Upregulated E-cadherin Expression Research Articles

Suppression of epithelial-mesenchymal transition and SIRT1/AKT signaling pathway in breast cancer by montelukast

Breast cancer is usually associated with metastatic features, poor prognosis, and high mortality. The epithelial-mesenchymal transition (EMT) process has been implicated in the initiation and metastasis of breast cancer. The study aimed to investigate the possible role of montelukast (Mont), the cysteinyl leukotriene receptor (CystLT1R) antagonist, in mitigating EMT in triple-negative breast cancer (TNBC) (in vitro study) and solid Ehrlich carcinoma (SEC) bearing mice (in vivo study) as well as to clarify the underlying molecular mechanisms in the presence and absence of sirtuin-1 inhibitor (sirtinol; Sirt). TNBC MDA-MB-231 cells were treated with either 5μM Mont or 25μM Sirt or both for 48h. Alternatively, SEC cells were inoculated in mice to induce breast cancer. After 12days, the mice were divided into four groups: Untreated SEC group (vehicle), Sirt group (1mg/kg), Mont group (10mg/kg), and cotreatment Sirt/Mont group. The mice groups received the assigned treatment for the consequent 16days. Mont and/or Sirt decreased cell proliferation, migration and suppressed EMT in both in vitro and in vivo experiments. All treatments downregulated sirtuin-1 and vimentin expression but upregulated E-cadherin expression. Furthermore, all treatments retarded angiogenesis as evidenced by decreased VEGF expression. These findings were associated with suppressing active protein kinase B (p-AKT). Cotreatment with Sirt and Mont proved more effective anti-tumor activity in TNBC cell line and in SEC bearing mice than either treatment alone, which could be attributed to the inhibition of sirtuin-1 and AKT- activated pathways, with the subsequent inhibition of EMT.

Effect of AR gene-specific knockout on the process of radiation-induced pulmonary fibrosis and its mechanism.

Numerous studies have proved that epithelial-mesenchymal transition (EMT) of lung epithelial cells is one of the important causes of radiation-induced pulmonary fibrosis (RIPF). Aldose reductase (AR) is a monomer enzyme in the polyglycolic metabolic pathway and belongs to the aldo-keno reductase protein superfamily. Our previous studies have found that AR as one of the most significantly up-regulated genes was associated with the development of bleomycin-induced PF in rats. It is not clear whether aldose reductase is related to the regulation of radiation-induced EMT and mediates RIPF. AR-knockout mice, wild-type mice and lung epithelial cells were induced by radiation to establish a RIPF animal model and EMT system, to explore whether AR is mediation to RIPF through the EMT pathway. In vivo, AR deficiency significantly alleviated radiation-induced histopathological changes, reduced collagen deposition and inhibited collagen I, matrix metalloproteinase 2 (MMP2) and Twist1 expression. In addition, AR knockout up-regulated E-cadherin expression and up-regulated α-SMA and Vimentin expression. In vitro, AR, collagen I and MMP2 expression were increased in lung epithelial cells after radiation, which was accompanied by Twist1 expression up-regulation and EMT changes evidenced by decreased E-cadherin expression and increased α-SMA and Vimentin expression. Knockdown or inhibition of AR inhibited the expressions of Twist1, MMP2 and collagen I, and reduced cell migration and reversed radiation-induced EMT. These results indicated that aldose reductase may be related to radiation-induced lung epithelial cells EMT, and that inhibition of aldose reductase might be a promising treatment for RIPF.

Eribulin mesylate exerts antitumor effects via CD103

ABSTRACT Eribulin mesylate (ERB) is a synthetic analog of halichondrin B, inhibiting tumor cell growth by disrupting microtubule function. Recently, anticancer drugs have been shown to not only act directly on tumor cells but also to exert antitumor effects by modifying the tumor environment. Although ERB has also been speculated to modify the tumor microenvironment including the immune response to tumors, the precise mechanism remains unclear. In our study, ERB suppressed the tumor growth of MC38 colon cancer in wildtype mice, whereas ERB failed to inhibit the tumor growth in Rag1-deficient mice which lack both B and T cells. Moreover, depletion of either CD4+ or CD8+ T cells abrogated the antitumor effect of ERB, indicating that both CD4+ and CD8+ T cells play an important role in ERB-induced antitumor effects. Furthermore, ERB treatment increased the number of tumor infiltrating lymphocytes (TILs) as well as the expression of activation markers (CD38 and CD69), immune checkpoint molecules (LAG3, TIGIT and Tim3) and cytotoxic molecules (granzyme B and perforin) in TILs. ERB upregulated E-cadherin expression in MC38. CD103 is a ligand of E-cadherin and induces T-cell activation. ERB increased the proportion of CD103+ cells in both CD4+ and CD8+ TILs. The ERB-induced antitumor effect with the increased TIL number and the increased expression of activation markers, inhibitory checkpoint molecules and cytotoxic molecules in TILs was abrogated in CD103-deficient mice. Collectively, these results suggest that ERB exerts antitumor effects by upregulation of E-cadherin expression in tumor cells and subsequent activation of CD103+ TILs.

Aloin inhibits gastric cancer cell proliferation and migration by suppressing the STAT3/HMGB1 signaling pathway

To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells.Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining.Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01).Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.

Role of Mir-452-5p Overexpression in Epithelial-Mesenchymal Transition (EMT) in Early-stage Colorectal Cancer.

The microRNA miR-452-5p holds a critical role in the progression of multiple tumor formations, but there is limited understanding regarding the epithelial-mesenchymal transition (EMT) progression and its underlying mechanisms in the early-stage colorectal cancer (CRC). We aimed to explore the change in miRNA expression in early-stage CRC and examine the role of these miRNAs in CRC. The expression levels of miR-452-5p in tissues and cells of early-stage CRC were determined by real-time quantitative polymerase chain reaction. Additionally, the biological effects of miR-452-5p on CRC were investigated by in vitro functional experiments. The expression levels of miR-452-5p were found increased in early-stage CRC tissue. We found that miR-452-5p promoted CRC cell proliferation but inhibited epithelial-mesenchymal transition. Furthermore, miR-452-5p promoted cell proliferation through activation of the extracellular signal-regulated kinase pathway, and inhibited cell invasion through suppression of Slug (Snail2) expression and up-regulation of E-cadherin expression. The expression of miR-452-5p is up-regulated in early CRC and suppresses epithelial-mesenchymal transition in CRC. These discoveries suggest that miR-452-5p has the potential to serve as a viable therapeutic target for CRC.

LncRNA SSTR5-AS1 as a Prognostic Marker Promotes Cell Proliferation and Epithelial-to-Mesenchymal Transition in Prostate Cancer.

This study is aimed to investigate the clinical significance and biological function of long non-coding RNA somatostatin receptor 5 antisense RNA 1 (SSTR5-AS1) in prostate cancer (PCa). Here, we found that SSTR5-AS1 expression was upregulated in PCa tissues compared with adjacent tissues using quantitative real time PCR analysis. The results from Chi-square test showed that increased SSTR5-AS1 expression levels were correlated with preoperative prostate specific antigen, tumor stage and lymph node metastasis. Kaplan-Meier survival curve described patients with high SSTR5-AS1 expression level showed poor survival. Univariate and multivariate cox regression analysis further identified SSTR5-AS1 expression as a poor independent prognostic factor for PCa patients. Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine incorporation assay, wound-healing assay and Transwell assay were performed to investigate the functional role of SSTR5-AS1 in PCa cells. The in vitro results indicated that SSTR5-AS1 knockdown inhibited, while SSTR5-AS1 overexpression promoted the proliferation, migration, and invasion of PCa cells. At molecular level, SSTR5-AS1 knockdown downregulated the protein levels of proliferating cell nuclear antigen, N-cadherin and vimentin, and upregulated E-cadherin expression in PC-3 cells. SSTR5-AS1 overexpression obtained opposite results on these protein markers in DU145 cells. In conclusion, these findings indicated that SSTR5-AS1 promotes PCa cell behaviors, which might provide a potential therapeutic target for PCa patients.

LMCD1-AS1 Facilitates Cell Proliferation and EMT by Sponging miR-873-3p in Cervical Cancer.

Long non-coding RNA LMCD1 antisense RNA 1 (LMCD1-AS1) has recently been reported to participate in the pathogenesis of several tumors, including thyroid cancer and osteosarcoma. However, the clinical significance of LMCD1-AS1 and the related biological function have not been reported in cervical cancer (CC). In this study, we observed that LMCD1-AS1 expression was highly expressed in CC specimens compared with adjacent normal specimens using quantitative real-time PCR. Chi-square test showed that high LMCD1-AS1 expression was correlated with FIGO stage and lymph node metastasis. Kaplan-Meier survival analysis showed poor prognosis with high LMCD1-AS1 expression. Moreover, FIGO stage, lymph node metastasis and high LMCD1-AS1 expression could be independent prognostic factors for the patients with CC. Functionally, knockdown of LMCD1-AS1 suppressed the proliferation, migration and invasion of two CC cell lines (HeLa and CaSki) cells by CCK-8 assay, colony formation assay, and Transwell assay. Knockdown of LMCD1-AS1 upregulated E-cadherin expression and downregulated the expression of PCNA, N-cadherin, and imentin in HeLa and CaSki cells. Luciferase reporter assay and RIP assay were conducted to evaluate the downstream molecular mechanisms of LMCD1-AS1. LMCD1-AS1 possesses a putative miR-873-3p-binding site and confirmed the negative correlation between them in CC tissues. Moreover, overexpression of LMCD1-AS1 promoted CC cell proliferation and EMT process through the regulation of miR-873-3p. In addition, depletion of LMCD1-AS1 reduced tumor growth and Ki-67 protein expression. In summary, our findings indicate that LMCD1-AS1 might exert an oncogenic role in CC and targeting LMCD1-AS1 might be a promising therapeutic target for CC treatment.

Inhibition of focal adhesion kinase/paxillin axis by caffeic acid phenethyl ester restrains aggressive behaviors of head and neck squamous cell carcinoma in vitro

ObjectiveCaffeic acid phenethyl ester (CAPE), one of the components of propolis that is produced by honeybees, reportedly suppresses multiple diseases, including bacterial infection, inflammation, and cancer. We aimed to investigate the inhibitory effects of CAPE on epithelial–mesenchymal transition (EMT) status and aggressive behaviors of human head and neck squamous cell carcinoma (HNSCC) in vitro and the underlying signaling pathway. DesignTo examine the cell growth and in vitro tumorigenic potential of HNSCC cells, cell viability and soft agar colony formation assays, respectively, were performed. Transwell migration and invasion assays were conducted to monitor HNSCC cells’ aggressive behaviors. Western blotting and immunocytochemistry analyses were done to investigate the signaling pathway responsible for relieving EMT progression and HNSCC cell aggressiveness. ResultsCAPE inhibited the in vitro tumorigenic potential of SNU-1041 cells stimulated by epidermal growth factor and suppressed the migratory and invasive capacities of SNU-1041 cells, irrespective of their cell proliferation state. CAPE was, at least partially, capable of inhibiting EMT progression by upregulating E-cadherin expression, which was accompanied by the reduction of phosphorylated focal adhesion kinase (FAK) and Paxillin. The inhibition of the FAK/Paxillin axis by PF-562271 was sufficient to alleviate the EMT progression through the induction of E-cadherin and aggressive behaviors of SNU-1041 cells. ConclusionsCAPE has a therapeutic potential as an anti-metastatic drug candidate for HNSCC therapy targeting the FAK/Paxillin axis.

HEY1-mediated cisplatin resistance in lung adenocarcinoma via epithelial-mesenchymal transition.

Lung cancer is one of the most common malignancies and the leading cause of cancer-related death in the world. In patients with advanced lung adenocarcinoma who are negative for driver gene mutations, platinum-based chemotherapy represented by cisplatin remain the standard of care. Therefore, studying the mechanism behind inevitable cisplatin resistance in lung adenocarcinoma is still important. In this study, the potentially related differential expression gene for cisplatin resistance in lung adenocarcinoma was screened in the GEO database. The expression level of HEY1 in cell lines of lung adenocarcinoma was detected and HEY1 expression was up-regulated in cisplatin-resistant lung adenocarcinoma tissues and cell lines A549/DDP. Patients with high HEY1 expression have poor prognosis after cisplatin therapy. Gain and loss function assays uncovered that HEY1 could regulate the cisplatin sensitivity of NSCLC cells. In vivo experiments have confirmed that silence of HEY1 expression can induce cisplatin resistance, and epithelial-mesenchymal transition (EMT) changes occur during this process. Mechanically, HEY1 silencing significantly up-regulated E-cadherin expression and down-regulated Vimentin in A549/DDP cells. While up-regulation of HEY1 resulted in down-regulation of E-cadherin and up-regulation of Vimentin in A549 cells. Immunohistochemical experiments confirmed that E-cadherin was significantly decreased, and Vimentin expression was significantly up-regulated in cisplatin-resistant lung adenocarcinoma tissues. HEY1 can mediate the occurrence of cisplatin-acquired resistance in lung adenocarcinoma, and the possible mechanism is to regulate the EMT. The results of this study can provide a new direction and target for clinical research on the reversal of cisplatin resistance in lung adenocarcinoma.

The significance of EphA2-regulated Wnt/β-catenin signal pathway in promoting the metastasis of HBV-related hepatocellular carcinoma.

Hepatitis B virus (HBV) infection is closely associated with the malignant progression of hepatocellular carcinoma (HCC). However, the mechanism involved in the HBV-related HCC development remains poorly understood. Hence, the aim of this study is to investigate the regulatory mechanism of EphA2-induced epithelial-mesenchymal transition (EMT) in the metastasis of HBV-related HCC cells. The expression level of EphA2 was determined in HBV-related human HCC cells. Then, the effects of EphA2 silencing on the EMT-associated proteins, the Wnt/β-catenin signal pathway and the metastatic potential of HBV-related HCC cells were evaluated. Finally, the inhibitory role of Entecavir (a potent antiviral drug for HBV) on EphA2-induced EMT was explored. The present study revealed that the EphA2 expression level was increased in HBV-related HCC cells compared with non-related HCC cells. Following EphA2 knockdown, the downregulation of Vimentin, β-catenin and p-GSK-3βSer9 expressions, the upregulation of E-cadherin expression, and the suppressed migration and invasion ability of HBV-related HCC cells were found. Additionally, Entecavir was proved to have a significant inhibitory effect on EphA2-induced EMT via attenuating the Wnt/β-catenin signal pathway. In this study, we found that EphA2-induced EMT was involved in the enhanced metastatic potential of HBV-related HCC cells through the activation of the Wnt/β-catenin signal pathway.

Resveratrol alleviates bleomycin-induced pulmonary fibrosis by inhibiting epithelial-mesenchymal transition and down-regulating TLR4/NF-κB and TGF-β1/smad3 signalling pathways in rats

BackgroundPulmonary fibrosis (PF) is a difficult clinical condition with no effective treatment and a high mortality rate. Patients usually die of respiratory failure. In the present, we hypothesized that resveratrol (Res) could suppress bleomycin (BLM)-induced PF in rats and examined the detailed mechanism. MethodsThe successfully established BLM-induced PF rat models and normal healthy rats were randomly assigned into the control, model, Res-low, Res-medium, and Res-high groups. The extent of PF in rats was determined by Masson and H&E staining. ELISA was used to detect changes in the inflammatory factors IL-1β, IL-6, TNF-α, SOD, and GSH-PX in lung tissue. Microscopic lung fibrosis scoring was done using the Ashcroft scale. Western blotting and RT-qPCR assays were used to analyze the effects of Res on the epithelial-mesenchymal transition (EMT). ResultsThe administration of Res ameliorated the BLM-induced PF in vivo. Res could inhibit pro-inflammatory factors and regulates oxidative stress levels. It also revealed that Res downregulated Vimentin and upregulated E-cadherin expression. Res inhibited fibrosis and inflammatory responses in rat lung tissue. The activation of the TLR4/NF-κB and TGF-β1/smad3 signaling pathways were inhibited following Res treatment. ConclusionThe present study suggested that Res may protect against BLM-induced PF by inhibiting the EMT and down-regulating the TLR4/NF-κB and TGF-β1/smad3 signaling pathways.

FAM201A Promotes Cervical Cancer Progression and Metastasis through miR-1271-5p/Flotillin-1 Axis Targeting-Induced Wnt/β-Catenin Pathway

This study investigated the role of the family with sequence similarity 201-member A (FAM201A), as previously reported oncogenic, in cervical cancer (CC). FAM201A expression in CC was analyzed through bioinformatics analyses, and its distribution in CC tissues/cells was determined by in situ hybridization. CC cells were transfected/cotransfected with FAM201A/flotillin-1 (FLOT1) overexpression plasmids and miR-1271-5p mimics, followed by functional analysis on viability, migration and invasion. Pearson's correlation tests were performed to analyze the correlation between FAM201A and miR-1271-5p in CC tissues. The targeting relationship between miR-1271-5p and FLOT1 was confirmed by dual-luciferase reporter assay. The expressions of FAM201A, miR-1271-5p, FLOT1, matrix metalloproteinases (MMP)-9, MMP-2, E-cadherin, N-cadherin, and the Wnt/β-catenin pathway-related molecules (Wnt1, β-catenin and p-β-catenin) in CC cells or tissues were assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and/or western blot. The results showed that FAM201A was abundantly expressed and miR-1271-5p expression was downregulated in CC. FAM201A was enriched in CC cell cytoplasm and negatively correlated with miR-1271-5p in CC tissues. FAM201A overexpression enhanced the cell viability, migration, invasion, and tumorigenesis of CC in vivo and increased FLOT1 expression. These trends were all reversed by upregulating miR-1271-5p, which induced opposite effects to FAM201A overexpression. MiR-1271-5p upregulation depleted the levels of MMP-9, MMP-2, N-cadherin, and the Wnt/β-catenin pathway-related molecules and upregulated E-cadherin expression. FLOT1 was a direct target of miR-1271-5p. FLOT1 overexpression induced effects contrary to the upregulation of miR-1271-5p and abolished miR-1271-5p upregulation-induced effects in CC cells. Overall, this study showed that FAM201A promoted cervical cancer progression and metastasis by targeting the miR-1271-5p/FLOT1 axis-induced Wnt/β-catenin pathway.

New triterpenoids from the Cyclocarya paliurus (Batalin) Iljinskaja and their anti-fibrotic activity

Cyclocarya paliurus, a Chinese herbal medicine and new food resource, contains a triterpenic-acid-rich extract that demonstrated ameliorative effect on diabetic nephropathy (DN). A more in-depth discovery of functional components led to the isolation of seven new triterpenoids including two pentacyclic triterpenes, 1α,2α,3β,23-tetrahydroxyolean-12-en-28-oic acid and 2α,3β,22α-tirhydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranoside, and five tetracyclic triterpenoid glycosides (cypaliurusides N-R), together with twelve known compounds from the leaves of C. paliurus. Their structures were determined using a comprehensive analysis of chemical and spectroscopic data. Partial compounds were assessed for anti-fibrotic activities in high-glucose and TGF-β1 induced HK-2 cells. Compound 16 remarkably decreased the level of fibronectin with an inhibition rate of 37.1%. Furthermore, 16 effectively alleviated the epithelial-mesenchymal transformation (EMT) process by upregulating E-cadherin expression and downregulating α-SMA expression, and it significantly decreased the level of the transcriptional inhibitors (Snail and Twist) of E-cadherin. The discovery of anti-fibrotic compounds from C. paliurus provides the potential utilization and functional candidates for the DN prevention.

Bone marrow mesenchymal stem cells combined with estrogen synergistically promote endometrial regeneration and reverse EMT via Wnt/β-catenin signaling pathway

BackgroundIntrauterine adhesion (IUA) is a clinical disease characterized by the uterine cavity occlusion caused by the damage of the endometrial basal layer. Bone marrow mesenchymal stem cells (BMSCs) transplantation have the potential to promote endometrial regeneration mainly through paracrine ability. Estrogen is an indispensable and important factor in the repair of endometrial damage, which has been reported as a promising and adjunctive therapeutic application for stem cell transplantation therapy. This study aims to investigate the synergistic effect of BMSCs and estrogen on improving the endometrial regeneration and restoring the endometrium morphology in a dual damage model of IUA in rabbits and the underlying molecular mechanisms.MethodsBMSCs were isolated and identified by adipogenic and osteogenic differentiation and flow cytometry assays. The rabbit IUA animal model was established by a dual damage method of mechanical curettage and lipopolysaccharide infection. Additionally, we investigated the therapeutic impact of both BMSCs and estrogen either separately or in combination in a rabbit model. The retention of PKH26-labeled BMSCs was observed by vivo fluorescence imaging.The number of endometrial glands and the degree of fibrosis were observed by H&E and Masson staining respectively. Western blotting, Immunohistochemistry and immunofluorescence staining were performed to detect biomarkers related to endometrial epithelium, endometrial fibrosis and EMT. Finally, the protein expression of core molecules of Wnt/β-catenin pathway was detected by Western blotting.ResultsPKH26-labeled fluorescence results revealed that BMSCs appeared and located in the endometrial glands and extracellular matrix area when orthotopic transplanted into the uterine cavity. Histological assays showed that remarkably increasing the number of endometrial glands and decreasing the area of endometrial fibrosis in the BMSCs combined with estrogen treatment group. Moreover, downregulated expression of fibrosis markers (fibronectin, CollagenI, a-SMA) and interstitial markers (ZEB1, Vimentin, N-cadherin), as well as upregulated E-cadherin expression were found in the combined group. Further study of in vivo staining revealed that fluorescence intensity of CK7 was stronger in the combined group than that of direct BMSCs intrauterine transplantation, while vimentin showed the opposite results. Moreover, the protein levels of β-catenin, Axin2, C-myc, CycinE of Wnt/β-catenin signaling pathway increased in the BMSCs combined with estrogen group than in the other treatment groups.ConclusionBMSCs combined with estrogen can promote the differentiation of stem cells into endometrial epithelial cells to facilitate the regeneration of damaged endometrium. The potential mechanism of the synergistic effect may inhibit the occurrence of EMT by activating the Wnt/β-catenin signaling pathway.

Organochlorine pesticides induce epithelial as well as inflammatory mediators following exposure to human ovarian surface epithelial cells: An in vitro study.

Although studies have suggested organochlorine pesticides (OCPs) exposure increased the risk of epithelial ovarian cancer, the mechanisms underlying its potential tumorigenic effects in the human ovary are not well understood. In this study, we investigated the impact of dichlorodiphenyldichloroethylene (DDE), endosulfan, and heptachlor exposure on epithelial cadherin (E-cadherin) and proinflammatory mediators in human ovary surface epithelial (HOSE) cells. We found that DDE, endosulfan, and heptachlor exposure resulted in epithelial differentiation accompanied by upregulation of E-cadherin expression and overexpression of proinflammatory cytokines (TNFα,IL-1β, and IL-6) in HOSE cells. The epithelial differentiation may accelerate HOSE cells to inclusion body formation, a common site for ovarian cancer initiation and persistent exposure to OCPs creates a chronic inflammatory microenvironment that may promote the neoplastic transformation of HOSE cells within the inclusion cyst.

A novel oncogenic seRNA promotes nasopharyngeal carcinoma metastasis

Nasopharyngeal carcinoma (NPC) is a common malignant cancer in southern China that has highly invasive and metastatic features and causes high mortality, but the underlying mechanisms of this malignancy remain unclear. In this study, we utilized ChIP-Seq to identify metastasis-specific super enhancers (SEs) and found that the SE of LOC100506178 existed only in metastatic NPC cells and powerfully aggravated NPC metastasis. This metastatic SE transcribed into lncRNA LOC100506178, and it was verified as a seRNA through GRO-Seq. Furthermore, SE-derived seRNA LOC100506178 was found to be highly expressed in metastatic NPC cells and NPC lymph node metastatic tissues. Knockdown of seRNA LOC100506178 arrested the invasion and metastasis of NPC cells in vitro and in vivo, demonstrating that seRNA LOC100506178 accelerates the acquisition of NPC malignant phenotype. Mechanistic studies revealed that seRNA LOC100506178 specifically interacted with the transcription factor hnRNPK and modulated the expression of hnRNPK. Further, hnRNPK in combination with the promoter region of MICAL2 increased Mical2 transcription. Knockdown of seRNA LOC100506178 or hnRNPK markedly repressed MICAL2, Vimentin and Snail expression and upregulated E-cadherin expression. Overexpression of seRNA LOC100506178 or hnRNPK markedly increased MICAL2, Vimentin and Snail expression and decreased E-cadherin expression. Therefore, seRNA LOC100506178 may promote MICAL2 expression by upregulating hnRNPK, subsequently enhancing EMT process and accelerating the invasion and metastasis of NPC cells. seRNA LOC100506178 has the potential to serve as a novel prognostic biomarker and therapeutic target in NPC patients.

Targeting kinesin family member 21B by miR-132-3p represses cell proliferation, migration and invasion in gastric cancer

ABSTRACT Recently, kinesin family member 21B (KIF21B) has been reported to be an oncogene in non-small cell lung cancer and hepatocellular carcinoma. However, the functional role of KIF21B and related molecular mechanisms in gastric cancer (GC) remain largely uncovered. In this study, online bioinformatics analysis showed that KIF21B was overexpression in GC and predicted poor prognosis. Consistently, we found that the protein expression of KIF21B was upregulated in GC tissues compared with adjacent tissues by immunohistochemistry. Knockdown of KIF21B significantly suppressed cell proliferation, migration and invasion in GC cell lines (AGS and SNU-5) using Cell counting kit‑8 (CCK-8) assay, colony formation and transwell assay. KIF21B was confirmed as the target of miR-132-3p in GC cells by luciferase reporter assay. Moreover, miR-132-3p was down-regulated and KIF21B expression was upregulated in GC tissues. Overexpression of KIF21B reversed the miR-132-3p-mediated suppressive effects on GC cell proliferation, migration and invasion. Furthermore, miR-132-3p overexpression downregulated the protein levels of Wnt1, c-Myc, β-catenin, proliferating cell nuclear antigen (PCNA) and N-cadherin, and upregulated E-cadherin expression in GC cells, which were all alleviated after KIF21B overexpression. In conclusion, our findings indicate that down-regulation of KIF21B by miR-132-3p suppresses cellular functions in GC, which might be linked to reduced Wnt/β-catenin signaling.

Abstract P3-06-04: Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer

Abstract Introduction: The Notch and Wnt signaling pathways play critical role in mammary development and tumourigenesis. The epithelia-mesenchymal transition (EMT) is an important process of breast cancer development. Growing evidence shows that Glycogen Synthase Kinase 3 Beta (GSK 3β) is considered as an EMT suppressor of its downregulation of protein snail, a zinc-finger transcriptional repressor that regulates epithelia marker E-cadherin. Previous studies by our group demonstrated that Notch3 may inhibit the emergence and progression of breast cancer. In mammalian cell, GSK3β has been show to physically bind and phosphorylate the intracellular domain of two Notch paralogues. But the relationship between Notch3,GSK3β and EMT in breast cancer still unclear. Intriguingly, we found that knockdown of Notch3 in luminal subtype of breast cancer cells MCF-7 downregulated GSK3β and promoted EMT. In contrast, overexpression of Notch3 in triple-negative cell lines MDA-MB-231 upregulated GSK3βand inhibited EMT. Mechanistically, our results revealed that Notch3 specifically binds to the CSL binding element of the GSK3β promoter and trans-activates its expression. Furthermore, Notch3 and GSK3β expression were positively associated in human breast cancer. Moreover, the mRNA expression of both Notch3 and GSK3βpredicted a better Overall-Survival for breast cancer patients. Our findings demonstrate that Notch3 inhibits breast cancer cells EMT via trans-activating GSK3β expression. Methods: 1. Western blot and/or qRT-PCR were performed to study the expressions of Notch3,GSK3β, β-catenin and EMT related-molecules in breast cancer cell line. 2. Chromatin immunoprecipitation (ChIP) and dual-luciferase assays were carried out to investigate whether Notch3 bound to GSK3β promoter and mediatesGSK3β expression on transcriptional level. 3. Wound healing assay, migration and invasion assays were performed to analyze the effect of Notch3 and GSK3β on breast cancer cell lines migration abilities. 4. The association of Notch3 and GSK3β expression in human breast cancer samples and correlation between Notch3 or GSK3β expression and patient survival in a cohort of human breast cancer patients in Kaplan Meier Plotter database were analyzed. Results: 1. Notch3 up-regulates GSK3β expression in mRNA and protein levels. 2. Notch3 regulates GSK3β expression by binding to the CSL antisense binding site (TTCCCA) positions -308bp to -303bp in the GSK3β promoter. 3. Notch3 intracellular domain (N3ICD) down-regulates the expression of Vimentin, β-catenin, but up-regulates E-cadherin expression. 4. Notch3 inhibits the ability of migration and invasion in breast cancer cell lines. 5. Notch3 and GSK3β expression were positively associated in human breast cancer. The mRNA expression of both Notch3 and GSK3β predicted a better Overall-Survival for breast cancer patients. Summary: We have identified a positive correlation between Notch3 and GSK3β in breast cancer and confirmed a role for the Notch3- GSK3β axis in inhibiting EMT. In addition, we explored in detail and first clarified the possible molecular mechanism of regulating this axis. Our results provide new insights into the complex regulation of breast cancer proliferation and tumorigenesis. We believe that the Notch3- GSK3β axis may be a prospective prognostic predictor for breast cancer. Citation Format: Chen Wei-Ling, Yong-Qu Zhang, Wen-He Huang, Min Chen, Yao-Chen Li, Guo-Jun Zhang. Notch3 inhibits epithelia-mesenchymal transition by trans-activating GSK3β in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-06-04.

LncRNA LINC01315 silencing modulates cancer stem cell properties and epithelial-to-mesenchymal transition in colorectal cancer via miR-484/DLK1 axis

ABSTRACT Long non-coding RNA long intergenic non-protein coding RNA 01315 (LncRNA LINC01315) has been found to be implicated in various cancers, but its role and functions in colorectal cancer (CRC) remain to be addressed. Data on LINC01315 expression in CRC were gathered using bioinformatics analysis, and cancer stem cells (CSCs) were sorted by aldehyde dehydrogenase (ALDH) assay and flow cytometry. Migration, invasion, and stemness of CSCs isolated from CRC cells after transfection were determined by scratch, Transwell, and sphere-formation assays, respectively. Tumor xenograft model was constructed. Target genes and potential-binding sites were predicted using online databases and further confirmed via dual-luciferase reporter assay. Relative factors expressions were determined via quantitative real-time polymerase-chain reaction and Western blot as needed. LINC01315 was high-expressed in CRC and ALDH+ cells. LINC01315 silencing suppressed the migration, invasion, and sphere formation of CRC cells and tumor growth, and downregulated expressions of CSC molecules (ALDH, cluster of difference 44 (CD44), Prominin, and sex determining region Y-box 2 (SOX2)), Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Vimentin but upregulated E-Cadherin expression. MiR-484 could competitively bind with LINC01315, and LINC01315 silencing promoted miR-484 expression. The level of Delta Like Non-Canonical Notch Ligand 1 (DLK1), the target gene of miR-484, was enhanced by overexpressed LINC01315 yet was suppressed by LINC01315 silencing. Also, DLK1 silencing reversed the effects of downregulated miR-484 on migration, invasion, sphere formation, and CSC molecules expressions in CRC cells. LINC01315 silencing modulated CSC properties and epithelial-to-mesenchymal transition via miR-484/DLK1 axis.

Oridonin represses epithelial-mesenchymal transition and angiogenesis of thyroid cancer via downregulating JAK2/STAT3 signaling

Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been reported to exert anticancer activity in various cancers. However, the molecular mechanism of oridonin in thyroid cancer has not yet been elucidated. In the present study, oridonin was found to significantly inhibit migration and invasion of thyroid cancer TPC-1 and BCPAP cells, as evidenced by wound healing assay, transwell migration assay and Matrigel invasion assay. In addition, oridonin could partially impede epithelial-mesenchymal transition by upregulating E-Cadherin expression and downregulating N-Cadherin and vimentin expressions in a concentration-dependent manner. Accumulating evidence indicated that JAK2 (Janus kinase-2)/STAT3 (Signal Transducer and Activator of Transcription 3) signaling pathway was associated with epithelial-mesenchymal transition. As expected, the protein levels of phosphorylated-JAK2 and phosphorylated-STAT3 were dramatically reduced upon oridonin treatment in thyroid cancer TPC-1 and BCPAP cells. Subsequently, the findings revealed that JAK2 overexpression could weaken the anti-metastatic effect and partially attenuate MET (mesenchymal-to-epithelial transition) by oridonin, while AG490, a JAK2 antagonist, enhanced the above process in thyroid cancer cells. The subsequent results showed that oridonin inhibited angiogenesis and VEGFA expression in thyroid cancer cells by tube formation assay, western blot and ELISA assay. Meanwhile, AG490 could further attenuate oridonin-treated VEGFA protein level. In addition, the in vivo results further confirmed that oridonin inhibited tumorigenicity in thyroid cancer xenograft. In conclusion, the results demonstrated that oridonin repressed metastatic phenotype, angiogenesis and modulated EMT (epithelial-mesenchymal transition) of thyroid cancer cells via the inactivation of JAK2/STAT3 signaling pathway, suggesting that JAK2 may be a novel therapeutic target of oridonin against thyroid cancer.