Inhibition Of Epithelial-mesenchymal Transition Research Articles

MiR-150-5p Alleviates Renal Tubule Epithelial Cell Fibrosis via the Inhibition of Epithelial-Mesenchymal Transition by Targeting ZEB1.

Although microRNA (miR)-150-5p participates in the progression of renal fibrosis, its mechanism of action remains elusive. A mouse model of unilateral ureteral obstruction was used. The in vitro renal fibrosis model was established by stimulating human kidney 2 (HK-2) cells with transforming growth factor beta 1 (TGF-β1). The expression profiles of miR-150-5p, zinc finger E-box binding homeobox 1 (ZEB1), and other fibrosis- and epithelial-mesenchymal transition (EMT)-linked proteins were determined using Western blot and quantitative reverse transcription polymerase chain reaction. The relationship between miR-150-5p and ZEB1 in HK-2 cells was confirmed by a dual-luciferase reporter assay. Both in vivo and in vitro renal fibrosis models revealed reduced miR-150-5p expression and elevated ZEB1 level. A significant decrease in E-cadherin levels, as well as increases in alpha smooth muscle actin (α-SMA) and collagen type I (Col-I) levels, was seen in TGF-β1-treated HK-2 cells. The overexpression of miR-150-5p ameliorated TGF-β1-mediated fibrosis and EMT. Notably, miR-150-5p acts by directly targeting ZEB1. A significant reversal of the inhibitory impact of miR-150-5p on TGF-β1-mediated fibrosis and EMT in HK-2 cells was observed upon ZEB1 overexpression. MiR-150-5p suppresses TGF-β1-induced fibrosis and EMT by targeting ZEB1 in HK-2 cells, providing helpful insights into the therapeutic intervention of renal fibrosis.

Capsaicin enhances cisplatin-induced anti-metastasis of nasopharyngeal carcinoma by inhibiting EMT and ERK signaling via SERPINB2.

Cisplatin (DDP)-based combined chemotherapy or concurrent chemoradiotherapy is the mainstay treatment for advanced-stage nasopharyngeal carcinoma (NPC), but needs improvement due to its severe side effects. Capsaicin (CAP) can enhance the anti-tumor activity of cytotoxic drugs. The aim of this study was to investigate the anti-metastasis activity of CAP in combination with DDP in NPC. Herein, CAP and DDP showed synergistic cytotoxic effects on NPC cells. CAP alone and DDP alone inhibited NPC migration and invasion in vitro and in vivo, and the combination of CAP and DDP had the greatest effect. Moreover, CAP upregulated the mRNA and protein expressions of SERPINB2. Further results showed that both SERPINB2 mRNA and protein expressions were downregulated in NPC cell lines and tissues and SERPINB2 overexpression inhibited NPC migration and invasion in vitro and in vivo, while silencing SERPINB2 acted oppositely. In addition, SERPINB2 was abnormally expressed in head and neck squamous cell carcinoma (HNSC) and other multiple cancers and downregulation of SERPINB2 predicted poor prognosis in HNSC according to the Cancer Genome Atlas (TCGA) database. We further found that SERPINB2 overexpression inhibited epithelial-mesenchymal transition (EMT) and the phosphorylated ERK (p-ERK), and the inhibitory effect was enhanced by CAP and DDP. Altogether, our results suggest that the combined inhibition of CAP and DDP on NPC metastasis may be related to the inhibition of EMT and ERK signals mediated by SERPINB2, and CAP may help to improve the efficacy of DDP in the treatment of NPC and develop new therapeutic approaches.

Abstract PO1-24-02: Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer

Abstract Introduction: Claudin-low breast cancer is mostly classified as triple-negative breast cancer and is known to possess cancer stem cell properties with high expression of epithelial-to-mesenchymal transition (EMT) markers. However, specific therapeutic targets for this subtype have not yet been identified. While reports suggest that inhibition of EMT using EMT inhibitors can suppress cell and tumor proliferation, monotherapy may not be sufficient. The expression of CD44, a surface marker of cancer stem cells, has been reported to be involved in multiple signaling pathways, promoting cancer growth and invasion. In this study, we focus on CD44 and the TGF-β receptor, which is involved in EMT, to investigate the effect of inhibiting TGF-β receptor through CD44 inhibition in Claudin-low breast cancer and validate its potential as an effective treatment. Methods: SUM159 and MDA-MB-231 cell lines were used as Claudin-low breast cancer cell models. CD44 knockdown cells were established to examine the effects of TGF-β receptor inhibition and downstream signaling. Results: CD44 knockdown did not affect the expression of TGF-β receptor. Both SUM159 and MDA-MB-231 cells showed comparable cell growth inhibition through CD44 knockdown and TGF-β receptor inhibition, with enhanced effects observed in CD44 knockdown cells in response to TGF-β receptor inhibition. Furthermore, CD44 knockdown combined with TGF-β receptor inhibition significantly reduced the expression of Smad2/3. Conclusion: In Claudin-low breast cancer cells, CD44 knockdown enhances the effectiveness of TGF-β receptor inhibition and significantly influences the expression level of Smad2/3. Citation Format: Ryoichi Matsunuma, Sae Imada, Shoko Sato, Ryosuke Hayami, Michiko Tsuneizumi. Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-02.

Abstract PO5-05-11: The effect of eribulin treatment for patients with HER2-low breast cancer

Abstract Background: Eribulin, a nontaxane, synthetic microtuble dynamics inhibitor, induces longer overall survival (OS) in patients with advanced or metastatic breast cancer (MBC). Interestingly, it also improves the tumor immune microenvironment via vascular remodeling. DESTINY-Breast04 trial showed that trastuzumab deruxtecan resulted in significantly longer OS of human epithelial growth receptor 2 (HER2)-low breast cancer patients, and this mechanism may also be related to tumor microenvironment. In this study, we investigated the effect of eribulin for patients with HER2-low breast cancer. Materials and Methods: This retrospective study comprised 97 MBC treated with eribulin from August 2011 to April 2019 at our institute. HER2 levels were determined through tumor biopsy specimen at first medical examination. We evaluated the correlations between HER2 levels and clinicopathological features, including the prognosis and immune related markers in each breast cancer subtype. We also classified the progressive disease (PD) into progression due to pre-existing lesion (PPL) and progression due to new metastasis (PNM) based on systemic computed tomography findings, and investigated the relationships between HER2 expression and PD types. Results: HER2-high group had significantly better OS than other groups (p=0.021, log-rank). Hormone receptor (HR)-negative/HER2-low patients had significantly better progression-free survival (PFS) than HR-negative/HER2-negative (p=0.024, log-rank). Though we also examined HER2 levels of recurrent tumor specimen, it was not a significantly useful prognostic marker in patients with HR-negative breast cancer (p=0.085, log-rank). There was no significant correlation between HER2 expression and any tested clinicopathological parameter, including absolute lymphocyte count, neutrophil-to-lymphocyte ratio and tumor infiltrating lymphocytes. PD were observed in 64 patients (PPL: n=47, PNM: n=17). PD type of all HR-positive/HER2-high patients was PNM, whereas all HR-negative/HER2-high patients was PPL (p=0.035). HER2-low group tended to have a higher PPL rare than negative group (83.3% vs 69.2%). Conclusions: In HR-negative/HER2-low group, PFS was improved by eribulin treatment, which might be due to unique anticancer effect of eribulin, suppression of epithelial-mesenchymal transition. Further study of this mechanism through basic research is important. Also, assessment of HER2 from primary tumor may be more useful than recurrent specimen. Citation Format: Wataru Goto, Saeko Henmi, Mariko Nishikawa, Asuka Kouchi, Rika Kouhashi, Akimichi Yabumoto, Koji Takada, Yuka Asano, Yukie Tauchi, Kana Ogisawa, Tamami Morisaki, Shinichiro Kashiwagi. The effect of eribulin treatment for patients with HER2-low breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-11.

Novel cancer-fighting role of ticagrelor inhibits GTSE1-induced EMT by regulating PI3K/Akt/NF-κB signaling pathway in malignant glioma

BackgroundGlioma is the most common malignant brain tumor of the central nervous system. Despite of the improvement of therapeutic strategy, the prognosis of malignant glioma patients underwent by STUPP strategy is still unexpected. Previous studies have suggested that ticagrelor exerted chemotherapeutic effects by inhibition of epithelial-mesenchymal transition (EMT) in various diseases including tumors. However, whether ticagrelor can exhibit the antitumor efficiency in glioma by affecting the EMT process is still unclear. In this study, we investigated the cancer-fighting role of ticagrelor and demonstrated its chemotherapeutic mechanism in glioma. Materials and methodsThe MTT assay was performed to detect the cytotoxicity of ticagrelor in glioma cells. We evaluated the expression of Ki67 in glioma cells by immunofluorescence assay after ticagrelor treatment. We conducted wound healing assay and transwell assay to determine the effects of ticagrelor on the migration and invasion of glioma cells. RNA-seq analysis was conducted to examine potential target genes and alternative signaling pathways for ticagrelor treatment. The expression levels of key EMT -related proteins were examined by Western blot experiment. ResultsTicagrelor inhibited the proliferation, migration and invasion of glioma cells with a favorable toxicity profile in vitro. Ticagrelor downregulated the expression of GTSE1 in glioma cells. RNA-seq analysis explored that GTSE1 acted as the potential target gene for ticagrelor treatment. Upregulation of GTSE1 antagonized the inhibitory effect of ticagrelor on the invasion of glioma and EMT progression by regulation of PI3K/Akt/NF-κB signaling pathway. And ticagrelor also exhibited the similar chemotherapeutic effect of glioma in vivo. ConclusionsTicagrelor as a potential chemotherapeutic option induced the inhibition of the GTSE1-induced EMT progression by regulation of PI3K/AKT/NF-κB signaling pathway.

Abstract P24: Elucidating the Mechanism of Action of Lenvatinib in Estrogen Receptor-Positive (ER+) Breast Cancer

Abstract Background: ER+ breast cancer is the most common subtype of breast cancer. Although most patients respond initially to endocrine therapy (ET), endocrine resistance inevitably develops, leading to disease progression. Lenvatinib, a multi-kinase inhibitor, demonstrated clinical benefit in the treatment of patients with advanced ER+ breast cancer who have progressed on ET in a phase Ib/II clinical trial from our group. Our study aims to elucidate the molecular mechanism of action of lenvatinib in ER+ breast cancer. Methods: A cell viability assay was performed to assess the sensitivity of four ER+ cell lines to lenvatinib. Western blot was used to elucidate its effect on downstream pathway signalling, and cell cycle arrest investigated through flow cytometry. A series of functional assays, including BrdU, colony formation and wound-healing assays assessed proliferative, clonogenic and migratory activity respectively. The effect of lenvatinib on epithelial-mesenchymal transition (EMT) was analysed using RT-qPCR, and compared with siRNA knockdown (KD) of Fgfr1 and Fgfr2. Results: ER+ cell lines demonstrated sensitivity to lenvatinib in the micromolar range. Western blot showed downregulation of FGFR1, FGFR2, pERK and pAKT expression post-treatment. Lenvatinib was found to induce a G1 phase arrest, suppressed cell proliferation in a dose-dependent manner, and diminished both the clonogenic ability and migratory activity of ER+ cells. Lenvatinib treatment reduced expression of Fgfr1, Fgfr2 and the mesenchymal markers N-cad and Fibronectin, while increasing the expression of the epithelial markers E-cad and Claudin-1, indicating potential inhibition of EMT. A similar change in EMT marker expression was observed in Fgfr1 KD but not Fgfr2 KD cells. Conclusion: Lenvatinib demonstrated potent anti-cancer activity and induction of G1 phase arrest, leading to decreased cell proliferation, reduction of clonogenic ability and cell migration. EMT inhibition was also observed, possibly through the FGFR1 signalling pathway. Citation Format: Natasha Gandasasmita, Gauri Vaidya, Arpita Datta, Charlie Marvalim, Joline Si Jing Lim, Soo Chin Lee. Elucidating the Mechanism of Action of Lenvatinib in Estrogen Receptor-Positive (ER+) Breast Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P24.

Ganglioside GD3 Regulates Inflammation and Epithelial-to-Mesenchymal Transition in Human Nasal Epithelial Cells.

Chronic sinusitis with nasal polyps (CRSwNP) is one of the most common chronic inflammatory diseases, and involves tissue remodeling. One of the key mechanisms of tissue remodeling is the epithelial-mesenchymal transition (EMT), which also represents one of the pathophysiological processes of CRS observed in CRSwNP tissues. To date, many transcription factors and forms of extracellular stimulation have been found to regulate the EMT process. However, it is not known whether gangliosides, which are the central molecules of plasma membranes, involved in regulating signal transmission pathways, are involved in the EMT process. Therefore, we aimed to determine the role of gangliosides in the EMT process. First, we confirmed that N-cadherin, which is a known mesenchymal marker, and ganglioside GD3 were specifically expressed in CRSwNP_NP tissues. Subsequently, we investigated whether the administration of TNF-α to human nasal epithelial cells (hNECs) resulted in the upregulation of ganglioside GD3 and its synthesizing enzyme, ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialytransferase 1 (ST8Sia1), and the consequently promoted inflammatory processes. Additionally, the expression of N-cadherin, Zinc finger protein SNAI2 (SLUG), and matrix metallopeptidase 9 (MMP-9) were elevated, but that of E-cadherin, which is known to be epithelial, was reduced. Moreover, the inhibition of ganglioside GD3 expression by the siRNA or exogenous treatment of neuraminidase 3 (NEU 3) led to the suppression of inflammation and EMT. These results suggest that gangliosides may play an important role in prevention and therapy for inflammation and EMT.

MiR-194-3p regulates epithelial-mesenchymal transition in embryonic epicardial cells via p120/β-catenin signaling.

The epicardium is integral to cardiac development and facilitates endogenous heart regeneration and repair. While miR-194-3p is associated with cellular migration and invasion, its impact on epicardial cells remains uncharted. In this work we use gain-of-function and loss-of-function methodologies to investigate the function of miR-194-3p in cardiac development. We culture embryonic epicardial cells in vitro and subject them to transforming growth factor β (TGF-β) treatment to induce epithelial-mesenchymal transition (EMT) and monitor miR-194-3p expression. In addition, the effects of miR-194-3p mimics and inhibitors on epicardial cell development and changes in EMT are investigated. To validate the binding targets of miR-194-3p and its ability to recover the target gene-phenotype, we produce a mutant vector p120-catenin-3'UTR-MUT. In epicardial cells, TGF-β-induced EMT results in a notable overexpression of miR-194-3p. The administration of miR-194-3p mimics promotes EMT, which is correlated with elevated levels of mesenchymal markers. Conversely, miR-194-3p inhibitor attenuates EMT. Further investigations reveal a negative correlation between miR-194-3p and p120-catenin, which influences β-catenin level in the cell adhesion pathway. The suppression of EMT caused by the miR-194-3p inhibitor is balanced by silencing of p120-catenin. In conclusion, miR-194-3p directly targets p120-catenin and modulates its expression, which in turn alters β-catenin expression, critically influencing the EMT process in the embryonic epicardial cells via the cell adhesion mechanism.

S100A6 mediated epithelial-mesenchymal transition affects chemosensitivity of colorectal cancer to oxaliplatin

PurposeTo investigate the mechanism by which S100 calcium-binding protein A6 (S100A6) affects colorectal cancer (CRC) cells to oxaliplatin (L-OHP) chemotherapy, and to explore new strategies for CRC treatment. MethodsS100A6 expression was assessed in both parental and L-OHP-resistant CRC cells using western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assays (ELISA). Lentiviral vectors were utilized to induce the knockdown of S100A6 expression, followed by comprehensive evaluations of cell proliferation, apoptosis, and epithelial-mesenchymal transition (EMT). Additionally, RNA-seq analysis was conducted to identify genes associated with the knockdown of S100A6. ResultsElevated S100A6 expression in CRC tissues correlated with an adverse prognosis in patients with CRC. Higher expression of S100A6 was also observed in L-OHP-resistant CRC cells, which showed enhanced proliferation, migration, invasion, and antiapoptotic capabilities. Notably, the knockdown of S100A6 expression resulted in decreased proliferation, increased apoptosis, and suppression of EMT and tumorigenicity in L-OHP-resistant CRC cells. Transcriptome sequencing reveals a noteworthy association between S100A6 and vimentin expression. Application of the EMT agonist, transforming growth factor β (TGF-β), induces EMT in CRC cells. S100A6 expression positively correlates with TGF-β expression. TGF-β facilitated the expression of EMT-related molecules and reduced the chemosensitivity of L-OHP in S100A6-knockdown cells. ConclusionIn conclusion, the knockdown of S100A6 may overcome the L-OHP resistance of CRC cells by modulating EMT.

Regulatory mechanism of the Glabrene against non-small cell lung cancer by suppressing FGFR3.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with limited effective treatment options. This study aimed to investigate the regulatory mechanism of Glabrene on NSCLC through its interaction with FGFR3. HCC827 cells were implanted into nude mice and treated with Glabrene. Tumor volume was monitored at 0, 3, 6, and 9 days after medical treatment. Tissue analysis included Hematoxylin and Eosin (HE) and Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) staining, as well as immunohistochemistry for Ki67, ERK1/2, and p-ERK1/2 expression. Cell viability was determined with the CCK8 method. We utilized immunofluorescence techniques to observe apoptosis, as well as the levels of E-cadherin and Vimentin expression. Cellular proliferation was determined via plate cloning assay and cellular mobility was determined via scratch assay. Cellular invasion ability was assessed via a transwell assay. mRNA and protein levels of FGFR3, MMP1, MMP9, vimentin, E-cadherin, ERK1/2, and p-ERK1/2 were detected via qPCR and Western blot. IGF-1, VEGF, and Estradiol (E2) levels were measured through Enzyme linked immunosorbent assay (ELISA). This study verified that Glabrene was capable of suppressing tumor growth in NSCLC mice, reversing tumor tissue's pathological morphology, attenuating the capacities of cancerous cells' proliferation, migration, and invasion, and leading to apoptosis. Besides, Glabrene could reduce the FGFR3 expression in HCC827 cells. Over-expression of FGFR3 promotes the proliferation of HCC827 cells, increase both contents of IGF-1, VEGF, and E2, and expressions of MMP1, MMP9, vimentin, and p-ERK1/2, while Glabrene inhibited FGFR3. Glabrene, and inhibition of FGFR3 expression were capable of decreasing FGFR3, MMP1, MMP9, vimentin, and p-ERK1/2 expression, as well as contents of IGF-1, VEGF, and E2 in model mice and HCC827 cells, and promoting the expression of E-cadherin. Glabrene has the potential as a therapeutic agent for NSCLC by reducing cancer invasion and migration through the inhibition of ERK1/2 phosphorylation and suppression of epithelial-mesenchymal transition (EMT).

Abstract 1438: Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response

Abstract In this study, we investigated the contributions of the epithelial-mesenchymal transition (EMT) program to small cell lung cancer (SCLC) tumorigenesis and chemoradiation (CRT) response. SCLC is a highly aggressive and deadly neuroendocrine malignancy. Two major factors that contribute to the high mortality of SCLC are (1) early metastasis and (2) rapid development of therapy resistance. Recent research suggests upregulation of EMT and the EMT transcription factor Twist1 correlated with accelerated tumor progression and CRT resistance in SCLC. Due to the lack of suitable animal models, the causal relationship between the EMT program and SCLC tumorigenesis and therapy response has not been rigorously investigated. To this end, we have generated a novel genetically engineered mouse model (GEMM) termed RPGT (Rb1Flox; Trp53Flox; ROSA26LSL-rtTA-IRES-EGFP; Twist1-TetO7-Luc). The RPGT GEMM enables the generation of autochthonous SCLC tumors after induction with Cre recombinase adenovirus. By withdrawing or providing doxycycline to mice, we can control Twist1 expression to activate or inactivate EMT in tumors. Upon characterizing mouse tumor samples with histological and molecular analyses, we observed that both control (no Twist1 overexpression) and Twist1-overexpressing RPGT mice developed neuroendocrine SCLC tumors. While SCLC-A was the predominant subtype in both cohorts, RPGT tumors exhibited more heterogeneity. Importantly, although no difference in tumorigenesis was observed, Twist1 overexpression dramatically increased the metastatic incidence in RPGT (87-100%) compared to control (25-50%) animals, indicating an important role of EMT in SCLC dissemination. Furthermore, transcriptomic profiling of primary tumors and matching metastases in RPGT mice revealed downregulation of Twist1 and EMT in metastases, suggesting that EMT suppression was necessary for metastatic outgrowth. To evaluate the impact of EMT activation on SCLC sensitivity to CRT, we performed in vitro viability assays on primary tumor cell lines established from RPGT tumors. We found that repressing Twist1 expression enhanced SCLC susceptibility to CRT. To validate these results in vivo, we treated RPGT mice with vs. without Twist1 overexpression with CRT. Consistent with our in vitro results, our in vivo data indicated that Twist1 overexpression promoted CRT resistance in SCLC tumors. Overall, our data suggest that the EMT program plays an important role in SCLC differentiation, metastasis, and resistance against chemo-radiotherapy. Citation Format: Triet Nguyen, Jinhee Chang, Kathleen Gabrielson, Amol Shetty, Yang Song, Audrey Lafargue, Shreya Jagtap, Danielle Council, Aaron Chan, Dipanwita Dutta Chowdhury, Muhammad Ajmal Khan, Nick Connis, Francesca Carrieri, Eddie Imada, Daniel Sforza, Eric Gardner, Christopher McFarland, Luigi Marchionni, Mohammad Rezaee, Christine Hann, Phuoc Tran. Investigating the roles of EMT in small cell lung cancer tumorigenesis and chemoradiation response [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 1438.

Astaxanthin suppresses the metastasis of clear cell renal cell carcinoma through ROS scavenging

Clear cell renal cell carcinoma (ccRCC) represents a prevalent form of kidney cancer, and its poor prognosis is closely tied to the occurrence of metastasis. ROS can drive epithelial-mesenchymal transition (EMT), thereby promoting cancer metastasis. Astaxanthin (AXT), renowned for its exceptional antioxidant properties, has been shown in this study to suppress ccRCC metastasis by scavenging ROS. AXT dose-dependently reduced ROS levels and attenuated migratory and invasive capabilities of ccRCC cells. Additionally, ccRCC cells treated with AXT exhibited phenotypic characteristics indicating EMT inhibition, which was reflected by the increased E-cadherin and decreased vimentin. Furthermore, AXT-induced suppressed metastatic potential was also corroborated in mouse lung metastasis model. Notably, artificially elevating ROS levels reversed the AXT-induced reduction in migratory and invasive capacity. Taken together, these findings highlight the anti-metastatic function of AXT in ccRCC, suggesting its therapeutic potential in the management of ccRCC metastasis.

LINC00158 modulates the function of BEAS-2B cells via targeting BCL11B and ameliorates OVA-LPS-induced severe asthma in mice models

Persistent type (T) 2 airway inflammation plays an important role in the development of severe asthma. However, the molecular mechanisms leading to T2 severe asthma have yet to be fully clarified. Human normal lung epithelial cells (BEAS-2B cells) were transfected with LINC00158/BCL11B plasmid/small interfering RNA (siRNA). Levels of epithelial-mesenchymal transition (EMT)-related markers were measured using real-time qPCR (RT-qPCR) and western blot. A dual luciferase reporter assay was used to validate the targeting relationship between LINC00158 and BCL11B. The effects of LINC00158-lentivirus vector-mediated overexpression and dexamethasone on ovalbumin (OVA)/lipopolysaccharide (LPS)-induced severe asthma were investigated in mice in vivo. Our study showed that overexpression of LINC00158/BCL11B inhibited the levels of EMT-related proteins, apoptosis, and promoted the proliferation of BEAS-2B cells. BCL11B was a direct target of LINC00158. And LINC00158 targeted BCL11B to regulate EMT, apoptosis, and cell proliferation of BEAS-2B cells. Compared with severe asthma mice, LINC00158 overexpression alleviated OVA/LPS-induced airway hyperresponsiveness and airway inflammation, including reductions in T helper 2 cells factors in lung tissue and BALF, serum total- and OVA-specific IgE, inflammatory cell infiltration, and goblet cells hyperplasia. In addition, LINC00158 overexpression alleviated airway remodeling, including reduced plasma TGF-β1 and collagen fiber deposition, as well as suppression of EMT. Additionally, overexpression of LINC00158 enhanced the therapeutic effect of dexamethasone in severe asthmatic mice models. LINC00158 regulates BEAS-2B cell biological function by targeting BCL11B. LINC00158 ameliorates T2 severe asthma in vivo and provides new insights into the clinical treatment of severe asthma.

Cryptochrome 2 Suppresses Epithelial-Mesenchymal Transition by Promoting Trophoblastic Ferroptosis in Unexplained Recurrent Spontaneous Abortion

Unexplained recurrent spontaneous abortion (URSA) is a serious reproductive issue that affects women of childbearing age. Studies have shown a close association between disrupted circadian rhythm and impaired epithelial-mesenchymal transition (EMT) process in trophoblasts during URSA, though the underlying mechanism has not been elaborated. This study aims to investigate the regulatory relationship between circadian rhythm gene cryptochrome 2 (CRY2) and ferroptosis on the migratory ability of trophoblast cells. Cell proliferation experiments, wound healing assays, and expression of related markers were conducted to verify the EMT efforts. Trophoblastic ferroptosis was confirmed by the expressions of malondialdehyde, glutathione, mitochondrial membrane potential, divalent iron ions, and expression level of related genes. The results showed significant increased expression of CRY2 and decreased expression of brain and muscle ARNT-like protein 1 (BMAL1) in the URSA villous tissues, accompanied by iron-dependent oxidative changes and abnormal expression of ferroptosis related proteins. It was observed that CRY2 and BMAL1 was co-localized and function as a feedback loop, which regulated the dynamic changes of EMT-related markers in trophoblast cells. CRY2 promoted trophoblastic ferroptosis, while BMAL1 had the opposite effect. Particularly, the ferroptosis inhibitor (Ferrostatin-1) effectively reversed the trophoblastic ferroptosis and EMT inhibition caused by CRY2 overexpression. Collectively, these results suggest that CRY2 regulates trophoblastic ferroptosis and hinders cellular EMT and migratory ability by suppressing BMAL1 expression.

HIF-1 inhibition reverses opacity in a rat model of galactose-induced cataract.

Cataract is an eye disease, in which the lens becomes opaque, causing vision loss and blindness. The detailed mechanism of cataract development has not been characterized, and effective drug therapies remain unavailable. Here, we investigated the effects of Hypoxia-inducible factor 1 (HIF-1) inhibitors using an ex vivo model, in which rat lenses were cultured in galactose-containing medium to induce opacity formation. We found that treatment with the HIF-1 inhibitors 2-Methoxyestradiol (2ME2), YC-1, and Bavachinin decreased lens opacity. Microarray analysis on 2ME2-treated samples, in which opacity was decreased, identified genes upregulated by galactose and downregulated by inhibitor treatment. Subsequent STRING analysis on genes that showed expression change by RT-qPCR identified two clusters. First cluster related to the cytoskeleton and epithelial-mesenchymal transition (EMT). Second cluster related to the oxidative stress, and apoptosis. ACTA2, a known marker for EMT, and TXNIP, a suppressor of cell proliferation and activator of apoptosis, were present in each cluster. Thus, suppression of EMT and apoptosis, as well as activation of cell proliferation, appear to underlie the decrease in lens opacity.

Cytotoxic and Antitumoral Effects of Methanolic Extracts of Avocado Fruit Mesocarp in Colorectal Cancer Cell Line HT29.

Colorectal cancer is a widespread neoplasia with high ratios of chemoresistance. Phytochemicals in plant-based extracts could be useful to treat colorectal cancer, and/or reduce chemoresistance. Methanolic extract of avocado mesocarp (MEAM) has demonstrated antitumoral properties, depending on the fruit ripening stage (RS). The aim of this study was to analyze the effects of methanolic extracts of "Hass" avocado fruit at different RS on cytotoxicity, antioxidative, anti-inflammatory, anti-invasive, cell cycle, and epithelial-mesenchymal transition inhibition in colorectal adenocarcinoma cell line HT29. The MEAM showed an increasing concentration of total phenolic compounds as the RS progressed, which was correlated with antioxidant capacity measured by the Ferric Reducing Antioxidant Power assay but not with the 2.2-diphenyl-1-picrylhydrazyl assay. The specific phenolic compounds of MEAM were determined by high-performance liquid chromatography, and it was found that concentrations of epicatechin decreased while concentrations of chlorogenic acid increased as the RS progressed. The HT29 cell line was treated with MEAM for 48 h, and all MEAM had a cytotoxic effect, reported by MTT assay, nevertheless, the strongest effect was associated with the presence of chlorogenic acid. MEAM induced apoptosis and cell cycle arrest in phase G0/G1, reported by flow cytometry. Moreover, MEAM inhibited cell migration evidenced by the wound healing assay. On the other hand, MEAM significantly reduced expression of mRNA of tumor necrosis factor-alpha and cyclooxygenase 2. These effects comprise important inhibition of some hallmarks of cancer. This, in turn, may provide interesting guidelines for developing antitumoral intervention agents.

Krüppel-like factor 4 modulates the miR-101/COL10A1 axis to inhibit renal fibrosis after AKI by regulating epithelial–mesenchymal transition

Acute kidney injury (AKI) can progress to renal fibrosis and chronic kidney disease (CKD), which reduces quality of life and increases the economic burden on patients. However, the molecular mechanisms underlying renal fibrosis following AKI remain unclear. This study tested the hypothesis that the Krüppel-like factor 4 (KLF4)/miR-101/Collagen alpha-1X (COL10A1) axis could inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis after AKI in a mouse model of ischemia-reperfusion (I/R)-induced renal fibrosis and HK-2 cells by gene silencing, overexpression, immunofluorescence, immunohistochemistry, real-time quantitative PCR, Western blotting, dual-luciferase reporter assay, fluorescence in situ hybridization (FISH) and ELISA. Compared with the Sham group, I/R induced renal tubular and glomerular injury and fibrosis, and increased the levels of BUN, serum Scr and neutrophil gelatinase-associated lipocalin (NGAL), Col10a1 and Vimentin expression, but decreased E-cadherin expression in the kidney tissues of mice at 42 days post-I/R. Similarly, hypoxia promoted fibroblastic morphological changes in HK-2 cells and enhanced NGAL, COL10A1, Vimentin, and α-SMA expression, but reduced E-cadherin expression in HK-2 cells. These pathological changes were significantly mitigated in COL10A1-silenced renal tissues and HK-2 cells. KLF4 induces miR-101 transcription. More importantly, hypoxia upregulated Vimentin and COL10A1 expression, but decreased miR-101, KLF4, and E-cadherin expression in HK-2 cells. These hypoxic effects were significantly mitigated or abrogated by KLF4 over-expression in the HK-2 cells. Our data indicate that KLF4 up-regulates miR-101 expression, leading to the downregulation of COL10A1 expression, inhibition of EMT and renal fibrosis during the pathogenic process of I/R-related renal fibrosis.

Investigation of in-vitro Anti-Cancer and Apoptotic Potential of Garlic-Derived Nanovesicles against Prostate and Cervical Cancer Cell Lines.

Investigate the anti-cancerous potential of garlic-derived nanovesicles (GDNVs), exploring their cytotoxic effects on HeLa and PC-3 cell lines, and elucidate the underlying mechanisms, including apoptosis induction and inhibition of epithelial-mesenchymal transition (EMT). GDNVs were isolated using differential centrifugation and ultracentrifugation. Characterization was performed through dynamic light scattering (DLS), field-emission scanning electron microscopy (FESEM), and Fourier-transform infrared spectroscopy (FTIR). Cytotoxicity assessments on HeLa and PC-3 cell lines using MTT assay. Apoptosis induction was evaluated through nuclear morphology changes and quantification of apoptotic cells using DAPI and PI/annexin V analysis. Western blot of apoptosis-related proteins (bcl-2, bax, caspase-3) was analysed. Anti-metastatic potential was assessed using wound healing assay and EMT transition inhibition. Garlic-derived nanovesicles (GDNVs), characterized by a size of 134.2 nm, demonstrated a substantial and dose- as well as time-dependent anti-proliferative impact on HeLa and PC-3 cell lines. The induction of apoptosis was unequivocally established through discernible modifications in nuclear morphology. The apoptotic cell count in HeLa and PC-3 cells increased by 42.4 ± 4.2% and 38.2 ± 3.2%, respectively. Comprehensive Western blot demonstrated alterations in the expression of key apoptotic regulators, namely bcl-2, bax, and caspase-3, providing robust evidence for the initiation of apoptosis. Furthermore, GDNVs exerted a significant inhibitory effect (p < 0.001) on the migratory potential of both HeLa and PC-3 cells. Moreover, there was a discernible association between GDNVs and the suppression of Epithelial-Mesenchymal Transition (EMT), emphasizing their role in impeding the metastatic potential of these cancer cell lines. This study establishes, for the first time, the anti-cancerous potential of GDNVs. The observed dose- and time-dependent anti-proliferative effects, selective cytotoxicity, apoptosis induction, and anti-migratory potential highlight GDNVs as a promising candidate for cancer treatment.

P121 Characterisation of the miRNAome in Crohn’s Disease patients reveals transcriptomic changes associated with tissue composition and drug treatment

Abstract Background Gene regulation plays a major role in Crohn's Disease (CD) susceptibility and drug response. MicroRNA (miRNA)-mediated mechanisms tune gene expression at the post-transcriptional level. MiRNAs are 20-to-25-nt long non-coding RNAs that repress protein expression by direct inhibition or cleavage of the targets. Evaluating the miRNAome is emerging as a promising tool to clarify pathogenesis and identify promising biomarkers. We characterised the regulatory miRNAome across disease subtypes, gut tissue locations and infliximab treatment in CD. Methods We generated 120 transcriptomic profiles from terminal ileum and left colon biopsies from Hospital Universitario de la Princesa (Madrid). This cohort is composed of 10 patients with active endoscopy, 10 quiescent CD cases, and 10 controls (30 in total). In a balanced setting, half of the samples were incubated with infliximab for 18 hours at 37ºC and the rest with basal medium (Table 1). We carried out an exploratory analysis to gain insights into the determinants of miRNAome variability, including variance partition analyses and linear mixed modelling to detect significantly altered miRNAs. We also harnessed results from several publications including GSE16879 dataset to perform a comparative study with infliximab response signatures. Results Tissue location is the primary determinant of miRNA expression variability. The Variance Partition model identified 30 top miRNAs whose variability is maximised across regions. However, patient-specific effects also play a relevant role. We also identified 96 differentially expressed miRNAs between terminal ileum and left colon, with an overrepresentation of molecules involved in stress and developmental pathways (Fig.1). Among these, we find miR200s and miR429, that are associated with suppression of epithelial-mesenchymal transition and are up-regulated in colon more than in ileum. Down-regulation of miR196 is also shown in terminal ileum. Regarding the effects of infliximab treatment, we detected nine deregulated miRNAs which are associated with cellular response pathways. These include miR200s and miR192, which targets NOD2 and down-regulates NF-κB. Our results show an up-regulation of miR192 upon infliximab incubation (log2FC=2.37), which is associated with a reduction in TNFα expression and, consequently, with induced overexpression of miR192. We conclude that this miRNA is a potential target for therapy development. Conclusion In short, tissue location, and to a lesser extent disease subtype, are the main determinants of miRNAome in CD. A few treatment-responsive miRNAs like miR192 and miR200s are identified, in spite of detection challenges, emphasizing the need to improve our comprehension of the miRNA dynamics associated with drug response.

The role of alternative polyadenylation in epithelial-mesenchymal transition of non-small cell lung cancer.

The metastatic non-small cell lung cancer (NSCLC) is one of the cancers with high incidence, poor survival, and limited treatment. Epithelial-mesenchymal transition (EMT) is the first step by which an early tumor converts to an invasive one. Studying the underlying mechanisms of EMT can help the understanding of cancer metastasis and improve the treatment. In this study, 1013 NSCLC patients and 123 NSCLC cell lines are deeply analyzed for the potential roles of alternative polyadenylation (APA) in the EMT process. A trend of shorter 3'-UTRs (three prime untranslated region) is discovered in the mesenchymal samples. The identification of EMT-related APA events highlights the proximal poly(A) selection of CARM1. It is a pathological biomarker of mesenchymal tumor and cancer metastasis through losing miRNA binding to upregulate the EMT inducer of CARM1 and releasing miRNAs to downregulate the EMT inhibitor of RBM47. The crucial role of this APA event in EMT also guides its effect on drug responses. The patients with shorter 3'-UTR of CARM1 are more benefit from chemotherapy drugs, especially cisplatin. A stratification of NSCLC patients based on this APA event is useful for chemotherapy design in future clinics.