E-cadherin Protein Expression Research Articles

Dibenzolium induces apoptosis and inhibits epithelial-mesenchymal transition (EMT) in bladder cancer cell lines

Bladder cancer treatments are highly aggressive and have strong side effects. Safer and more effective treatments are needed. In this study, Dibenzolium (DIB), a potent NADPH oxidase inhibitor, was evaluated for its anti-tumor effects. KK-47 (non-invasive), T24 and 5637 (invasive) cells were used in experiments. Cell proliferation, apoptosis and wound healing assays and western blotting were conducted. In addition, DIB was intratumorally administered to mice bearing KK-47, T24 and 5637 tumors, and tumor size and weight were observed over time. After removing tumors, immunohistochemistry (IHC) staining was conducted. Cell proliferation was significantly suppressed in all cell lines, and apoptotic cells increased in the KK-47 and T24 cell lines after DIB. Wound healing was suppressed in all cell lines by DIB. In KK-47 and T24, DIB increased the protein expression of the epithelial marker E-cadherin. In vivo, DIB safely suppressed tumor growth in all cell lines-bearing mice. Cleaved-Caspase-3 and E-cadherin expression increased in KK-47 and T24 tumors after DIB. In conclusion, DIB inhibited tumor growth by inducing apoptosis through the Caspase-3 pathway and reduced migration and invasion by suppressing epithelial mesenchymal transition (EMT) in bladder cancer similarly shown as our previous study of prostate cancer.

MicroRNA-34a negatively regulates Netrin1 and mediates MEK/ERK pathway to regulate chemosensitivity of gastric cancer cells.

To explore the mechanism of action of MicroRNAs-34a (miR-34a) and Eurite growth guiding factor 1 (Netrin1) in cisplatin resistance in gastric cancer (GC), providing new clues for overcoming tumor resistance and optimizing anti-tumor therapy for GC. The Cancer Genome Atlas (TCGA), Differentially Expressed MicroRNAs (miRNAs) in human cancers (dbDEMC), and Starbase online databases were used to analyze the correlation between miR-34a and Netrin-1 and prognosis in GC, and to predict and verify the targeted binding of miR-34a to Netrin-1. The experimental methods including Cell transfection, real-time polymerase chain reaction (RT-PCR), Cell-Counting-Kit-8 (CCK8) assay, flow cytometry, wound scratch assay, transwell assay, and western blotting were used to investigate the effects of miR-34a and Netrin1 on chemotherapy resistance and biological characteristics in cisplatin-resistant GC cells (HGC27/DDP), and to analyze the molecular mechanism of cisplatin resistance. miR-34a expression was downregulated in gastric cancer clinical samples and cisplatin-resistant cells, while Netrin1 was upregulated, and was related to overall survival (OS). Upregulation of miR-34a can significantly reduce the IC50 value of cisplatin(0.65 vs 1.6 ng/mL) and Multidrug Resistance 1 (MDR-1) protein level, inhibit the proliferation activity, reduce the expression levels of proliferating cell nuclear antigen (PCNA) and ki-67 protein, and induce the increase of apoptosis rate and the enhancement of cycle arrest. Upregulation of miR-34a can also significantly reduce the expression level of Matrix metalloproteinase 9 (MMP9) protein, promote the expression of E-cadherin protein, reduce the wound healing rate and invasion number to inhibit migration and invasion ability in drug-resistant gastric cancer cells. Moreover, overexpression of Netrin1 on the basis of upregulation of miR-34a can weaken the above changes caused by upregulation of miR-34a. In addition, upregulation of miR-34a can significantly inhibit the Mitogen-activated protein kinase kinase (MEK) / Extracellular regulated protein kinases (ERK) pathway, while overexpression of Netrin1 can activate the MEK/ERK pathway, and inhibition of MEK/ERK pathway can effectively counteract the protein expression of Netrin1, and reverse changes in the expression of cisplatin IC50 and MDR-1 proteins caused by co-upregulation of miR-34a/Netrin1 in HGC27/DDP, as well as changes in proliferation, apoptosis, migration and invasion. In addition, upregulation of miR-34a can significantly inhibit the MEK/ERK pathway, while overexpression of Netrin1 can activate the MEK/ERK pathway. If the MEK/ERK pathway was inhibited, it can effectively counteract the protein overexpression of Netrin1, and reverse the changes in the expression of cisplatin IC50 and MDR-1 proteins in HGC27/DDP induced by co-upregulation of miR-34a / Netrin1, as well as changes in proliferation, apoptosis, migration and invasion. miR-34a targets and negatively regulates Netrin1 to mediate the proliferation, apoptosis, apoptosis, migration, and invasion of drug-resistant gastric cancer cells via the MEK/ERK pathway, and change the chemosensitivity in GC cells. miR-34a/Netrin1/MEK/ERK axis may serve as a novel therapeutic target for chemoresistance in GC, it is of great significance for overcoming drug resistance and developing new therapeutic strategies for GC.

Chrysene contribution to bronchial asthma: Activation of TRPA1 disrupts bronchial epithelial barrier via ERK pathway

BackgroundElevated polycyclic aromatic hydrocarbon (PAH) levels are associated with exacerbation of asthma. Chrysene is one of the most prevalent unsubstituted PAHs in the environment. Transient receptor potential ankyrin 1 (TRPA1) can be used as a chemoreceptor to detect inhaled stimuli and plays an important role in the occurrence and deterioration of asthma. Whether exposure to a high concentration of chrysene in the environment can activate TRPA1 and contribute to the development of asthma, potentially through the dysfunction of the bronchial epithelial barrier, remains unclear. MethodsA cell-based assay was performed to verify the downregulation of the expression of E-cadherin and tight junction (TJ) proteins by chrysene in bronchial epithelial cells to explore the role of chrysene-mediated TRPA1 activation in the regulation of TJ protein expression through the extracellular signal-regulated protein kinase (ERK) pathway. Animal tests were conducted to determine whether chrysene could enhance airway hyperresponsiveness (AHR) induced by house dust mites (HDMs) and disrupt barrier function, thereby contributing to asthma. ResultsThe cell-based assay revealed that chrysene could disrupt the function of the bronchial epithelial barrier and decrease the expression levels of E-cadherin, zonula occludens-1 (ZO-1), occludin, and claudin-5 through the ERK pathway. Chrysene induced airway epithelial barrier dysfunction primarily through TRPA1 instead of transient receptor potential vanilloid 1. TRPA1 knockdown was able to attenuate chrysene-induced downregulation of TJ protein expression and downregulate ERK activation (p-ERK). Compared with exposure to HDM alone, coexposure to chrysene and HDM resulted in an increased incidence of AHR, disruption of barrier function, and eosinophilic inflammatory responses in a mouse model of asthma. Coexposure to chrysene and HDM increased TRPA1 expression. The animal test verified that the TRPA1 inhibitor HC030031 could suppress chrysene and HDM-induced asthma in mice. ConclusionsOur findings showed that chrysene contributed to the breakdown of the function of the bronchial epithelial barrier through the TRPA1–ERK axis and therefore acted as an adjuvant to contribute to asthma.

Comparative Immunohistochemistry Study Of Expression Of E- Cadherin In Benign Prostatic Hyperplasia And Prostate Cancer

Benign Prostatic Hyperplasia (BPH) and Prostate Cancer (PC ) are the most common diseases in men, especially in older men than 50 years old, BPH is a non-cancerous enlargement of the prostate as a result of the increased number of smooth muscle cells. Tumors can be benign (not cancerous), or malignant (cancerous), prostate cancer is the abnormal and random growth of cells in the prostate gland to the extent that it loses its shape and function. this study involved 120 samples of tissues taken from men suffering from benign and malignant tumours with ages ranging between (40- 85) years. For detection of the expression of E-cadherin in BPH and PC using immunohistochemistry (IHC) method. The results obtained after microscopic examination showed that protein expression of E-cadherin was highly positive in BPH comparison with the expression of E-cadherin was weakly positive in low-grade cancer and negative in high-grade cancer (advanced prostate cancer).

Nebulization of Hypoxic hUCMSC-EVs Attenuates Airway Epithelial Barrier Defects in Chronic Asthma Mice by Transferring CAV-1.

Nebulization of hypoxic human umbilical cord mesenchymal stem cell-derived extracellular vesicles (Hypo-EVs) can suppress airway inflammation and remodeling in a chronic asthmatic mouse; however, the exact mechanism remains unclear. Recently, airway epithelial barrier defects have been regarded as crucial therapeutic targets in asthma. The aim of this study was to investigate whether and how Hypo-EVs protect against the disruption of the airway epithelial barrier under asthmatic conditions. The therapeutic effects of Hypo-EVs on airway epithelial barrier defects were evaluated in ovalbumin (OVA)-induced asthmatic mice and in IL-4 and IL-13-induced HBE135-E6E7 cell models by detecting cell monolayer leakage and junctional protein expression. The protein levels in Hypo-EVs were determined by Western blotting, and a gene knockdown approach was used to investigate the biofactors in Hypo-EVs. Nebulization of Hypo-EVs directly alleviated airway epithelial barrier defects in asthmatic mice, as evidenced by colocalization with bronchial epithelial cells, decreased albumin concentration, and increased ZO-1 and E-cadherin expression. In vitro, Hypo-EV treatment dramatically rescued the increase in airway cell permeability, and upregulated the ZO-1 and E-cadherin protein expressions. Based on WB analysis, we found that caveolin-1 (CAV-1) was strongly enriched in Hypo-EVs. The knockdown of CAV-1 protein levels in Hypo-EVs significantly impaired Hypo-EV-mediated barrier protection in vitro and in vivo. Moreover, CAV-1 knockdown significantly abolished the beneficial effects of Hypo-EVs on airway inflammation and remodeling in asthmatic mice. In addition, we showed that IL-4/IL-13-induced airway epithelial barrier defects were mainly related to activation of STAT6 phosphorylation (p-STAT6), and overexpression of CAV-1 or Hypo-EV treatment inhibited the levels of p-STAT6 in IL-4/IL-13-induced HBE135-E6E7 cells. Nebulization of Hypo-EVs can attenuate airway epithelial barrier defects in asthma by delivering CAV-1 to inhibit p-STAT6 expression and may be used to treat other barrier defect diseases.

Serinc2 Drives the Progression of Cervical Cancer Through Regulating Myc Pathway.

As one of the most common malignancies, cervical cancer (CC) seriously affects women's health. This study aimed to investigate the biological function of Serinc2 in CC. Serinc2 expression was surveyed utilizing immunohistochemistry, western blot, and qRT-PCR. CC cell viability, invasion, proliferation, migration, and apoptosis, were detected via CCK-8, Transwell assay, colony formation, wound healing assay, and flow cytometry. Glucose consumption, lactate production, and ATP levels were determined by the corresponding kit. The protein expression of c-Myc, PDK1, HK2, PFKP, LDHA, Snail, Vimentin, N-cadherin, and E-cadherin was detected via western blot. The interaction between the promoter of PFKP and Myc was confirmed through luciferase reporter assay and Chip assay. Invivo, to evaluate the function of Serinc2 on tumor growth, a xenograft mouse model was used. In CC tissues and cells, Serinc2 was upregulated. In CC cells, knockdown of Serinc2 suppressed cell invasion, proliferation, migration, decreased the expression of Snail, Vimentin, N-cadherin, HK2, PFKP, LDHA, and PDK1, increased E-cadherin expression, reduced glucose consumption and the production of lactate and ATP, and induced cell apoptosis; Serinc2 overexpression led to the opposite results. Mechanically, Serinc2 promoted Myc expression, and Myc induced PFKP expression. Furthermore, overexpressed Myc abolished the inhibitive influences of Serinc2 knockdown on the malignant behaviors of CC cells. Additionally, knockdown of Serinc2 inhibited tumor growth and reduced the protein expression of c-Myc, PFKP, LDHA, and PDK1 invivo. Knockdown of Serinc2 inhibited the malignant progression of CC, which was achieved via Myc pathway. Our study provides novel insight into CC pathogenesis.

Effects of Platycodon grandiflorus on doxorubicin resistance and epithelial-mesenchymal transition of breast cancer cells via the p38 mitogen-activated protein kinase pathway.

With the increase of chemotherapy frequency for breast cancer, the drug resistance rate of patients is rising, accompanied by cell invasion and metastasis, thus causing mortality. We aimed to explore the mechanism by which Platycodon grandiflorus affects breast cancer cells in terms of the doxorubicin (Dox) resistance and epithelial-mesenchymal transition (EMT) via the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway. MCF-7/R cell lines with resistance to Dox were established. After 24h of culture with DMEM (blank group), they were divided into Platycodon grandiflorus, Platycodon grandiflorus + Ophiopogon japonicus, Platycodon grandiflorus + Curcumae Rhizoma, Platycodon grandiflorus + Curcumae Rhizoma + U46619 groups. Flow cytometry, colony formation assay, as well as Transwell assay were performed to examine the cells for apoptosis, proliferation, and invasion, respectively. Western blotting was performed to measure the phosphorylated (p)-p38 MAPK-to-p38 MAPK ratio together with N-cadherin, vimentin, β-catenin, and E-cadherin protein expressions. Compared with the blank group, the half maximal inhibitory concentration (IC50), number of cell colonies, number of invading cells and expressions of proteins related to EMT (i.e. N-cadherin, vimentin, and β-catenin) significantly reduced, but increases in apoptosis rate, p-p38 MAPK/p38 MAPK ratio and E-cadherin protein expression were observed in different groups (P < 0.05). Compared with the Platycodon grandiflorus + Curcumae Rhizoma group, the Platycodon grandiflorus + Curcumae Rhizoma + U46619 group had significantly decreased IC50, cell colony count, invading cell count and β-catenin, N-cadherin, and vimentin expressions, in addition to elevated E-cadherin protein expression, apoptosis rate, and p-p38 MAPK/p38 MAPK ratio (P < 0.05). Platycodon grandiflorus can reverse the resistance of breast cancer cells to Dox and regulate their biological activities by activating the p38 MAPK signaling pathway.

Diterpene glycosides from Fructus Rubi ameliorates benign prostatic hyperplasia in rats through the androgen and TGF-β/Smad signaling pathway

Fructus Rubi (FR), a food material with medicinal value, is used in traditional Chinese medicine (TCM) for treatment of various kidney-related problems, such as impotence, spermatorrhea, and frequent urination. It is also frequently used to produce diverse functional foods in China. The purpose of this research was to assess the therapeutic effects of FR diterpene glycosides on RWPE-1 epithelial cell (RWPE-1), a human normal prostatic epithelial cell, and benign prostate hyperplasia (BPH) rats, both of which had been exposed to dihydrotestosterone (DHT) and testosterone propionate (TP), respectively, and to investigate the mechanism of action. Target proteins that could stably bind to certain diterpene glycosides were screened through drug affinity responsive target stability combined with mass spectrometry (DARTS/MS). DHT-induced RWPE-1cells were used to detect drug activity. TP was subcutaneously injected to induce BPH in rats. The extract of diterpene glycosides from FR (FDS) was orally administered for 28 days. The DHT levels in the serum and prostate tissue of the rats were measured through enzyme-linked immunosorbent assay (ELISA), and to analyze cell proliferation and epithelial-mesenchymal transition (EMT), the protein expression of prostate-specific antigen (PSA), androgen receptor (AR), steroid 5α-reductase type 2 (SRD5A2), proliferating cell nuclear antigen (PCNA), S100 calcium-binding protein A2 (S100A2), transforming growth factor-β1 (TGF-β1), E-cadherin, vimentin, and Smad4 was determined through western blotting (WB), immunohistochemistry (IHC), or immunofluorescence (IF). FDS reduced the proliferation of DHT-induced RWPE-1cells. It also significantly inhibited rat prostate enlargement; decreased DHT levels in the serum and prostate tissue; inhibited the protein expression of AR, PSA, PCNA, S100A2, TGF-β1, E-cadherin, and Smad4; and increased the protein expression of E-cadherin. The present study is the first to report that diterpene glycosides isolated from FR inhibited BPH at the cellular level, regulated the proliferation of prostate cells through the androgen signaling pathway, and prevented EMT in the prostate through the S100A2-mediated TGF-β/Smad signaling pathway. These results indicate that FDS is a promising multitarget therapy for BPH.

Diagnostic Values of Ki67, Cox2, CD31, E-cadherin, VEGF, MMP2, and MMP9 Protein Expression in Human Melanoma

Background: Cutaneous melanoma, the most severe form of skin cancer, has an unpredictable behavior and a high mortality rate. Recent research has identified new biomarkers and their associations with certain histopathological features, offering essential insights into diagnosis, prognosis, and therapeutic strategies. Material and method: In this meticulously designed and executed study, we analyzed 85 formalin-fixed, paraffin-embedded (FFPE) tissue samples using the gold standard technique of immunohistochemistry (IHC) to characterize the protein expression profiles. We used a comprehensive panel of Ki67, Cox2, CD31, VEGF, MMP2, MMP9, and E-cadherin antibodies. Clinical stages were meticulously determined according to TNM classification, the thickness of the Breslow depth, and Clark levels. We employed robust statistical methods such as one-way ANOVA and curve estimation regression to analyze the data. The STRING database, a trusted resource, was used to identify protein-protein interactions and related pathways. Results: The results of our study unveiled novel pairwise positive correlations between CD31 and Ki67 (r = 0.72), and between VEGF and Ki67 protein expression (r = 0.63). We also found a significant inverse relationship between the expression of E-cadherin and some biomarkers, such as Ki67, CD31, and VEGF (p &lt; 0.001 for all). Additionally, our findings revealed a strong positive association between the extent of tumor invasion and the expression levels of Ki67, CD31, and VEGF. The overexpression of these proteins was significantly correlated with advanced clinical stages (p &lt; 0.001 for all). Conclusion: Our study suggests that the biomarkers we examined could be reliable potential diagnostic and prognostic biomarkers for cutaneous melanoma. These findings have the potential to significantly impact the diagnosis, prognosis, and treatment of this deadly disease, offering new avenues for improved patient care.

Boronic ester bonding integrated dextran-based hydrogel patch for diabetic peripheral nerve injury treatment

Peripheral nerve injury (PNI) is a common clinical complication of diabetes, however, lacks effective clinical treatment due to complex pathological microenvironment. Considering both biomimetic remodeling and pathological reversal, we proposed a boronic ester bonding integrated strategy to develop dextran-based hydrogel patch with dopamine methacrylate-conjugated exosomes (mExo) immobilization. Aldehyde/phenylboronic acid difunctional dextran was synthesized to establish hydrogel skeleton and provide self-adhesion and ROS/glucose modulation capacities for neuroprotection. Meanwhile, mExo as therapeutic factors were immobilized into hydrogels via boronic ester bonding and released in pathologic response to target mitochondrial dysfunction and achieve neurotherapy. Through in vitro assays, the composite hydrogel patch was shown to reduce oxidative stress by ROS scavenging, thereby protecting the mitochondrial morphological and functional homeostasis of healthy RSC96 cells. Indeed, the sustained released exosome conjugates regulated bioenergy metabolism and alleviated mitochondrial dysfunction in hyperglycemia damaged RSC96 cells by the up-regulated expression of E-cadherin and AMPK proteins. In vivo experiments proved that through correcting diabetic pathology and targeted treatment of damaged mitochondria, the hydrogel patch inhibited neuronal apoptosis and finally promoted axon regeneration, remyelination and motor function recovery after PNI in diabetic rats. The synergistic “two-pronged” strategy of biomimetic remodeling and pathological reversal opens a promising opportunity for non-invasive treatment of diabetic PNI.

Conbercept reverses TGF-β2-induced epithelial-mesenchymal transition in human lens epithelial cells by regulating the TGF-β/Smad signaling pathway

To investigate the mechanism by which conbercept reverses transforming growth factor-β2 (TGF-β2)-induced epithelial-mesenchymal transition (EMT) in human lens epithelial cells (HLECs). Cultured HLEC SRA01/04 cells were treated with TGF-β2, conbercept, or both, and the changes in cell proliferation, apoptosis, and migration were observed using MTT assay, flow cytometry, scratch assay, and Transwell assay. Western blotting and qRT-PCR were used to detect the changes in the expression of EMT-related epithelial cell markers (E-Cadherin, α-SMA, and Snail), extracellular matrix components, and genes related to the TGF-β/Smad signaling pathway. Conbercept significantly reduced TGF-β2-induced EMT of SRA01/04 cells, decreased the expression levels of mesenchymal and extracellular matrix markers α-SMA, Snail, collagen I, collagen IV, and FN1, and upregulated the protein and mRNA expressions of E-cadherin (P <0.05). Transwell assay showed significantly lower cell migration ability in TGF-β2+conbercept group than in TGF-β2 group (P <0.05). Conbercept also inhibited the increase in Smad2/3 phosphorylation levels in HLEC-SRA01/04 cells with TGF-β2-induced EMT (P <0.01). Conbercept inhibits TGF-β2 induced EMT by downregulating the expression of pSmad2/3 in TGF-β/Smad signaling pathway, indicating a potential therapeutic strategy against visual loss induced by posterior capsule opacification.

Vitexin Induces Apoptosis and Ferroptosis and Suppresses Malignant Proliferation and Invasion of Bladder Urothelial Carcinoma through PI3K/AKT-Nrf2 Axis

Background: Bladder urothelial carcinoma (BUC) is a type of malignant urinary system. Although several strategies have been applied in the treatment of BUC, its survival remains unsatisfactory, especially in the patients with advanced BUC. Vitexin, a natural flavonoid has exhibited the inhibitory effect on various tumors, however, its effect on BUC is still unclear. Objective: This study aimed to explore the effect of vitexin on the progression of BUC. Methods: The toxicity of vitexin on T24 and 5637 cells was detected by cell counting kit-8 (CCK-8). The effects of vitexin on proliferation, apoptosis, invasion, epithelial-mesenchymal transition (EMT) and ferroptosis in BUC cells were determined by CCK-8, flow cytometry, western blot, transwell and immunofluorescence assays. Additionally, the related mechanism was explored by examining the expression of the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (AKT)-nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway. Besides, in vivo validation was performed in the xenografted mice. Results: Vitexin reduced the BUC cell viability and enhanced the apoptosis rate and the relative protein expression of p53 and cleaved-caspase3. Also, vitexin decreased the invasion number, and increased the relative protein expression of E-cadherin with the decreased N-cadherin protein level in T24 and 5637 cells. Besides, vitexin promoted the levels of ROS and MDA, while reduced the GSH level. Vitexin also increased the level of iron, but decreased the relative protein expression of xCT and GPX4. Erastin further increased the vitexin-induced iron levels, whereas inverse outcomes were observed in the application of ferrostatin-1. Additionally, vitexin decreased the relative protein levels of PI3K, p-AKT/AKT, and nuclear Nrf2, while increased the relative protein level of cytoplasmic Nrf2. Overexpression of PI3K notably inverted the effect of vitexin on cell viability, apoptosis, invasion, level of ROS and iron. Furthermore, vitexin reduced the tumor volume and weight of xenografted mice. Vitexin decreased the protein level of N-cadherin, while increased apoptosis rate of xenografted mice. In addition, vitexin reduced the relative protein levels of PI3K, p-AKT/AKT, and nuclear Nrf2 with the enhanced relative protein expression of cytoplasmic Nrf2 in xenografted mice. Moreover, vitexin decreased the relative protein expression of xCT and GPX4 and the GSH level, whereas increased the MDA level in xenografted mice. Conclusion: Vitexin suppressed malignant proliferation and invasion and induced apoptosis and ferroptosis of BUC involving in PI3K/AKT-Nrf2 pathway.

MiR-129–2-3p binds SEMA4C to regulate HCC development and inhibit the EMT

BackgroundAmong primary liver cancers, HCC is the most prevalent. Small noncoding RNAs called miRNAs control the expression of downstream target genes to take part in a variety of physiological and pathological processes, including those related to cancer. MethodsmiR-129–2–3p and SEMA4C expression levels were assessed using RT-qPCR. The CCK-8, invasion, and wound healing assays were used to confirm the capacity of HCC cells for proliferation, invasion and migration respectively. Serum SEMA4C levels were detected via ELISA. The RIP and dual-luciferase reporter assays were used to confirm the existence of intergenic binding sites. Cell apoptosis assay and cell cycle assay were performed to detect the apoptosis rate and cycle distribution of cells, and WB was performed to detect the protein expression of SEMA4C, RhoA, ROCK1, E-cadherin, N-cadherin, and vimentin. Furthermore, cancer-inhibiting role of miR-129–2–3p were further confirmed by animal tests. ResultsmiR-129–2–3p expression was reduced in HCC tissues and cells. Overexpression of miR-129–2–3p decreased the proliferation, invasion, migration, and EMT in HCC cells, whereas inhibition of miR-129–2–3p had the opposite effects. Our research also showed that SEMA4C was increased in HCC tissues, serum and cells, and that SEMA4C knockdown prevented HCC cell invasion, migration, proliferation, and EMT. Overexpression of SEMA4C reversed the inhibitory effect of miR-129–2–3p on HCC. ConclusionsOverall, we discovered that through binding to SEMA4C, miR-129–2–3p regulates HCC cell proliferation, invasion, migration, and EMT.

Mechanism of electroacupuncture in treating uterine endometrial fibrosis in intrauterine adhesions rats based on Wnt/β-catenin pathway-mediated epithelial-mesenchymal transition.

To observe the effect of electroacupuncture (EA) on the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition (EMT)-related proteins in rats with intrauterine adhesions (IUA), so as to explore the possible mechanisms of EA in repairing endometrial damage in IUA. Female SD rats were randomly divided into blank, model, EA, and ICG-001 groups, with 10 rats in each group. The IUA model was established by using mechanical scraping combined with lipopolysaccharide infection for double injury. In the EA group, "Guanyuan" (CV4) was needled and EA (2 Hz/15 Hz, 1-2 mA) was applied to "Zusanli" (ST36) and "Sanyinjiao"(SP6) on both sides. In the ICG-001 group, ICG-001 (5 mg/kg), the inhibitor of β-catenin was intraperitoneally injected. After intervention, samples were taken from 5 rats in each group, and uterine endometrium morphology, endometrial thickness, and gland counts were observed using HE staining. Masson staining was used to assess the degree of fibrosis in the endometrial tissue. Immunohistochemistry was used to detect the positive expression of transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMA), fibronectin (FN), connective tissue growth factor (CTGF), type I collagen (Col- Ⅰ), glycogen synthase kinase-3β (GSK-3β), β-catenin, E-cadherin, N-cadherin, and Vimentin in the endometrial tissue. Western blot was used to detect the relative expression of GSK-3β, β-catenin, E-cadherin, N-cadherin, and Vimentin proteins in the endometrial tissue. Another 5 rats from each group were placed in cages with male rats after intervention to record the number of embryo implantations. Necrosis and loss of endometrial tissue in the model group observed after HE staining were alleviated in the EA group, better than those in the ICG-001 group. Compared with the blank group, the numbers of glands and endometrial thickness in the uterine endometrial tissue, relative expression and positive expression of E-cadherin and GSK-3β proteins in the uterine endometrial tissue, and embryo implantation numbers were reduced(P<0.000 1, P<0.001, P<0.01) in the model group, while fibrosis area ratio in the uterine endometrial tissue, TGF- β 1, α -SMA, FN, CTGF, Col- Ⅰ positive expressions, N-cadherin, Vimentin, and β-catenin proteins expression and positive expression were increased(P<0.000 1, P<0.001, P<0.01). Compared with the model group, the number of glands and endometrial thickness, E-cadherin and GSK-3β proteins expression and positive expression, and embryo implantation numbers were increased (P<0.001, P<0.05, P<0.01) in the EA and ICG-001 groups, while the fibrosis area ratio in the uterine endometrial tissue, TGF-β1, α-SMA, FN, CTGF, Col- Ⅰ positive expression, and N-cadherin, Vimentin, and β-catenin proteins expression and positive expression were decreased(P<0.001, P<0.01, P<0.05). Compared with the EA group, the differences of the above-mentioned indicators in the ICG-001 group were not statistically significant. EA may reverse the EMT process and reduce the degree of fibrosis in endometrial tissue by inhibiting the Wnt/β-catenin signaling pathway, thereby promoting the repair of endometrial damage in IUA.

18β-glycyrrhetinic acid ameliorates bleomycin-induced idiopathic pulmonary fibrosis via inhibiting TGF-β1/JAK2/STAT3 signaling axis

Idiopathic pulmonary fibrosis (IPF) is a debilitating and progressive lung disease with an unknown cause that has few treatment options. 18β-Glycyrrhetinic acid (18β-GA) is the main bioactive component in licorice, exhibiting anti-inflammatory and antioxidant effects, while also holding certain application value in the metabolism and regulation of steroids. In this study, we demonstrated that 18β-GA effectively alleviates bleomycin (BLM)-induced IPF by inhibiting the TGF-β1/JAK2/STAT3 signaling axis. In vivo experiments demonstrate that 18β-GA significantly attenuates pulmonary fibrosis progression by reducing lung inflammation, improving lung function, and decreasing collagen deposition. In vitro experiments reveal that 18β-GA inhibits the activation and migration of TGF-β1-induced fibroblasts. Furthermore, it regulates the expression of vimentin, N-cadherin and E-cadherin proteins, thereby inhibiting TGF-β1-induced epithelial-mesenchymal transition (EMT) in lung alveolar epithelial cells. Mechanistically, 18β-GA ameliorates pulmonary fibrosis by modulating the TGF-β1/JAK2/STAT3 signaling pathway in activated fibroblasts. Taken together, our findings demonstrate the potential and underlying mechanisms of 18β-GA in ameliorating IPF, emphasizing its potential as a novel therapeutic drug for the treatment of this devastating disease.

MiR-141-3p attenuates inflammation and oxidative stress-induced pulmonary fibrosis in ARDS via the Keap1/Nrf2/ARE signaling pathway.

The present research aimed to investigate the effects and mechanisms of microRNA (miR)-141-3p on pulmonary fibrosis of acute respiratory distress syndrome (ARDS). A rat ARDS model was established by the intratracheal drip of 10mg/kg lipopolysaccharide (LPS). miR-141-3p and Kelch-like ECH-associated protein 1 (Keap1) expression was detected using RT-qPCR assay. Inflammatory factors in bronchoalveolar lavage fluid (BALF) and lung tissues were measured with enzyme-linked immunosorbent assay (ELISA). Lung fibrosis was evaluated using Masson's trichrome staining and hydroxyproline assay kits. Tissue oxidative stress marker levels were assessed by a commercial kit. Protein variations in the EMT pathway and Keap1/nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway were investigated by Western blot analysis. Targeting relationship verified by dual-luciferase reporter assay. The expression of miR-141-3p was significantly upregulatedin LPS-induced ARDS rats, while Keap1 was downregulated. Overexpression of miR-141-3p decreased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, superoxide dismutase (SOD), and glutathione (GSH) while elevating malondialdehyde (MDA) expressionin LPS-induced ARDS rats. Elevation of miR-141-3p reduced fibrosis scores, enhanced E-cadherin protein expression, and decreased vimentin and α-SMA protein expressionin LPS-induced ARDS rats. This elevation of miR-141-3p also upregulated Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxido-reductase-1 (NQO1) proteinslevels. Moreover, Keap1 overexpression reversed the inhibitory effects of miR-141-3p on LPS-triggered inflammation, oxidative stress, and fibrosis. miR-141-3p may attenuate inflammation and oxidative stress-induced pulmonary fibrosis in ARDS via the Keap1/Nrf2/ARE signaling pathway. Our study provides new ideas for the treatment of ARDS.

Mechanism of Zhenwu Decoction modulating TLR4/NF-κB/HIF-1α loop through miR-451 to delay renal fibrosis in type 2 CRS

BackgroundType 2 cardiorenal syndrome (CRS) is a progressive renal insufficiency in patients with chronic heart failure, but its pathophysiology is still unclear. The Chinese medicine Zhenwu Decoction plays an important role in the prevention and treatment of 2-CRS, however, its mechanism of action remains unknown. PurposeThe aim of this study was to investigate whether the ameliorative effect of ZWD on 2-CRS renal fibrosis is related to the modulation of miR-451 expression and thus mediating the TLR4/NF-κB/HIF-1α loop. Study design and methodsA type 2 CRS rat model was constructed using ligation of the left anterior descending branch of the coronary artery + 3/4 nephrectomy, and randomly divided into Control, Sham, Model, Captopril, ZWD-L, ZWD-M and ZWD-H groups.After 4 weeks of ZWD intervention, its effects on cardiac and renal functions of type 2 CRS rats were observed by hematuria and cardiac ultrasonography. Changes in kidney tissue morphology were observed by HE, Masson and PASM staining. The protein and mRNA expression of TLR4, NF-κB, HIF-1α and IκBα in kidney tissues were detected by immunohistochemistry and qPCR. Immunofluorescence was used to detect the protein expression of NF-κB and HIF-1α in renal tissues. Western blot and qPCR were used to detect the protein expression of MCP-1, ICAM-1, IL-1β, IL-6, TGF-β, α-SMA, FN, Smad2, Smad3, and E-cadherin in renal tissues. PCR was used to detect the protein expression of miR-451mRNA expression level in kidney tissues. ResultsIn this study, we found that ZWD was able to reduce the expression of Scr, BUN, NT-proBNP, and 24-hour quantitative urine protein, elevate LVEF, FS, CO, and reduce the level of LVIDS in type 2 CRS rats, as well as attenuate renal interstitial fibrosis and improve tubular swelling. In addition, Zhenwu Decoction up-regulated the expression of miR-451 in renal tissues and inhibited the expression of TLR4, NF-κB, and HIF-1α proteins and genes, which in turn inhibited the expression of inflammatory factors and fibrosis-related factors. ConclusionZWD was able to up-regulate the expression of miR-451 in renal tissues, inhibit the TLR4/NF-κB/HIF-1α response loop, and then inhibit the expression of inflammatory factors and fibrosis-related factors, improve renal fibrosis, and delay the pathological process of type 2 CRS.

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

ARHGAP17 Inhibits Hepatocellular Carcinoma Progression by Inactivation of Wnt/β-Catenin Signaling Pathway

Abstract GS03-04: Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics

Abstract Background: Invasive lobular carcinoma (ILC) of the breast is the second most common histologic subtype of breast cancer (BC), following invasive ductal carcinoma of no special type (IDC-NST). The hallmark histologic feature of ILC is cellular discohesiveness, the result of bi-allelic inactivation of CDH1, and represents an important genotypic-phenotypic correlation in BC. Although most ILCs harbor CDH1 loss-of function mutations associated to loss-of-heterozygosity (LOH) of the wild type-allele, a subset of ILCs lack these alterations despite displaying a typical lobular phenotype. Here, we sought to identify alternative molecular mechanisms converging on CDH1 inactivation by employing an integrative artificial intelligence (AI) and genomics approach. Materials and Methods: A genomics-driven AI-based algorithm using hematoxylin and eosin (H&amp;E) whole slide images (WSIs) as input, previously developed to detect bi-allelic CDH1 mutations (inactivating mutation associated to LOH) in BC was employed. WSIs of 1,057 BCs including ILCs (n=187) and non-lobular BCs (n=870) previously subjected to FDA-cleared tumor/normal targeted sequencing were subjected to analysis with the AI-based algorithm. Cases predicted to harbor CDH1 bi-allelic mutations by the AI-model but lacking CDH1 bi-allelic mutations by targeted sequencing were assessed through targeted sequencing data re-analysis, CDH1 gene promoter methylation evaluation and/or whole genome sequencing analysis. Results: AI-based analysis WSIs corresponding to 1,057 BCs resulted in the identification of 34 cases found to lack CDH1 bi-allelic mutations by targeted sequencing but predicted to harbor these genetic alterations by the AI-based model. CDH1 gene promoter methylation assessment revealed CDH1 promoter methylation in 18 cases. Targeted sequencing data reanalysis revealed other genetic mechanisms of CDH1 inactivation including CDH1 homozygous deletions (n=3), intragenic deletion with LOH (n=1), and likely pathogenic non-coding CDH1 alterations associated with LOH (n=2). WGS analysis of an ILC revealed a novel deleterious CDH1 fusion stemming from translocation t(13;16), resulting in loss of the 5’UTR, transcription start site and exons 1 and 2 of CDH1, associated with complete loss of E-cadherin protein expression. Taken together, we identified alternative/novel mechanisms of bi-allelic CDH1inactivation in 74% (25/34) cases analyzed. Conclusions: By applying an AI-based algorithm trained to detect a genetic alteration (i.e., CDH1 bi-allelic mutations), we were able to identify alternative epigenetic and genetic molecular mechanisms of CDH1 inactivation in ILCs, including novel non-coding CDH1 genetic alterations and a new inactivating CDH1 fusion gene. These findings indicate that molecular mechanisms affecting a single gene or process converging on the same phenotype can be unveiled by the integration of AI and genomics, highlighting the robustness of this approach for the discovery of novel biology. Citation Format: Fresia Pareja, Higinio Dopeso, Yikan Wang, Andrea Gazzo, David Brown, Pier Selenica, Jan Bernhard, Fatemeh Derakhshan, Edaise M. da Silva, Lorraine Colon-Cartagena, Thais Basili, Antonio Marra, Jillian Sue, Qiqi Ye, Arnaud Da Cruz Paula, Selma Yeni, Xin Pei, Hunter Green, Kaitlyn Gill, Yingjie Zhu, Matthew Lee, Ran Godrich, Adam Casson, Britta Weigelt, Nadeem Riaz, Hanna Y Wen, Edi Brogi, Matthew Hanna, Diana Mandelker, Jeremy Kunz, Brandon Rothrock, Sarat Chandarlapaty, Christopher Kanan, Gerard Oakley III, David Klimstra, Thomas Fuchs, Jorge Reis-Filho. Novel Mechanisms of CDH1 Inactivation in Breast Invasive Lobular Carcinoma Unveiled by the Integration of Artificial Intelligence and Genomics [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 GS03-04.

Overexpression of SOCS2 Inhibits EMT and M2 Macrophage Polarization in Cervical Cancer via IL-6/JAK2/STAT3 Pathway.

SOCS2 is a member of the suppressor of cytokine signaling (SOCS) protein family associated with the occurrence and development of multiple cancers. This study revealed the expression and molecular mechanisms of SOCS2 in cervical cancer. In this study, RT-qPCR, Western Blot, and immunohistochemistry were used to detect the expression level of SOCS2 in cervical cancer tissues and tumor cells. We overexpressed SOCS2 in SiHa cells via lentivirus. In-vitro experiments were used to investigate the changes in cervical cancer cell proliferation, migration, and invasion ability before and after SOCS2 overexpression. Western Blot was used to detect the expression of IL-6/JAK2/STAT3 pathway and EMTrelated proteins. M0 macrophages were co-cultured with the tumor-conditioned medium. The effect of SOCS2 on macrophage polarization was examined by RT-qPCR. SOCS2 expression level was significantly downregulated in cervical cancer tissues. SOCS2 was negatively correlated with CD163+M2 macrophages. Overexpression of SOCS2 inhibited the proliferation, migration, and invasion of cervical cancer cells. The expressions of Twist- 2, N-cadherin, and Vimentin were decreased, while the expression of E-cadherin was increased. Moreover, the expression of IL-6, p-JAK2, and p-STAT3 were decreased. After the addition of RhIL-6, the expression of E-cadherin protein in the LV-SOCS2 group was reversed. CM in the LV-SOCS2 group inhibited the polarization of M2 macrophages. SOCS2 acts as a novel biological target and suppressor of cervical cancer through IL- 6/JAK2/STAT3 pathway.