeutic agents for patients suffering from degenerative diseases. Studies have demonstrated that the therapeutic effects of hAECs mainly depend on their paracrine components. Currently, appropriate pretreatment is a widely confirmed strategy for enhancing the repair potential of stem cells; however, the effect of proinflammatory factor pretreatment on hAECs and their secretome is still unclear. In this study, we used the well-characterized proinflammatory factors tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) to stimulate hAECs and analyzed the effect of TNF-α and IFN-γ on hAECs, including gene expression profile, paracrine proteins and miRNAs in exosomes. Results showed that TNF-α and IFN-γ pretreatment improved the viability of hAECs, but inhibited the proliferation of hAECs. TNF-α and IFN-γ pretreatment altered the gene expression profile of hAECs, and upregulated differentially expressed genes (DEGs) were predominantly enriched in biological adhesion, antioxidant activity and response to IFN-beta. In addition, TNF-α and IFN-γ pretreatment enhanced the paracrine secretion of cytokines by hAECs. The upregulated differentially expressed proteins (DEPs) were mainly enriched in tissue remodeling proteins and cytokine-cytokine receptor. Notably, the expression of miRNAs in exosomes from hAECs was also changed by TNF-α and IFN-γ pretreatment. The target genes of upregulated exosomal miRNAs substantially contributed to the response to stimulus, metabolic pathways and PI3K-Akt signaling pathway. Our findings improve our understanding of the biological characteristics of hAECs after proinflammatory factor pretreatment and provide novel insights to strengthen and optimize the therapeutic potential of hAECs and their secretome in regenerative medicine.

EPPK1 as a Prognostic Biomarker in Type I Endometrial Cancer and Its Correlation with Immune Infiltration.

Purpose: Approximately 20% of patients with type I endometrial cancer (EC) of the uterus experience recurrence and metastasis. However, existing data do not provide sufficient evidence for the utility of protein levels as prognostic biomarkers in type I EC. This study aims to determine whether epiplakin1 (EPPK1) and progesterone receptor (PR) play a role in the recurrence and metastasis of type I EC. Methods: Following the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) for assessing the quality of biomarker research results, a retrospective analysis was conducted on clinical information and tissue samples of type I EC patients. Protein expression data and clinical data for type I EC were downloaded from The Cancer Proteome Atlas (TCPA) database. We utilized the Kaplan-Meier (K-M) method and Cox proportional hazards regression analyses to evaluate whether epiplakin1 (EPPK1), progesterone receptor (PR) and certain clinical parameters can serve as independent prognostic factors. The Immune Cell Abundance Identifier (ImmuCellAI) and Cancer Immunome Atlas (TCIA) were employed to predict responses to immunotherapy. Immunohistochemistry was carried out to assess the expression of EPPK1 in type I EC. Results: Type I EC patients with high EPPK1 and low PR expression had higher International Federation of Gynecology and Obstetrics (FIGO) stage, recurrence, and metastasis rates. Furthermore, EPPK1 was identified as an independent prognostic factor, and low expression of EPPK1 was predominantly observed in the POLE ultramutated (POLEmut) group, indicating a favorable prognosis. Additionally, the high EPPK1 expression group had a lower Immune Prognostic Score (IPS), suggesting that the high-expression group may not benefit from immune checkpoint inhibitors. Conclusion: High expression of EPPK1 is an independent prognostic factor in type I EC patients with low PR expression. It can identify a subgroup of patients at high risk of recurrence. A more aggressive treatment approach is recommended for these patients.

Identification and characterization of multipotential stem cells in immortalized normal ovarian surface epithelial cells.

The ovarian surface epithelium (OSE) is a single layer of squamous-to-cuboidal epithelial cells that experience repetitive ovulatory rupture and subsequent repair. However, the characteristics of human immortalized ovarian surface epithelial cells (IOSE80) remain elusive. This study aims to determine whether IOSE80 cells have the characteristics of stem cell proliferation and multilineage differentiation and their application in regenerative medicine. IOSE80 cells are sequenced by high-throughput transcriptome analysis, and 5 sets of public data are used to compare the differences between IOSE80 cells and bone marrow mesenchymal stem cells, pluripotent stem cells, and oocytes in transcriptome profiling. The IOSE80 cells present a cobblestone-like monolayer and express the epithelial cell marker KRT18; the stem cell markers IFITM3, ALDH1A1, and VIM; lowly express stem cell marker LGR5 and germ cell markers DDX4 and DAZL. In addition, the GO terms "regulation of stem cell proliferation", "epithelial cell proliferation", etc., are significantly enriched ( P<0.05). IOSE80 cells have the potential to act as mesenchymal stem cells to differentiate into adipocytes with lipid droplets, osteoblasts, and chondroblasts in vitro. IOSE80 cells express pluripotent stem cell markers, including OCT4, SSEA4, TRA-1-60, and TRA-1-81, and they can be induced into three germ layers in vitro. IOSE80 cells also form oocyte-like cells in vitro and in vivo. In addition, IOSE80 cells exhibit robust proliferation, migration, and ovarian repair functions after in vivo transplantation. This study demonstrates that IOSE80 cells have the characteristics of pluripotent/multipotent stem cells, indicating their important role in tissue engineering and regenerative medicine.

TNF-α and IFN-γ prestimulation enhances the therapeutic efficacy of human amniotic epithelial stem cells in chemotherapy-induced ovarian dysfunction.

BACKGROUND: Exposure to a harsh ovarian microenvironment induced by chemotherapeutic agents seriously affects the remodeling of ovarian function and follicular development, leading to premature ovarian failure or insufficiency (POF/POI). For decades, the effectiveness of stem cell therapies in POI animal models has been intensively studied; however, strategies to enhance the therapeutic effect of stem cells remain challenging. METHODS: In this study, we first observed the pathological changes of the ovaries at different time points during chemotherapy, including the number of follicles, granulosa cell proliferation, oxidative stress damage, ovarian fibrosis, and inflammatory reaction. Moreover, we investigated whether activated hAECs stimulated by the proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) were more effective than native hAECs in repairing ovarian injury induced by chemotherapy. RESULTS: The inhibitory effect of chemotherapy drugs on ovarian granulosa cells (GCs) in growing follicles mainly occurred on day 3 after chemotherapy in a mouse model. Then, continued ovarian injury, including oxidative damage and cell death cascades, resulted in the depletion of follicular reserves and inflammation-related ovarian fibrosis. Cytokine array demonstrated that activated hAECs secreted high levels of paracrine cytokines related to extracellular matrix (ECM) remodeling, angiogenesis, and immunomodulation. An in vivo study showed that the engraftment rate of activated hAECs in damaged ovaries was higher than that of native hAECs. Furthermore, activated hAECs in damaged ovaries had significantly upregulated expression of the antioxidant proteins thioredoxin1/2. In addition, activated hAECs had increased numbers of mature follicles and ameliorated the ovarian microenvironment by promoting angiogenesis and reducing ovarian fibrosis. CONCLUSIONS: These results indicated that secondary ovarian damage induced by chemotherapy, including oxidative stress damage, chronic inflammatory response, and ovarian tissue fibrosis should be attended. Prestimulation with the proinflammatory factors TNF-α and IFN-γ could enhance the therapeutic efficacy of hAECs against chemotherapy-induced ovarian dysfunction, which may become a new feasible strategy to improve the therapeutic potential of hAECs in regenerative medicine.

Paris saponins â ¦ inhibits glycolysis of ovarian cancer via the RORC/ACK1 signaling pathway.

Rhizoma Paridis is a traditional Chinese medicine commonly used for treatment of malignant tumors. Paris saponins Ⅶ (PSⅦ) is one of the components of Rhizoma Paridis, but the role of PSⅦ in glucose metabolism in ovarian cancer remains elucidated. A series of experiments in the current study demonstrated that PSⅦ inhibites glycolysis and promotes cell apoptosis in ovarian cancer cells. Expression levels of glycolysis-related proteins and apoptosis-related proteins were significantly altered by upon treatment with PSⅦ, as determined from western blot analyses. Mechanistically, PSⅦ exerted its anti-tumor effects by targeting the RORC/ACK1 signaling pathway. These findings indicate that PSⅦ inhibits glycolysis-induced cell proliferation and apoptosis through the RORC/ACK1 pathway, supporting its potential development as a candidate chemotherapeutic agent for ovarian cancer.

Correlation between vaginal microbiota and different progression stages of cervical cancer.

The process from high-risk human papillomavirus (HR-HPV) infection to cervical cancer is a continuous and long-term process, but the pathogenesis of the whole process is not completely clear. Here, 59 Chinese women were engaged in this study, and divided into five groups: normal healthy group, HR-HPV infections group, low-grade intraepithelial neoplasia (LSIL) group, high-SIL(HSIL) group, and cervical cancer group. With the occurrence of HR-HPV infection and the development of cervical lesions, the diversity of vaginal microbiota species was increased, and the relative abundance of Lactobacillus (L.), the dominant bacteria in maintaining vaginal microecological balance, was decreased gradually. In contrast, the abundance of Actinobacteria in the four disease groups was significantly higher than that in normal group. Furthermore L. iners may be related to the serious progression of cervical cancer. After analyzing the whole process, we found that Gardnerella(G.), Atopobium(A.) and Dialister(D.) have important effects on both persistent HR-HPV infection and the pathogenesis of cervical cancer. In addition, PICRUSt2 and KEGG results showed that the KEGG pathways enriched by the predicted genes of vaginal microbiota in cancer group included metabolic diseases, endocrine system and immune systems when compared with that in normal group. These findings may provide insights into the pathogenesis of cervical cancer, and help to improve the early detection and prevention of cervical precancerous lesions.

Transcytosis mechanisms of cell-penetrating peptides: Cation-independent CC12 and cationic penetratin.

Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the development and further application of CPPs. Flow cytometry and laser confocal fluorescence microscopy were performed to detect the effects of different endocytosis inhibitors on the internalization of CC12 and penetratin in ARPE-19 cells. The co-localization of CPPs with the lysosome and macropinosome was detected via an endocytosis tracing experiment. The flow cytometry results showed that chlorpromazine, wortmannin, cytochalasin D, and the ATP inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. The laser confocal fluorescence results showed that oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. For penetratin, cytochalasin D and oligomycin had obvious inhibitory effects. The laser confocal fluorescence results indicated that oligomycin had the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, and they are typically dependent on ATP. The transport of penetratin could be partly achieved through the direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway.

Hsa_Circ_0001860 Promotes Smad7 to Enhance MPA Resistance in Endometrial Cancer via miR-520h.

Background: Medroxyprogesterone acetate (MPA) is one of the most commonly prescribed progestin for the treatment of endometrial cancer (EC). Despite initial benefits, many patients ultimately develop progesterone resistance. Circular RNA (circRNA) is a kind of noncoding RNA, contributing greatly to the development of human tumor. However, the role of circular RNA in MPA resistance is unknown. Methods: We explored the expression profile of circRNAs in Ishikawa cells treated with (ISK/MPA) or without MPA (ISK) by RNA sequencing, and identified a key circRNA, hsa_circ_0001860. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify its expression in MPA-resistant cell lines and tissues. CCK8, Transwell, and flow cytometry were used to evaluate the functional roles of hsa_circ_0001860 in MPA resistance. The interaction between hsa_circ_0001860 and miR-520 h was confirmed by bioinformatics analysis, luciferase reporter assay, and RNA pull-down assay. Results: The expression of hsa_circ_0001860 was significantly downregulated in MPA-resistant cell lines and tissues, and negatively correlated with lymph node metastasis and histological grade of EC. Functional analysis showed that hsa_circ_0001860 knockdown by short hairpin RNA (shRNA) promoted the proliferation, inhibited the apoptosis of Ishikawa cells, and promoted the migration and invasion of Ishikawa cells treated with MPA. Mechanistically, hsa_circ_0001860 promoted Smad7 expression by sponging miR-520 h. Conclusion: Hsa_circ_0001860 plays an important role in the development of MPA resistance in EC through miR-520h/Smad7 axis, and it could be targeted to reverse the MPA resistance in endometrial cancer.

RDM1 plays an oncogenic role in human ovarian carcinoma cells.

Ovarian cancer is one of the deadliest gynecological cancer, with a low overall 5-year survival rate. RDM1, RAD52 motif-containing protein 1, is sensitive to cisplatin, a common chemotherapy drug and it has an important role inDNA damage repair pathway. Until now, the effect of RDM1 in ovarian cancer is undiscovered. Here, clinical data shows that the tumour tissues of ovarian carcinoma patients with higher mRNA and protein expression of RDM1. Knockdown of RDM1 in ovarian carcinoma cells reduces cell proliferation and promotes apoptosis, consistent with the role RDM1 in the overexpression experiments. The research of xenograft mouse model shows stable knockdown of RDM1 significantly inhibits ovarian cancer tumour growth. These in vitro and in vivo results conclude that RDM1 plays an oncogenic role in human ovarian carcinoma. Interestingly, p53/RAD51/RAD52 signalling pathway can be regulated by RDM1, and the negative regulation of p53 by RDM1 may be one of major mechanisms for RDM1 to accomplish its oncogenic functions in ovarian carcinoma. Therefore, RDM1 may be a new target for the treatment of ovarian carcinoma.

AMPH1 functions as a tumour suppressor in ovarian cancer via the inactivation of PI3K/AKT pathway.

AMPH1, an abundant protein in nerve terminals, plays a critical role in the recruitment of dynamin to sites of clathrin-mediated endocytosis. Recently, it is reported to be involved in breast cancer and lung cancer. However, the impact of AMPH1 on ovarian cancer is unclear. In this study, we used gain-of-function and loss-of-function methods to explore the role of AMPH1 in ovarian cancer cells. AMPH1 inhibited ovarian cancer cell growth and cell migration, and promoted caspase-3 activity, resulting in the increase of cell apoptosis. In xenograft mice model, AMPH1 prevented tumour progression. The anti-oncogene effects of AMPH1 on ovarian cancer might be partially due to the inhibition of PI3K/AKT signalling pathway after overexpression of AMPH1. Immunohistochemistry analysis showed that the staining of AMPH1 was remarkably reduced in ovarian cancer tissues compared with normal ovarian tissues. In conclusion, our study identifies AMPH1 as a tumour suppressor in ovarian cancer in vitro and in vivo. This is the first evidence that AMPH1 inhibited cell growth and migration, and induced apoptosis via the inactivation of PI3K/AKT signalling pathway on ovarian cancer, which may be used as an effective strategy.

Medroxyprogesterone acetate causes the alterations of endoplasmic reticulum related mRNAs and lncRNAs in endometrial cancer cells.

BACKGROUND: Progestin is effective to promote endometrial cancer (EC) cells apoptosis, however, continuous progestin administration causes low level of progestin receptor B (PRB), further resulting in progestin resistance. Here, we performed microarray analysis on Ishikawa cells (PRB+) treated with medroxyprogesterone acetate (MPA) to explore the molecular mechanism underlying the inhibitory influence of MPA on PRB+ EC cells. METHODS: Microarray analysis was performed by using Ishikawa cells (PRB+) treated with MPA. Differentially expressed mRNA and long noncoding RNAs (lncRNAs) were identified. Furthermore, the functions of these mRNAs and lncRNAs were predicted by functional enrichment analysis. QRT-PCR was further performed to verify the microarray data. RESULTS: A total of 358 differentially expressed genes and 292 lncRNAs were identified in Ishikawa cells (PRB+) treated with MPA. QRT-PCR verified these data. Functional enrichment analysis identified endoplasmic reticulum (ER) stress as the key pathway involved in the inhibitory effect of MPA on EC cells. And the ER stress apoptotic molecule CHOP and ER stress related molecule HERPUD1 were both highly expressed in Ishikawa cells (PRB+) treated with MPA. Co-expression analysis showed lnc-CETP-3 was highly correlated with CHOP and HERPUD1, suggesting it might participate in ER stress pathway-related EC cell apoptosis caused by MPA. In addition, compared with untreated cells, lnc-CETP-3, CHOP and HERPUD1 were significantly up-regulated in Ishikawa cells (PRB+) treated with MPA, whereas they have no statistical significance in KLE cells (PRB-). CONCLUSIONS: MPA may activate ER stress by progesterone-PRB pathway to up-regulate CHOP expression, which may be one of the molecular mechanisms underlying the inhibitory effect of MPA on EC cells with PRB+. Lnc-CETP-3 might be involved in this process. These findings may provide therapeutic targets for EC patients with PRB-, and resistance-related targets to increase the sensitivity of MPA on EC cells.

RDM1 plays an oncogenic role in human lung adenocarcinoma cells.

RAD52 motif containing 1 (RDM1) is involved in DNA damage repair pathway and RDM1-/- cells increase sensitivity to cisplatin, a common chemotherapy drug. Lung cancer is the leading cause of cancer death worldwide. However, the role of RDM1 in lung cancer is unknown. Here, we find that the mRNA and protein expression levels of RDM1 are significantly increased in human lung tumors, especially in lung adenocarcinoma. The lung adenocarcinoma patients with higher mRNA expression of RDM1 show the worse clinical outcomes. Knockdown of RDM1 in lung adenocarcinoma cells reduces cell proliferation and promotes apoptosis, consistent with the role RDM1 in the overexpression experiments. Xenograft mouse model shows stable knockdown of RDM1 significantly inhibits lung adenocarcinoma tumor growth. These in vitro and in vivo results conclude that RDM1 plays an oncogenic role in human lung adenocarcinoma. Interestingly, P53/RAD51/RAD52 can be regulated by RDM1, and the negative regulation of P53 by RDM1 may be one of major mechanisms for RDM1 to accomplish its oncogenic functions in lung adenocarcinoma. Therefore, RDM1 may be a new target for the treatment of lung adenocarcinoma.

Preferentially Expressed Antigen of Melanoma Prevents Lung Cancer Metastasis.

Lung cancer is the most common cause of cancer death worldwide. The poor survival rate is largely due to the extensive local invasion and metastasis. However, the mechanisms underlying the invasion and metastasis of lung cancer cells remain largely elusive. In this study, we examined the role of preferentially expressed antigen of melanoma (PRAME) in lung cancer metastasis. Our results show that PRAME is downregulated in lung adenocarcinoma and lung bone metastasis compared with normal human lung. Knockdown of PRAME decreases the expression of E-Cadherin and promotes the proliferation, invasion, and metastasis of lung cancer cells by regulating multiple critical genes, most of which are related to cell migration, including MMP1, CCL2, CTGF, and PLAU. Clinical data analysis reveals that the expression of MMP1 correlates with the clinical features and outcome of lung adenocarcinoma. Taken together, our data demonstrate that PRAME plays a role in preventing the invasion and metastasis of lung adenocarcinoma and novel diagnostic or therapeutic strategies can be developed by targeting PRAME.

Identification of Preferentially Expressed Antigen of Melanoma as a Potential Tumor Suppressor in Lung Adenocarcinoma.

BACKGROUND Preferentially expressed antigen of melanoma (PRAME) is known as a tumor-associated antigen that is altered in a variety of malignancies, including lung cancer. However, the role of PRAME in lung cancer remains unclear. MATERIAL AND METHODS We analyzed the expression of PRAME in human lung adenocarcinomas and studied the function of PRAME using small interfering RNA (siRNA)-induced gene knockdown in lung cancer cell lines PC9 and A549. RESULTS We found that PRAME expression is down-regulated in lung adenocarcinomas. Knockdown of PRAME promoted proliferation and suppressed apoptosis of PC9 and A549 cells. CONCLUSIONS In line with its roles in controlling cell growth, RPAME regulates multiple critical cell-growth related genes, including IGF1R oncogene. IGF1R up-regulation contributes to increase of cell growth upon the knockdown of PRAME. Taken together, our results suggest that PRAME has inhibitory roles in lung cancer.