HEC-1B Cells Research Articles

Identification of GPNMB in endometrial cancer based on pan-cancer analysis and in vitro validation

BackgroundGPNMB is a type I transmembrane protein, and emerging evidence supports the relationship between GPNMB and cancers.ObjectiveThrough a comprehensive pan-cancer analysis, we examined the expression levels, prognostic significance, and mutation profiles of GPNMB in different cancer types. Subsequently, utilizing in vitro experiments, we elucidated the impact of GPNMB in endometrial cancer (EC).MethodsTIMER2, GEPIA2, UALCAN and cBioPortal were used to analyze the expression pattern, prognostic values, and mutation status of GPNMB. HEC-1B and Ishikawa cells were used to conduct in vitro analyses of GPNMB overexpression. GeneMANIA and TIMER2 were used to evaluate the potential functions and correlations between GPNMB expression and tumor-infiltrating immune cells in EC.ResultsGPNMB was found to be highly expressed in multiple cancers, where it was associated with poor prognosis. Additionally, GPNMB was downregulated at both mRNA and protein levels in EC. Overexpression of GPNMB inhibited the proliferation, migration, and invasion of HEC-1B and Ishikawa cells. Functional analysis showed that GPNMB was enriched in pathways associated with regulation of plasma lipoprotein particle levels. The expression of GPNMB was positively connected with B cell, CD8+ T cell, CD4+ T cell, Macrophage, Neutrophil, and Dendritic cell levels.ConclusionThrough pan-cancer analysis, we identified the antitumor effect of GPNMB in EC and predicted the potential mechanisms between GPNMB expression and EC.

MiR-200a-3p promotes the malignancy of endometrial carcinoma through negative regulation of epithelial-mesenchymal transition

BackgroundmiR-200a-3p is involved in the progression of malignant behavior in various tumors, and its mechanism of action in endometrial cancer is speculated to be related to epithelial-mesenchymal transition (EMT). Therefore, this study explored the metastatic mechanism of miR-200a-3p and EMT in endometrial cancer, with the aim of identifying potential therapeutic targets.MethodsqRT-PCR was used to analyze miR-200a-3p expression in HEC-1B and Ishikawa cell lines. The cell proliferation assay, transwell assay, and cell scratch test were used to assess changes in the malignant phenotypes of cells after regulating miR-200a-3p expression. Changes in EMT-related protein zinc finger E-box binding homeobox 1 (ZEB1) were detected after regulating miR-200a-3p expression. An endometrial carcinoma transplantation mouse tumor model was constructed, and multiple EMT-related proteins were examined.ResultsThe expression of miR-200a-3p and ZEB1 in the endometrial cancer cell lines was higher than in normal endometrial epithelial cell lines (P < 0.05). After silencing miR-200a-3p, the expression of EMT-related protein ZEB1 increased, indicating a negative correlation. Simultaneously, the proliferation, invasion, and metastasis of endometrial cancer cells were significantly enhanced. After miR-200a-3p overexpression, the corresponding malignant phenotype was reversed (P < 0.05). In in vivo experiments, the degree of tumor malignancy and the expression level of EMT-related proteins were significantly reduced in the miR-200a-3p mimic group (P < 0.05).ConclusionThis study found that miR-200a-3p is a promising target, regulating the EMT process and promoting endometrial cancer progression.

Abstract 7563: Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line

Abstract Objectives: PARP inhibitor is effective in treating DNA repair deficiency endometrial cancer. Endometrial cancer (EC) is one of the most common gynecological cancers in developed countries and its incidence is increasing, and the prognosis of patients with early stage and low-risk EC is very good, but the prognosis of progressive and recurrent EC is poor. Novel PARP inhibitor JPI-547 is a PARP and tankyrase dual inhibitor, and in cancer cells, tankyrase inhibitor plays an important role in DNA repair, telomere maintenance, and cell survival. This experiment was conducted to determine whether the reactivity of the PTEN mutant EEC was increased by using the Novel PARP inhibitor. Method: Human endometrioid endometrial cancer cell lines, Hec-1A, Hec-1B and Ishikawa were obtained. Cell viability assay for the drugs Olaparib and JPI-547 were assessed. Colonies consisting of at least 50 cells were counted. The number of dead cells was counted in five different areas of the hemocytometer, and the experiment was performed three times. The percentage of dead cells was analyzed compared to the untreated group. We confirmed the efficacy by siRNA transfection and immunofluorescence. Result: When treated with Olaparib, there was no change in cell viability in the rest of the cells except for Ishikawa cells, which are PTEN mutations. It decreased by 35.4% in Ishikawa cells, whereas when treated with JPI-547, all cell lines Hec-1A, Hec-1B, and Ishikawa cell viability decreased by 28%, 34.3%, and 48.7%, respectively. PTEN knock-down reduced cell visibility by 28% when treated with olaparib, whereas when treated with JPI-547, Hec-1A (siCont) and Hec-1A (siPTEN) decreased by 26% and 42%, respectively, in all cells regardless of whether PTEN knock-down is present. When treated with Olaparib, dead cells were identified only in PTEN knock-down cells, increasing by 22%. When treated with JPI-547, dead cells increased by 25.5% and 33% in all cells of Hec-1A (siCont) and Hec-1A (siPTEN), respectively. It was confirmed that JPI-547 can reduce cell viability regardless of the formation of Rad51 according to PTEN. Conclusion: JPI-547, a novel PARP inhibitor, showed good efficacy in PTEN mutation and PTEN-wild-type EEC cell lines than in conventional PARP inhibitor. The effect of reducing survival of ECC cell line with JPI-547 was confirmed with or without PTEN mutation. Further evaluation and in vivo experiments are needed to prove the efficacy of JPI-547 in EC. Citation Format: Shin-Wha Lee, So-Hyun Nam, Su-Bin Park. Efficacy of JPI-547, novel dual PARP inhibitor, in endometrial cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7563.

#39 : Identification of Pharmacologically Active Small Molecules that Induce Spheroid Attachment and Embryo Implantation In Vitro and In Vivo

Background and Aims: Human endometrium is receptive to embryo implantation during the mid-secretory phase of the menstrual cycle. The in vitro spheroid-endometrial epithelial cells co-culture model was used in this study. We hypothesized that the small molecules from thelibrary of pharmacologically active compounds (LOPAC) can effectively enhance embryo implantation. Method: We established a high-throughput in-vitro spheroid-endometrial epithelial (BeWo-Ishikawa) cells coculture model to screen 1280 LOPAC. The top 20 small molecules which increased spheroid attachment rate were further studied in the non-receptive cell lines (HEC-1B and AN3CA). Endometrial cells were either treated with 1% DMSO (vehicle control), AZA at 20 μM (positive control) or LOPAC at 10 μM for 48 h. The trophoblastic BeWo spheroids were generated by AggreWell and the number of attached spheroids after coculture were counted. The effect on mice was studied by trans-cervical injection of LOPAC on 1.5-day postcoital (dpc) and the number of implantation sites on 5.5 dpc were evaluated. Results: Screening of 1280 LOPAC identified 255 molecules increased, and 173 molecules significantly decreased the spheroid attachment rate. The top 20 potential molecules were further studied and three of them were found to significantly increase spheroid attachment in the HEC-1B cells. Another molecule J was found to suppress spheroid attachment in receptive Ishikawa and RL95-2 cells while increase spheroid attachment significantly in non-receptive HEC-1B and AN3CA cells. In mice, molecule J could significantly increase the number of implantation sites on the treated sides when compared with non-treated sides. Conclusion: Three molecules were found to increase spheroid attachment in receptive Ishikawa cells and non-receptive HEC-1B cells. Another molecule J can increase the spheroid attachment in non-receptive HEC-1B and AN3CA cells and increase embryo implantation in ICR mice. [This study is partially supported by the GRF grants 15162211 and 17120720 to KFL]

Exploring the Potential of Thymoquinone-Stabilized Selenium Nanoparticles: In HEC1B Endometrial Cancer Cells Revealing Enhanced Anticancer Efficacy.

The aim of this research is to examine the potential anticancer properties of thymoquinone (TQ)-encapsulated selenium nanoparticles (TQ-SeNPs) in HEC1B endometrial carcinoma cells. TQ-SeNPs were synthesized, and their size, morphology, and elemental analysis were characterized. Morphological changes were examined by using scanning electron microscopy (SEM). The cytotoxicity and viability of nanothymoquinone were assessed by the XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5 carboxanilide) assay. Gene expressions and protein levels of the mitogen-activated protein kinase (MAPK) signaling pathway were analyzed by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The decrease in the viability of HEC1B endometrial carcinoma cells was observed in a time- and dose-dependent manner. HEC-1B cells were treated with TQ-SeNP at 40-640 μg/mL concentrations and time intervals, and their viability was assessed by XTT assay. IC50 doses of TQ-SeNP in HEC1B cells were detected as 526.45 μg/mL at 48th hour. ELISA indicated that TQ-SeNP treatment reduced the level of p38 MAPK. ERK2, MEK2, and NFKB (p65) mRNA expressions were decreased in the dose group administered TQ-SeNP at the 48th hour compared to that in the control group. However, it was not significant. The novel nanoparticle showed an antiproliferative effect in endometrial cancer cells. However, further studies are needed to increase the anticancer activity of the cell in the TQ-SeNP interaction.

MiR-196a-5p facilitates progression of estrogen-dependent endometrial cancer by regulating FOXO1.

Estrogen-dependent endometrial cancer mainly occurs in younger pre-menopausal and post-menopausal women and threatens their health. Recently, microRNAs (miRNAs) have been considered as novel targets in endometrial cancer treatment. Therefore, we aimed to explore the effect of miRNA (miR)-196a-5p in estrogen-dependent endometrial cancer. 17β-estradiol (E2; 2.5, 5, 10 and 20 nM) was used to treat RL95-2, HEC-1B and ECC-1 cells followed by cell viability assessment using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The level of miR-196a-5p was measured by reverse transcription-quantitative PCR (RT-qPCR). We then transfected miR-196a-5p mimic/inhibitor and Forkhead box protein O1 (FOXO1) small interfering RNA (siRNA) into E2-treated cells. Apoptotic cells were measured by flow cytometry. Wound healing and Transwell assays were implemented to assess migration and invasion. Bioinformatics and luciferase reporter assays were applied to confirm the interaction between miR-196a-5p and FOXO1. Immunoblotting determined the levels of FOXO1, Bcl-2, Bax, Caspase 3. E2 promoted cell viability and miR-196a-5p expression in RL95-2 and ECC-1 cells. miR-196a-5p mimic enhanced cell viability, migration and invasion but suppressed apoptosis and FOXO1, whilst miR-196a-5p inhibitor blocked these processes. In addition, miR-196a-5p upregulated Bcl-2, but down regulated Bax and Caspase 3 expression, an effect that was reversed by miR-196a-5p inhibitor. We determined that miR-196a-5p targeted FOXO1, and that si-FOXO1 blocked the effects of miR-196a-5p inhibitor on viability, apoptosis, migration and invasion of E2-treated RL95-2 and ECC-1 cells. Our findings suggested potential diagnostic and therapeutic applications for miR-196a-5p and its FOXO1 target in patients suffering from estrogen-dependent endometrial cancer.

Vitexin suppresses the proliferation, angiogenesis and stemness of endometrial cancer through the PI3K/AKT pathway

Context Endometrial cancer is a common gynecologic malignancy. Vitexin is an active flavonoid compound with an antitumor function. Objective This study elucidated the role of vitexin in endometrial cancer development and clarified the potential mechanism. Materials and methods The toxicity of vitexin (0–80 μM) treatment for 24 h on HEC-1B and Ishikawa cells was tested utilizing the CCK-8 assay. Endometrial cancer cells were divided into vitexin 0, 5, 10, and 20 μM groups. Cell proliferation, angiogenesis and stemness in vitro after treatment with vitexin (0, 5, 10, 20 μM) for 24 h were evaluated using the EdU staining assay, tube formation assay and sphere formation assay, respectively. Twelve BALB/c mice were grouped into control and vitexin (80 mg/kg) groups to monitor tumour growth for 30 days. Results Vitexin suppressed cell viability of HEC-1B (IC50 = 9.89 μM) and Ishikawa (IC50 = 12.35 μM) cells. The proliferation (55.3% and 80% for HEC-1B; 44.7% and 75% for Ishikawa), angiogenesis (54.3% and 78.4% for HEC-1B; 47.1% and 68.2% for Ishikawa) and stemness capacity (57.2% and 87.3% for HEC-1B; 53.4% and 78.4% for Ishikawa) of endometrial cancer cells were inhibited by 10 and 20 μM vitexin. Furthermore, the inhibitory effects of vitexin on endometrial cancer were reversed by PI3K/AKT agonist 740Y-P (20 μM). Moreover, the xenograft tumour experiment lasting for 30 days proved that vitexin (80 mg/kg) blocked tumour growth of endometrial cancer in vivo. Discussion and conclusions Vitexin has therapeutic potential on endometrial cancer, which supports further clinical trials.

Niclosamide causes lysosome-dependent cell death in endometrial cancer cells and tumors.

Endometrial cancer is the most common female cancer showing continuous rise in its incidence and mortality rate. Despite the extensive research efforts in cancer therapeutics, still there is a lack of effective treatment options and the outcome is poor for patients with advanced and recurrent endometrial cancers. In this study, we aimed to evaluate the efficacy of niclosamide (NIC) against endometrial cancer. NIC is an FDA-approved anti-helminthic drug, which has been recently extensively studied as a potent anti-cancerous agent in several cancers. The anti-cancerous activity of NIC was analyzed in-vitro (ANC3A, Hec1B, and Ishikawa endometrial cancer cell lines) by cell viability-, soft agar-, invasion- and migration- assay. The action mechanism of NIC was demonstrated by western blot analysis and immune-fluorescence imaging and validated by specific inhibitors. The in-vivo efficacy of NIC was studied in the Ishikawa xenograft animal model. NIC effectively suppressed the viability (IC50<1 μM), colony formation ability, migration, and invasion of all endometrial cancer cells tested. We demonstrated that NIC inhibited AKT/mTOR signaling pathway and induced apoptosis and autophagy in endometrial cancer cells. Further study demonstrated that although NIC induced autophagosome formation, it inhibits autolysosome formation. In addition, we observed that NIC induced BAX co-localization with lysosome and inhibited Cathepsin B maturation from pro-cathepsin B, thereby inducing the lysosomal membrane permeability and release of hydrolytic enzymes from the lysosome to cytosol, which eventually contributed cell death. NIC also inhibited tumor weight and volume in the Ishikawa xenograft animal model without having any evidence of toxicity. Due to its potent anti-cancerous activity and safety profile, NIC seems to be a promising agent for human endometrial cancer therapeutics.

LncRNA XIST/miR‑129‑2‑3p axis targets CCP110 to regulate the proliferation, invasion and migration of endometrial cancer cells.

Centromere coiled-coil protein 110 (CCP110) plays a role in the development of several types of cancer; however, its regulatory mechanism and role in endometrial cancer is unclear. The present study revealed that CCP110 is regulated by a signaling pathway involving microRNA (miR/miRNA)-129-2-3p and the long non-coding RNA (lncRNA) X-inactive-specific transcript (XIST), and plays a role in controlling the proliferation, migration and invasion of endometrial cancer cells. CCP110 was upregulated in human endometrial cancer tissues, as revealed by immunohistochemistry, and high expression of the protein was related to reduced overall survival of the patients. Genetic knockdown of CCP110 by small interfering RNA promoted apoptosis and suppressed the proliferation, migration, invasion and colony formation of endometrial cancer cells significantly in the endometrial cancer Ishikawa and HEC-1B cell lines, as assessed by flow cytometry, and Cell Counting Kit-8, Transwell and colony formation assays. A bioinformatics analysis and luciferase reporter assay revealed that CCP110 is a target of miR-129-2-3p. Overexpression of miR-129-2-3p mimic fragments inhibited the proliferation, migration and invasion of endometrial cancer cells significantly, while co-overexpression of CCP110 counteracted these inhibitory effects. The expression level of the lncRNA XIST was upregulated significantly in endometrial cancer tissues, as assessed by reverse transcription-quantitative PCR assay, while that of miR-129-2-3p was downregulated significantly. A bioinformatics analysis and luciferase reporter assay showed that XIST could inhibit miR-129-2-3p via a miRNA sponge effect. Furthermore, co-overexpression of XIST antagonized the inhibitory effect of the miR-129-2-3p mimic on the luciferase reporter gene signal and protein expression of CCP110. Co-overexpression of XIST also abolished the inhibitory effect of the miR-129-2-3p mimic on the proliferation, migration and invasion of endometrial cancer cells. Overall, these data identified a novel regulatory mechanism of CCP110 involving XIST and miR-129-2-3p, which affected the development of endometrial carcinoma. CCP110, XIST and miR-129-2-3p could represent novel targets for the clinical treatment of endometrial cancer.

Apolipoprotein E enhances migration of endometrial cancer cells byactivating the ERK/MMP9 signaling pathway

To study the role of apolipoprotein E (APOE) in regulating endometrial cancer metastasis and explore the signaling pathway in the regulatory mechanism. Human endometrial cancer cell line HEC-1B was transfected with a control siRNA (siCtrl) or a specific siRNA targeting APOE (siAPOE) or with either pEGFP-N1 plasmid or an APOEoverexpressing plasmid. The changes in migration, proliferation, apoptosis and cell cycle of the transfected cells were examined using wound healing assay, Transwell migration assay, MTT assay, flow cytometry, and Hoechst staining. The activity of the ERK/MMP9 signaling pathway in the transfected cells was assessed using RT-qPCR and Western blotting. The expression level of APOE in clinical specimens of endometrial cancer tissues were detected using immunohistochemistry and its correlation with differentiation of endometrial cancer tissues was analyzed. Wound healing assay and Transwell migration assay showed that compared with those in siCtrl group, HEC-1B cells transfected with siAPOE showed significantly reduced migration ability (P < 0.05), whereas APOE overexpression significantly promoted the migration of the cells (P < 0.05). Neither APOE knockdown nor overexpression produced significant effects on HEC-1B cell proliferation as shown by MTT assay and flow cytometry. Hoechst staining revealed that transfection with siAPOE did not significantly affect apoptosis of HEC-1B cells. APOE knockdown obviously reduced and APOE overexpression enhanced ERK phosphorylation and MMP9 expression in HEC-1B cells (P < 0.05). Treatment with U0126 partially reversed the effects of APOE overexpression on ERK phosphorylation, migration and MMP9 expression in HEC-1B cells (P < 0.05). APOE is highly expressed in clinical samples of endometrial cancer tissues as compared with the adjacent tissues. APOE is highly expressed in endometrial cancer tissues to promote cancer cell migration by enhancing ERK phosphorylation and MMP9 expression.

Anti-tumor effect of Wasabi component, 6-(methylsulfinyl) hexyl isothiocyanate, against endometrial carcinoma cells

PurposeWasabi is a traditional plant seasoning with an anti-septic function. Recent studies revealed several functions of Wasabi, such as anti-inflammation; however, the anti-tumor effect against endometrial carcinoma (EMC) cells has not been examined. In the present study, we investigated the anti-tumor effect of 6-(methylsulfinyl) hexyl isothiocyanate (6-MITC), a major chemical compound of Wasabi, against various EMC cell lines in vitro and in vivo.MethodsThe effect of 6-MITC on cell viability was measured by the WST-1 assay in EMC and HUVEC cells. The impact of 6-MITC oral administration in nude mice was measured to assess the growth of the EMC xenograft and natural killer (NK) cell activity in the spleen.ResultsThe addition of 6-MITC suppressed the proliferation of EMC cells (Ishikawa, HEC265, HEC108, KLE, and HEC1B) dose-dependently, but not HUVEC cells. 6-MITC (5 µM) enhanced the cisplatin sensitivity of EMC cells. 6-MITC induced apoptosis in a dose-dependent fashion in EMC cells other than HEC1B cells and was associated with increased expression of cleaved-caspase3 and decreased expression of BCL2. Oral administration of 6-MITC (2 and 4 µmol/kg) to Ishikawa and HEC1B xenografting mice resulted in a reduced tumor volume compared with the control (P < 0.05, 4 µmol/kg). Immunohistochemical staining of resected tumors revealed increased expression of Ki-67 and reduced cleaved-caspase3. Furthermore, 6-MITC treatment enhanced NK cell activity, especially when administered before tumor xenografting.ConclusionThese results indicate that 6-MITC has a marked anti-tumor effect against EMC cells and a novel effect to enhance NK cell activity. These effects suggest the therapeutic potential of 6-MITC.

Tripartite motif containing 28 (TRIM28) promotes the growth and migration of endometrial carcinoma cells by regulating the AKT/mTOR signaling pathway.

Although the medical science has been developed for decades, the molecular mechanism of endometrial cancer (EC) is not yet completely clear. Previous studies have shown that the tripartite motif containing 28 (TRIM28) plays a crucial role in tumor development. However, TRIM28 is rarely studied in EC, and its role and mechanism need to be further determined. This study was aimed to delve into the related molecular mechanism underling the role of TRIM28 in EC cell growth and migration. qPCR assays and Western blot assays revealed that the expression level of TRIM28 was higher in EC tissues or cell lines (HEC1B, AN3CA, and Ishikawa) than normal tissue or human endometrial epithelial cells (hEEC), respectively. Then, CCK-8 cell viability assay and clone formation assay were performed in HEC1B and AN3CA cell lines after overexpression or knockdown of TRIM28. The results verified that suppression of TRIM28 expression inhibited the proliferation of EC cells. The wound scratch healing assay and transwell assay were performed in HEC1B and AN3CA cell lines after overexpression or knockdown of TRIM28. The results showed that suppression of TRIM28 expression inhibited the invasion and migration of EC cells. Finally, the Western blot assays hinted that overexpression or knockdown of TRIM28 in HEC1B and AN3CA cell lines would promote or inhibit the phosphorylation of AKT and mTOR protein. These findings indicated that TRIM28 promoted the growth and migration of EC cells via regulating the AKT/mTOR pathway.

Methyltransferase 3 beta (DNMT3B) inhibition by decitabine promotes endometrial carcinoma cell apoptosis by autophagy induction

Autophagy regulates endometrial cancer cell apoptosis and inhibits endometrial cancer development; however, the specific underlying mechanisms are unclear. The effects of DNA methylation on endometrial cancer cell autophagy were explored in HEC-1B cells using decitabine, a DNA methylase inhibitor. Real-time fluorescence quantitative PCR (RT-qPCR) and western blot (WB) analyses were used to detect the mRNA and protein expression of DNA methylase (DNMT3B), autophagy-related genes LC3B and Beclin-1, and apoptosis-related genes Bax and Bcl-2. High-purity and high-quality RNA was obtained by nano-magnetic bead extraction, to ensure highly accurate RT-qPCR and WB results. MTT cell viability assay, scratch assay, and Transwell migration assay were utilized to detect the proliferation and migration of HEC-1B cells. The results showed that decitabine significantly inhibited the expression of DNMT3B protein and mRNA, with 25 μM decitabine being the most effective. In subsequent experiments, this concentration of decitabine was used. The low DNMT3B expression significantly inhibited the expression of Beclin-1 and LC3B proteins (***P &lt;0.001 and *P &lt;0.05) and mRNA (*P &lt;0.05 and *P &lt;0.05). Decitabine promoted the mRNA and protein expression of apoptotic factor Bax (*P &lt; 0.05 and *P &lt; 0.05), while inhibiting the mRNA protein expression of anti-apoptotic factor Bcl-2 (***P &lt;0.001 and *P &lt;0.05). MTT assay results showed decreased proliferation of HEC-1B cells following inhibition of DNMT3B expression by decitabine, which was significantly different from that of the NC group (*P &lt;0.05). The results of the cell scratch and Transwell migration assays showed reduced migration ability of HEC-1B cells compared to that observed in the NC group (**P &lt;0.01 and *P &lt;0.05). These results show that decitabine inhibits DNMT3B expression, and low DNMT3B levels regulate the expression of autophagy- and apoptosis-related factors, thereby inhibiting the proliferation and migration of endometrial cancer cells.

Retracted: Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation.

[This retracts the article DOI: 10.1155/2022/7052066.].

C/D box small nucleolar RNA SNORD104 promotes endometrial cancer by regulating the 2ʹ-O-methylation of PARP1

BackgroundSmall nucleolar RNAs (snoRNAs) are dysregulated in many cancers, although their exact role in tumor genesis and progression remains unclear.MethodsThe expression profiles of snoRNAs in endometrial cancer (EC) tissues were analyzed using data from The Cancer Genome Atlas, and SNORD104 was identified as an upregulated snoRNA in EC. The tumorigenic role of SNORD104 in EC was established in CCK8, colony formation, EdU, apoptosis, Transwell, and in vivo xenograft experiments. The molecular mechanisms of SNORD104 were analyzed by RNA immunoprecipitation (RIP), Nm-seq, RTL-P assay, RNA stability assay, qRT-PCR, and western blotting.ResultsAntisense oligonucleotide (ASO)-mediated knockdown of SNORD104 in Ishikawa cells significantly inhibited their proliferation, colony formation ability, migration, and invasion in vitro and increased apoptosis. On the other hand, overexpression of SNORD104 promoted EC growth in vivo and in vitro. RIP assay showed that SNORD104 binds to the 2ʹ-O-methyltransferase fibrillarin (FBL), and according to the results of Nm-seq and RTL-P assay, SNORD104 upregulated PARP1 (encoding poly (ADP-ribose) polymerase 1) 2ʹ-O-methylation. The binding of FBL to PARP1 mRNA was also verified by RIP assay. Furthermore, SNORD104 expression was positively correlated with PARP1 expression in EC tissues. In the presence of actinomycin D, SNORD104 increased the stability of PARP1 mRNA and promoted its nuclear localization. Finally, silencing FBL or PARP1 in the HEC1B cells overexpressing SNORD104 inhibited their proliferative and clonal capacities and increased apoptosis rates.ConclusionsSNORD104 enhances PARP1 mRNA stability and translation in the EC cells by upregulating 2ʹ-O-methylation and promotes tumor growth.

Effects of glabridin on cell proliferation and long non-coding RNA expression in HEC-1B cells

Purpose: Endometrial cancer is one of the most common gynecological cancers in the world. Glabridin is a main isoflavone in Glycyrrhiza glabra (licorice) root. It has several therapeutic effects such as anti-proliferative and anti-inflammatory. Long non-coding RNAs (LncRNAs) play a role in a variety of cellular processes, and their abnormal expression may contribute to tumor development and progression. In this study, the effects of glabridin on LncRNAs gene expression and viability of HEC-1B human endometrial cancer cell lines have been investigated. &#x0D; Materials and methods: Glabridin was applied to HEC-1B cells in concentrations of 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, and 80 μM. Glabridin's effect on HEC-1B cell proliferation was also evaluated using MTS assay. Expression profiles of LncRNAs such as H19, RNU43, LNC-MYC-3:1 and ABCC5-AS1:1 were determined by real-time PCR. &#x0D; Results: Glabridin reduced the viability of HEC-1B cells in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) dose in HEC-1B cells was detected to be 21.32 μM and 13.5 μM at the 24th and 48 hours, respectively. Glabridin has been observed to cause a significant decrease in the expression of H19 and RNU43 while increasing in the expression of LNC-MYC-3:1 and ABCC5-AS1:1.&#x0D; Conclusion: Glabridin could induce HEC-1B cell death by regulating LncRNAs expression. As a result, glabridin is a potential candidate for a more effective therapeutic agent against human endometrial cancer.

Small Nucleolar RNA and C/D Box 15B Regulate the TRIM25/P53 Complex to Promote the Development of Endometrial Cancer.

Background Endometrial cancer is associated with a high mortality rate, which warrants the identification of novel diagnostic markers and therapeutic targets. The aim of this study is to evaluate the role of SNORD15B in the development of endometrial cancer and explore the potential underlying mechanisms. Methods Bioinformatics was used to analyze the expression level and prognostic relevance of SNORD15B in endometrial cancer. The Ishikawa and HEC-1B cells were respectively transfected with SNORD15B expression plasmid and an antisense oligonucleotide, or the corresponding empty vector and a nonspecific sequence. The malignant phenotype of the suitably transfected cells was assessed by standard in vitro functional assays and the establishment of in vivo xenografts. The expression levels of the specific markers were analyzed with RT-qPCR and western blotting. The subcellular localization of P53 was determined by analyzing the nuclear and cytoplasmic fractions. RIP, Co-IP, and immunohistochemistry were performed as per standard protocols. Results SNORD15B was overexpressed in the endometrial cancer tissues and correlated to a poor prognosis. Ectopic expression of SNORD15B in Ishikawa cells inhibited apoptosis, increased the proliferation, invasion, and migration in vitro, and enhanced their tumorigenicity in vivo. SNORD15B overexpression also upregulated TRIM25 and accelerated P53 accumulation in the cytoplasm of the endometrial cancer cells. Conclusion SNORD15B functions as an oncogene in endometrial cancer by targeting the TRIM25/P53 complex and blocking the nuclear translocation of P53.

DSCAM-AS1 Long Non-Coding RNA Exerts Oncogenic Functions in Endometrial Adenocarcinoma via Activation of a Tumor-Promoting Transcriptome Profile.

Accumulating evidence suggests that lncRNA DSCAM-AS1 acts tumor-promoting in various cancer entities. In breast cancer, DSCAM-AS1 was shown to be the lncRNA being most responsive to induction by estrogen receptor α (ERα). In this study, we examined the function of DSCAM-AS1 in endometrial adenocarcinoma using in silico and different in vitro approaches. Initial analysis of open-source data revealed DSCAM-AS1 overexpression in endometrial cancer (EC) (p < 0.01) and a significant association with shorter overall survival of EC patients (HR = 1.78, p < 0.01). In EC, DSCAM-AS1 was associated with endometrial tumor promotor gene PRL and with expression of ERα and its target genes TFF1 and PGR. Silencing of this lncRNA by RNAi in two EC cell lines was more efficient in ERα-negative HEC-1B cells and reduced their growth and the expression of proliferation activators like NOTCH1, PTK2 and EGR1. DSCAM-AS1 knockdown triggered an anti-tumoral transcriptome response as revealed by Affymetrix microarray analysis, emerging from down-regulation of tumor-promoting genes and induction of tumor-suppressive networks. Finally, several genes regulated upon DSCAM-AS1 silencing in vitro were found to be inversely correlated with this lncRNA in EC tissues. This study clearly suggests an oncogenic function of DSCAM-AS1 in endometrial adenocarcinoma via activation of a tumor-promoting transcriptome profile.

Exploration of the Molecular Mechanism of Danzhi Xiaoyao Powder in Endometrial Cancer through Network Pharmacology.

Endometrial cancer (EC) is a common malignant tumor of the female reproductive system. Current treatments such as surgery and long-term hormone therapy are ineffective and have side effects. Danzhi Xiaoyao powder (DXP) can inhibit the growth of EC cells and induce apoptosis, but the pharmacological and molecular mechanisms of anticancer effects are still unclear. In this study, active components and potential targets of DXP were obtained from public databases. Protein effects and regulatory pathways of common targets were analyzed by protein-protein interaction (PPI), GO and KEGG. The results of network pharmacology showed that there are 87 common targets between EC and DXP. GO enrichment analysis showed that these targets were associated with response to oxidative stress, response to nutrient levels, hormone receptor binding and nuclear hormone receptor binding, etc. The results of KEGG analysis indicated that IL-17, TNF, PI3K/AKT, and RAS/RAF/MEK/ERK (ERK) signaling pathway were enriched in the anti-EC of DXP. Additionally, we cultured HEC-1B and KLE cells for validate experiments. DXP showed an inhibition of proliferation, migration, and cell cycle of both cells. Moreover, the expression of RAS, p-RAF, p-MEK, ERK, and p-ERK related proteins were downregulated. In conclusion, DXP might inhibit the proliferation of EC cells via apoptosis. Furthermore, DXP-induced inhibition of EC development might involve RAS/RAF/MEK/ERK pathway.

Isoalantolactone exerts anticancer effects on human HEC-1-B endometrial cancer cells via induction of ROS mediated apoptosis and inhibition of MEK/ERK signalling pathway.

Isoalantolactone has been shown to inhibit the growth of different cancer cells. The objective of the present study was to evaluate the effects of isoalantolactone on the proliferation of endometrial cancer HEC-1-B cells. Results showed that isoalantolactone suppressed the proliferation of HEC-1-B cells in a concentration-dependent manner and exhibited an IC50 of 10 µM. The cytotoxic effects of isoalantolactone were relatively lower against the normal THESC cells. Mechanistic studies revealed apoptosis to be responsible for the isoalantolactone mediated antiproliferative effects. Annexin V/PI assay showed that the percentage of the apoptotic HEC-1B cells increased from 3.74% in untreated cells to 28.9% at 20 µM isoalantolactone. The expression of Bax was significantly increased and that of Bcl-2 was decreased in isoalantolactone treated HEC-1B cells. Analysis of ROS levels revealed that the ROS levels of HEC-1B cells increased with the increase in concentration of isoalantolactone. The ROS levels increased to 210% of the control at 20 µM isoalantolactone. The wound healing and the transwell assays showed that migration and invasion of the HEC-1B cells was significantly decreased upon isoalantolactone treatment. Finally, the effects of isoalantolactone were also evaluated on the MEK/ERK signalling pathway. It was found that isoalantolactone could concentration-dependently block the expression of p-MEK and p-ERK. Taken together, the results suggest that isoalantolactone could prove to be a lead molecule in the development of chemotherapy for endometrial cancer.