Cell Wound Scratch Assay Research Articles

Abstract 4633: Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is always diagnosed as stage IV with a 5 year survival rate of less than 3% due to its aggressive metastatic phenotype. ATC lacks differentiation and loses its thyroid-like functions making it a difficult cancer to treat due to its lack of targetable genetic lesions and unresponsiveness to conventional treatments. Therefore, we investigated genes that had aberrant expression in ATC in relation to differentiation. Using NCBI Gene expression omnibus (GSE33630, GSE85457), which provides publicly available transcriptomic datasets, and bioinformatics software, GEO2R, we found that Homeobox D4 (HOXD4) has 12.5FC higher expression in ATC vs. normal tissue (p=0.000552) and 4.36FC higher in ATC vs. papillary thyroid cancer (PTC) in human patients (p=6.69e-08). HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, we are exploring modulation of differentiation by all-trans retinoic acid (ATRA). ATRA was used to induce differentiation in ATC cell line T238 therefore abrogating the aggressive phenotype of this cancer. T238 treated with ATRA had a 26% decrease in proliferation at 24 hours, 46% at 48 hours, and 87% at 72 hours measured using a trypan blue exclusion assay. T238 treated with ATRA also had a 44% decrease in migratory capacity at 24 hours and 24% at 48 hours measured by a cell scratch wound assay. Immortalized normal thyroid cell line NTHY-ori-3-1 did not have a significant decrease in either proliferation or migratory capacity when treated with ATRA. T238 cells treated with ATRA for 24 hours had a 30% decrease in HOXD4 protein levels. Lastly, immunohistochemistry staining of HOXD4 in human patient tissue samples confirmed the over-expression of HOXD4 in ATC (19.5% staining) vs. normal tissue (2.5%) and PTC (7.5%). Overall, we hypothesize that the aberrant expression of HOXD4 in ATC plays a crucial role in the aggressive metastatic phenotype and therefore differentiation agents such as ATRA can target this biomarker to modulate the ATC phenotype. Therefore, HOXD4 can be used as a potential prognostic indicator for early detection of ATC. Future studies will involve CRISPRi based knockdown of HOXD4 in vitro and validation of these results in animal in vivo models. Citation Format: Kaci Kopec, Tara Jarboe, Nicole R. DeSouza, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. Differentiation driver gene HOXD4 as a potential prognostic indicator and therapeutic target in anaplastic thyroid cancer [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 4633.

Identification a novel Ganoderma FIP gene from Ganoderma capense and its functional expression in Pichia pastoris.

Ganoderma capense is a precious medicinal fungus in China. In this study, a novel fungal immunomodulatory protein gene, named as FIP-gca, was cloned from G. capense by homologous cloning. Sequencing analysis indicated that FIP-gca was composed of 336bp, which encoded a polypeptide of 110 amino acids. Protein sequence blasting and phylogenetic analysis showed that FIP-gca shared homology with other Ganoderma FIPs. FIP-gca was effectively expressed in Pichia pastoris GS115 at an expression level of 166.8mg/L and purified using HisTrap™ fast-flow prepack columns. The immunomodulation capacity of rFIP-gca was demonstrated by that rFIP-gca could obviously stimulate cell proliferation and increase IL-2 secretion of murine spleen lymphocytes. Besides, antitumor activity of rFIP-gca towards human stomach cancer AGS cell line was evaluated in vitro. Cell wound scratch assay proved that rFIP-gca could inhibit migration of AGS cells. And flow cytometry assay revealed that rFIP-gca could significantly induce apoptosis of AGS cells. rFIP-gca was able to induce 18.12% and 22.29% cell apoptosis at 0.3μM and 0.6μM, respectively. Conclusively, the novel FIP-gca gene from G. capense has been functionally expressed in Pichia and rFIP-gca exhibited ideal immunomodulation and anti-tumour activities, which implies its potential application and study in future.

Serine/threonine kinase 36 induced epithelial-mesenchymal transition promotes docetaxel resistance in prostate cancer

To investigate the role and potential mechanism of serine/threonine kinase 36 (STK36) in docetaxel resistance-prostate cancer (PCa). The expression of STK36 in PCa and the correlation with clinicopathological characteristics of PCa patients were analyzed using the data from different databases and tissue microarrays. To investigate the role of STK36 on cell proliferation, invasion, and migration, STK36 was overexpressed and silenced in DU-145 and PC-3 cell lines. Cell counting kit-8 (CCK8) was used to test cell proliferation. Cell invasion and migration were detected by cell wound scratch assay and trans well, respectively. The expression profile of STK36, E-Cadherin, and Vimentin was analyzed by Western blot. Cell apoptosis was detected by the TUNEL assay. STK36 expression was upregulated in PCa tissue compared with adjacent benign PCa tissue; it was higher in patients with advanced stages compared with lower stages and was significantly correlated with decreased overall survival. Up-regulation of STK36 significantly promoted the proliferation, invasion, and migration of DU-145 and PC-3 cells and compensated for the suppression caused by docetaxel treatment in vitro. A striking apoptosis inhibition could be observed when dealing with docetaxel, although the apoptosis of DU-145 and PC-3 cells was not affected by the STK36 exclusive overexpression. Besides, E-Cadherin expression was restrained while the expression levels of vimentin were all enhanced. The knockdown of STK36 reversed the above process. STK36 up-regulation could accelerate the biological behavior and docetaxel resistance of PCa by epithelial-mesenchymal transition (EMT) activation. STK36 may be potentially used as a target in PCa resolvent with docetaxel.

LncRNA MEG3 inhibits the proliferation and migration abilities of colorectal cancer cells by competitively suppressing MiR-31 and reducing the binding of MiR-31 to target gene SFRP1.

To explore the potential mechanism of long-chain non-coding ribonucleic acid (lncRNA) maternal expression gene 3 (MEG3) in colorectal cancer (CRC). The relationship between MEG3 and miR-31 was detected by dual-luciferase assay. Quantitative polymerase chain reaction was utilized to determine the expression of MEG3 in CRC cell lines. Cell Counting Kit-8 assay was performed to detect cell proliferation. Transwell, cell scratch wound assay, and monoclonal proliferation assay were used to detect the proliferation, migration, and invasion of cells. In addition, cell motility was evaluated by detecting the expression of cellular pseudopodia protein α-actinin via immunofluorescence assay, and cell proliferation and motility were judged by determining the expressions of Ki-67, MMP2 and MMP9 via Western blotting. The effect of MEG3 and miR-31 on the development of colorectal cancer was verified by nude mouse tumor-bearing assay and HE staining. Transient transfection with MEG3 overexpression plasmid revealed that MEG3 inhibited the proliferation and motility of cells. The results of dual-luciferase assay showed that MEG3 could specifically inhibit the expression of miR-31, which inhibits the development of colorectal cancer. Transwell, cell scratch wound assay, and monoclonal proliferation experiment showed that miR-31 enhanced cell proliferation, migration and invasion. MEG3 overexpression plasmid was capable of reversing the proliferation and motility of CRC cells enhanced by miR-31. MEG3 can inhibit the proliferation and motility of CRC cells by competitively suppressing the binding of miR-31 to the target gene SFRP1, thus playing an inhibitory role in the pathogenesis of CRC.

Down-regulation of EZH2 genes targeting RUNX3 affects proliferation, invasion, and metastasis of human colon cancer cells by Wnt/β-catenin signaling pathway.

In order to detect the effect of EZH2 genes on proliferation, migration, invasion, and apoptosis of colon carcinoma cell strains HCT116 and HT29 by the Wnt/β-catenin signaling pathway, qRT-PCR was applied to measure relative expressions of EZH2, RUNX3, CEA, CA199, MMP-9, VEGF, β-catenin, and CyclinD1 in each group; Western-blot was employed with the intention of exploring relative expressions of these proteins in vivo and in vitro; monoclonal proliferation experiments and CCK-8 assay was adopted so as to check cell proliferation; the effect on cell migration was investigated via Transwell assay and cell scratch wound assay; flow cytometry was applied with a view to determining the effect on cell apoptosis. Transfected HCT116 cells are injected subcutaneously into nude mice. In colon cell strains HCT-116 and HT29, contrasted to the si-NC group, the RUNX3 expression was prominently up-regulated in the si-EZH2 group. Besides, expressions of CEA, CA199, MMP-9, and VEGF were significantly reduced; down-regulation of EZH2 genes remarkably inhibited cell proliferation, invasion and migration when facilitating apoptosis; down-regulation of EZH2 genes also significantly reduced expressions of essential proteins β-catenin and CyclinD1 on the Wnt pathway. The subcutaneous tumor body of nude mice was reduced. EZH2-OE is the opposite trend to si-EZH2; The EZH2 gene may target regulatory RUNX3 regulation via that Wnt/β-catenin signaling pathway, hence affecting colon carcinoma cell proliferation, invasion, migration, and apoptosis. Therefore, EZH2 may become a promising target for the clinical therapy of colon carcinoma.

Dicer Inhibits Hepatocellular Carcinoma by Inhibiting the Interleukin 6 Pathway

Background: Inflammatory cytokines are related to the occurrence and development of hepatocellular carcinoma (HCC). Objectives: Interleukin 6 (IL-6) is associated with the occurrence and prognosis of HCC, while Dicer inhibits HCC; accordingly, we checked whether Dicer regulates HCC by modulating the IL-6 pathway. Methods: The HCC cells of SMMC-7721 transfected with Dicer overexpression (pCMV-Dicer) and control (pCMV-NC) lentivirus were divided into 3 groups: The SMMC-7721, pCMV-NC, and pCMV-Dicer groups. The assays of cell proliferation, migration, and invasion were performed using cell counting, wound scratch, and transwell chamber assay. The IL-6 level was measured by flow cytometry bead-based immunoassays. All statistical analyses were analyzed using SPSS version 21 by the student t test and 1-way analysis of variance (ANOVA). Results: Dicer inhibited the secretion of IL-6 from HCC cells, as well as the growth of HCC related to proliferation, migration, and invasion. IL-6 incubation with pCMV-NC cells increased proliferation (from 24 hours to 72 hours; P < 0.05), migration (P = 0.008), and invasion (P = 0.85). In addition, IL-6 could reverse the inhibitory effect of Dicer on HCC cells related to proliferation (P < 0.01) and migration (P = 0.006) when incubated with pCMV-Dicer cells, whereas an IL-6 blocker of tocilizumab could enhance the Dicer-induced inhibition related to proliferation (P < 0.05), migration (P = 0.007), and invasion (P = 0.001) when incubated with pCMV-Dicer cells. Furthermore, Dicer could increase the inhibition efficiency of apatinib on HCC treatment by their cooperation in IL-6 downregulation. Conclusions: Dicer could inhibit HCC by the IL-6 pathway, which would be a potential target for HCC treatment. The Dicer inducer might be an enhancer of apatinib for HCC treatment.

Addition of ellagic acid improved the immune ability and delayed the apoptosis of ovarian granulosa cells of Guizhou black goat

Context Follicular development plays an important role in the growth and reproduction of female mammals. Ellagic acid (EA), as a natural antioxidant, has been used in freezing protection of pig semen. However, the effects of EA on immunity and the anti-apoptotic ability of ovarian granulosa cells (GCs) are still unclear. Aims The aim of this study was to analyse the effects of different concentrations of EA on the immune and anti-apoptotic ability of ovarian GCs of Guizhou black goats. Methods In this study, different concentrations of EA (0, 50, 100, 150, 200 μmol/L) were added to the culture of ovarian GCs in vitro, and Cell-Counting Kit 8 (CCK8) assay, cell wound scratch assay, and real-time fluorescence quantitative polymerase chain reaction (RT–qPCR) assay were used to detect the effects of different concentrations of EA on the proliferation, migration, and reproductive marker genes of ovarian GCs. Then the optimal addition concentration of EA was selected and the effects of EA supplementation on immune factors, cytochrome P450 family 19 subfamily A member 1 gene (CYP19A1), estradiol concentrations, intracellular reactive oxygen species concentrations, and apoptosis-related protein expression were detected by RT–qPCR, enzyme-linked immunosorbent assay (ELISA), ROS, and western blotting on the basis of the optimal addition concentration. Key results The CCK8 test and cell scratch test showed that the addition of EA could significantly inhibit the proliferation and migration ability of ovarian GCs compared with the control group, and a dose effect was observed with the increase in concentration. RT–qPCR results showed that different concentrations of EA significantly increased the expression of genes associated with reproduction, including bone morphogenetic protein 15 (BMP15), bone morphogenetic protein receptor 1B (BMPR-1B), growth differentiation fFactor 9 (GDF9), and follicle-stimulating hormone β subunit (FSHβ), and the maximum increase was observed at 150 μmol/L EA. Further analyses using 150 μmol/L EA as the optimal concentration showed significantly increased expressions of CYP19A1, interleukin-10 (IL-10), and superoxide dismutase (SOD2) after EA supplementation, while the expression of IL-8 was significantly decreased compared with those of the control group. ELISA and ROS showed that both intracellular and extracellular estradiol concentrations were higher, while ROS concentrations were significantly lower than those in the control group. Western blotting results showed that 150 μmol/L EA significantly decreased the expression of Caspase-3 and Caspase-9 and the ratio of BCL2-associated X:B-cell lymphoma-2. Conclusions The supplementation of 150 μmol/L EA had significant effects on improving GC immunity and delaying GC apoptosis in goats. The addition of EA also increased the expression of BMP15, BMPR-1B, GDF9, FSHβ, and CYP19A1 and promoted the secretion of estradiol in GCs. Implications These results provided a preliminary lead for further research on the effect of EA on the maturation and development of goat oocytes in vitro.

Garlic Extract Participates in the Proliferation and Apoptosis of Nonsmall Cell Lung Cancer Cells Via Endoplasmic Reticulum Stress Pathway.

To investigate the effect of garlic extract (GE) on the proliferation and apoptosis of cell lines A549 and H1299 in lung cancer (LC). A549 and H1299 cells with well-developed logarithmic growth were added with GE at a concentration of 0 μg/ml, 25 μg/ml, 50 μg/M, 75 μg/ml, and 100 μg/ml, respectively. The inhibition of A549 cell proliferation was detected using CCK-8 after cultured for 24 h, 48 h, and 72 h. The apoptosis of A549 cells was analyzed via flow cytometry (FCM) after 24 h of cultivation. In vitro migration of A549 and H1299 cells was determined by cell wound scratch assay after 0 h and 24 h culture. The caspase-3 and caspase-9 protein expression levels in A549 and H1299 cells were evaluated through western blot after 24 h of cultivation. Colony formation and EdU assays revealed that Z-ajoene could inhibit cell viability and cell proliferation in NSCLC cells. After 24 h culture, there was no significant difference in the proliferation rate of A549 and H1299 cells with different GE concentrations (P > 0.05). A remarkable proliferation rate difference emerged between A549 and H1299 cells with different GE concentrations after 48 and 72 hours of cultivation. The proliferation rate of A549 and H1299 cells in the experiment group was significantly lower than that in the control group. With an elevated level of GE concentration, the proliferation rate of A549 and H1299 cells decreased (P < 0.05) while the apoptotic rate increased continuously. GE could exert toxic effects on A549 and H1299 cells, inhibit cell proliferation, promote apoptosis, and attenuate cell migration. Meanwhile, it might induce apoptosis of A549 and H1299 cells through the caspase signal pathway, which is positively correlated with the mass action concentration and is expected to be a new drug for LC treatment.

SPATA33 affects the formation of cell adhesion complex by interacting with CTNNA3 in TM4 cells

Communication between Sertoli cell is essential during spermatogenesis and testicular development in mice, and the dynamic balance of this communication is regulated by some adhesion proteins. In this study, we found that SPATA33 and CTNNA3 were involved in this process. Quantitative real-time PCR and western blotting showed similar trend of expression of two proteins in the testis of mice of different ages. Subsequently, CRISPR-Cas9 technique was used to prepare Spata33 knockout cell lines with TM4 cells, cell wound scratch assay showed that Spata33 gene knockout affected cell migration, and flow cytometry assay showed that Spata33 knockout resulted in a decreased percentage of G1 phase cells in TM4 cell line. In addition, phalloidin staining assay showed that Spata33 gene knockout disrupted the formation of F-actin. Moreover, the protein immunoprecipitation experiment showed the interaction between SPATA33 and CTNNA3, which affected the interaction between CTNNA3 and CTNNB1. SPATA33 inhibits the formation of CDH1-CTNNB1-CTNNA3 complex through its interaction with CTNNA3, thus weakening adhesion between Sertoli cell and promoting cell migration.

PLEK2 promotes the proliferation and migration of non-small cell lung cancer cells in a BRD4-dependent manner.

It has been reported that Pleckstrin 2 (PLEK2) acts as an oncogene in non-small cell lung cancer (NSCLC). Bromodomain containing protein 4 (BRD4), an important transcriptional regulator of tumorigenesis, has been shown to play a key role in NSCLC. However, whether BRD4 regulates the transcription of PLEK2 and further promotes the proliferation and migration of NSCLC remains unknown. In this study, we performed western blotting, real-time quantitative polymerase chain reaction, immunofluorescence, cell scratch wound assay and chromatin immunoprecipitation. According to these results, we found that PLEK2 plays a tumor‑promoting role in NSCLC via the PI3K/AKT signaling pathway. Moreover, BRD4 expression is significantly upregulated in NSCLC cell lines and suppression of BRD4 expression by siBRD4 and JQ-1 inhibits NSCLC cell lines proliferation and migration. Prominently, we first confirmed that BRD4 binds to the promoter region of the PLEK2 gene, which explains the mechanism by which BRD4 regulates the transcription of PLEK2 gene from the perspective of epigenetics. The present study suggested that PLEK2 promotes the proliferation and migration of NSCLC in a BRD4-dependent manner and provided key insights into the potential avenues for preventing and treating NSCLC.

MiR-139 inhibits proliferation, migration and invasion of osteosarcoma cell line MG63 via down-regulating integrin subunit alpha V(ITGAV)

ObjectivesOsteosarcoma is a relatively common primary malignant bone tumor in clinic, which frequently occurs in children and adolescents. It is essential to clarify the molecular mechanism of osteosarcoma to provide better diagnosis and treatment. Abnormal expression of miRNAs is closely related to the pathogenesis and progression of osteosarcoma. MiRNAs play a regulatory role in tumorigenesis and development of osteosarcoma. The purpose of this study is to reveal the working mechanism of miR-139/ITGAV axis in osteosarcoma progression. MethodsITGAV and miR-139 expression was detected in osteosarcoma tissues or paracancerous normal tissues. TargetScan and Double luciferase reporter gene assay were adopted to verify weather ITGAV was the target gene of miR-139. Western blot and qRT-PCR were used to evaluate the effects of miR-139 on ITGAV. CCK8, Flow cytometry, Transwell and Cell wound scratch assay were used to measure the effects of miR-139 and ITGAV on cell cycle, proliferation, migration and invasion of MG63, respectively. A nude mouse xenograft model of cervical cancer was constructed to observe the effects of miR-139 on the tumor growth. ResultsWe found that the expression of miR-139 in osteosarcoma tissue was significantly reduced, while the expression of ITGAV was significantly increased. MiR-139 could specifically bind to the 3'-UTR of ITGAV and negatively regulate its expression. Transfection of miR-139 mimic could inhibit the proliferation, S-phase arrest, invasion and migration of MG63 cells, and up-regulating the expression of ITGAV could reverse such inhibitory effect. In nude mouse xenograft model of osteosarcoma, overexpression of miR-139 could inhibit tumor growth, while down-regulation of miR-139 produced the opposite effect. ConclusionsThese results indicate that miR-139/ITGAV axis was related to osteosarcoma initiation. MiR-139 could inhibit the biological behavior of osteosarcoma cells and the tumor growth in nude mouse model via targeting ITGAV, and miR-139/ITGAV axis may impede the progression of osteosarcoma.

MiRNA-218 inhibits cell proliferation, migration and invasion by targeting Runt-related transcription factor 2 (Runx2) in human osteosarcoma cells.

PurposeThe deregulation of miRNA-218 has been found in a number of cancers. Using miRNA-218 as a target for Runt-related transcription factor 2 (Runx2), we sought to understand the role of miRNA-218 in osteosarcoma (OS).MethodsThe expression of miRNA-218 was detected in the OS tumor tissues and OS cells. The Runx2 expression level was evaluated in Saos-2, 143B, U2OS, and MG-63. miRNA-218 overexpressed U2OS cells were achieved by transfection with miRNA-218 mimics. The role of miRNA-218 in inhibiting OS tumorigenesis was explored by CCK8, colony formation, cell wound scratch and Transwell assay. TargetScan and dual-luciferase reporter assay identified the interaction between miRNA-218 and Runx2. The inhibitive effect of miRNA-218 on OS through targeting Runx2 was also evaluated.ResultsMiRNA-218 levels were remarkably down-regulated in OS tumor tissues and cell lines. The overexpression of miRNA-218 suppressed U2OS cell development and metastasis. The target interaction between miRNA-218 and Runx2 was validated, and their expression showed a negative correlation in U2OS cells. The suppressed U2OS cell development and metastasis were remarkably reversed by Runx2 overexpression.ConclusionMiRNA-218 showed an inhibitive effect on the development and metastasis of osteosarcoma cell proliferation by targeting Runx2. Our findings may provide novel clues for OS treatment.

MicroRNA-663 prevents monocrotaline-induced pulmonary arterial hypertension by targeting TGF-β1/smad2/3 signaling

ObjectivePulmonary vascular remodeling due to excessive growth factor production and pulmonary artery smooth muscle cells (PASMCs) proliferation is the hallmark feature of pulmonary arterial hypertension (PAH). Recent studies suggest that miR-663 is a potent modulator for tumorigenesis and atherosclerosis. However, whether miR-663 involves in pulmonary vascular remodeling is still unclear. Methods and resultsBy using quantitative RT-PCR, we found that miR-663 was highly expressed in normal human PASMCs. In contrast, circulating level of miR-663 dramatically reduced in PAH patients. In addition, in situ hybridization showed that expression of miR-663 was decreased in pulmonary vasculature of PAH patients. Furthermore, MTT and cell scratch-wound assay showed that transfection of miR-663 mimics significantly inhibited platelet derived growth factor (PDGF)-induced PASMCs proliferation and migration, while knockdown of miR-663 expression enhanced these effects. Mechanistically, dual-luciferase reporter assay revealed that miR-663 directly targets the 3’UTR of TGF-β1. Moreover, western blots and ELISA results showed that miR-663 decreased PDGF-induced TGF-β1 expression and secretion, which in turn suppressed the downstream smad2/3 phosphorylation and collagen I expression. Finally, intratracheal instillation of adeno-miR-663 efficiently inhibited the development of pulmonary vascular remodeling and right ventricular hypertrophy in monocrotaline (MCT)-induced PAH rat models. ConclusionThese results indicate that miR-663 is a potential biomarker for PAH. MiR-663 decreases PDGF-BB-induced PASMCs proliferation and prevents pulmonary vascular remodeling and right ventricular hypertrophy in MCT-PAH by targeting TGF-β1/smad2/3 signaling. These findings suggest that miR-663 may represent as an attractive approach for the diagnosis and treatment for PAH.

Extracellular signal-regulated kinase inhibition prevents venous adaptive remodeling via regulation of Eph-B4.

Vein graft adaptation (VGA) is a process that vein as a vascular graft conduits in arterial reconstructive surgery; VGA can lead to postoperative vein graft stenosis (VGS) and complications after coronary artery bypass graft and other peripheral artery bypass surgeries. VGA is characterized by vein graft loss the venous features without exhibiting arterial features; furthermore, the activation of ERK inhibited the maintenance of venous properties of the vein graft. We hypothesized that ERK inhibition can affect vein VGS through regulating the expression of EphB4. Rat vein transplantation model was established using wild-type and EphB4+/- Sprague-Dawley rats. Hematoxylin-eosin, Masson, Verhoeff, actin staining, and immunohistochemistry were applied to observe the structure of the vein grafts. Vascular smooth muscle cells (VSMCs) were isolated from the vein and vein grafts. Western blotting was used to determine the expression of p-ERK1/2 and EphB4, and immunofluorescence was applied to detect the expression and location of EphB4. Cell wound scratch assay and CCK8 assay were used to determine the migration and proliferation of VSMCs. Real-time polymerase chain reaction was used to determine the mRNA expression of EphB4. Western blotting in vein sample and vein graft sample detected p-ERK1/2 and ERK1/2 expression in both EphB4+/+ and EphB4+/- rats. The expression of p-ERK was increased in vein graft compared to vein. Immunofluorescence in VSMCs form EphB4+/+ and EphB4+/- rats detected EphB4 expression in both cells, and the expression of EphB4 was increased in VSMCs form EphB4+/+ rats. SCH772984 reduces the proliferation and migration of VSMCs. Inhibition of ERK suppressed the increase of vein graft wall thickness, and the expression of collagen fibers, elastic fibers, and α-actin was decreased. Vein graft from EphB4+/- rats reduces the expression of EphB4, and SCH772984 suppressed the decrease of EphB4 in vivo. Vein graft from EphB4+/- rats increased the expression of EphB4, and SCH772984 suppressed the increase of EphB4 in vivo. The inhibition of ERK1/2 suppressed the process of VGS by decreasing the proliferation of VSMCs. The ERK-inhibitor SCH772984 suppressed the level of VGS by extending the time of EphB4 expression during the process of VGA, thus maintaining the venousization of vein graft. The mechanism may be that the inhibitor SCH772984 suppresses the level of VGS by extending the time of EphB4 expression during the process of VGA. Therefore, our research provides a new target of VGS treatment by inhibiting the expression of ERK1/2 through the process of VGA.

Abstract 12535: MicroRNA-663 Ameliorates Monocrotaline-induced Pulmonary Arterial Hypertension by Targeting TGFβ1/smad2/3 Signaling

Objective: Pulmonary vascular remodeling due to excessive growth factor production and pulmonary artery smooth muscle cells (PASMCs) proliferation is the hallmark feature of pulmonary arterial hypertension (PAH). Recent studies suggest that miR-663 is a potent modulator for tumorigenesis and atherosclerosis. However, whether miR-663 involves in pulmonary vascular remodeling is still unclear. Methods and Results: By using quantitative RT-PCR, we found that miR-663 was highly expressed in normal human PASMCs. In contrast, circulating level of miR-663 dramatically reduced in PAH patients. In addition, in situ hybridization showed that expression of miR-663 was decreased in PAMSCs of PAH patients. Furthermore, MTT and cell scratch-wound assay showed that transfection of miR-663 mimics significantly inhibited platelet derived growth factor (PDGF)-induced PASMC proliferation and migration, while knockdown of miR-663 expression enhanced these effects. Mechanistically, dual-luciferase reporter assay revealed that miR-663 directly targets the 3’UTR of TGF-β1. Moreover, western blots and ELISA results showed that miR-663 decreased PDGF-induced TGF-β1 expression and secretion, which in turn suppressed the downstream smad2/3 phosphorylation and collagen I expression. Finally, intratracheal instillation of adeno-miR-663 dramatically attenuated pulmonary vascular remodeling and right ventricular hypertrophy, as well as right ventricular systolic pressure and mean pulmonary arterial pressure in MCT-induced PAH rat models. Conclusion: These results indicate that miR-663 is a potential biomarker for PAH. MiR-663 decreases PDGF-BB-induced PAMSCs proliferation and ameliorates pulmonary vascular remodeling and right ventricular hypertrophy in MCT-PAH by targeting TGF-β1/smad2/3 signaling. These findings suggest that miR-663 may represent as an attractive approach for the diagnosis and treatment of PAH.

CircHIPK3 regulates cardiac fibroblast proliferation, migration and phenotypic switching through the miR-152-3p/TGF-β2 axis under hypoxia.

BackgroundThe occurrence of pathological cardiac fibrosis is attributed to tissue hypoxia. Circular RNAs play significant regulatory roles in multiple cardiovascular diseases and are involved in the regulation of physiological and pathophysiological processes. CircHIPK3 has been identified as the one of the most crucial regulators in cardiac fibrosis. However, the mechanisms by which circHIPK3 regulates cardiac fibrosis under hypoxia remain unclear. Our study aimed to determine circHIPK3 expression in cardiac fibroblasts (CFs) and investigate the functions of circHIPK3 in hypoxia environment.MethodsThe expression level of circHIPK3 in CFs under hypoxia (1% O2) was analyzed by qRT-PCR. The role of circHIPK3 on the proliferation and migration of CFs were determined by EdU, cell wound scratch assay and cell cycle. The expression of proteins associated with phenotypic transformation in CFs in vitro was examined by immunofluorescence assay and western blot. Bioinformatics analysis, dual luciferase activity assay and RNA fluorescent in situ hybridization assay revealed that miR-152-3p was identified as a target of circHIPK3 and that TGF-β2 was targeted by miR-152-3p.ResultsCircHIPK3 expression was significantly upregulated in CFs in a hypoxic environment. In vitro, overexpressing circHIPK3 obviously promoted CF proliferation, migration and phenotypic changes under hypoxia, but those processes were suppressed by circHIPK3 silencing. CircHIPK3 acted as an endogenous miR-152-3p sponge and miR-152-3p aggravated circHIPK3 silencing induced inhibition of CF proliferation, migration, phenotypic transformation and TGF-β2 expression in vitro. In summary, circHIPK3 plays a pivotal role in the development of cardiac fibrosis by targeting the miR-152-3p/TGF-β2 axis.ConclusionsThese findings demonstrated that circHIPK3 acted as a miR-152-3p sponge to regulate CF proliferation, migration and phenotypic transformation through TGF-β2, revealing that modulation of circHIPK3 expression may represent a potential target to promote the transition of hypoxia-induced CFs to myofibroblasts.

CFTR Regulates the Proliferation, Migration and Invasion of Cervical Cancer Cells by Inhibiting the NF-κB Signalling Pathway.

BackgroundThe cystic fibrosis transmembrane conductance regulator (CFTR) and nuclear factor κB (NF-κB) signalling pathways are currently regarded as co-regulators of the occurrence of cervical cancer. However, the detailed mechanism of CFTR- and NF-κB-mediated effects in cervical cancer remains to be elucidated. This study aimed to investigate the mechanism by which CFTR and NF-κB influence the development of cervical cancer.Patients and MethodsCFTR ΔF508 mutation and CFTR promoter methylation were detected in cervical tissue samples. NF-κB p65 and IκBα protein levels were tested in HeLa cells with CFTR overexpression and knockdown by Western blotting. The effects of CFTR on cell proliferation, migration, and invasion were examined in HeLa cells by WST-1 and soft agar assays, cell wound scratch assay, and Matrigel invasion assays, respectively. The protein–protein interaction (PPI) network was constructed by using GeneMANIA. GeneCoDis3 was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis on genes in the PPI network.ResultsCFTR mutation and CFTR promoter methylation were not associated with the occurrence of cervical cancer. NF-κB p65 protein levels were decreased in CFTR overexpression lines and increased in CFTR knockdown lines, and IκBα levels were affected in the opposite manner, indicating that CFTR inhibited the NF-κB signalling pathway. CFTR also regulated the cell proliferation, migration, and invasion ability of cervical cancer cells. When CFTR was overexpressed, cell proliferation, migration, and invasion ability were decreased. There were 20 genes that interacted with CFTR. KEGG pathway analysis showed enrichment in the gastric acid secretion, chemokine signalling, bile secretion and apoptosis pathways.ConclusionCFTR plays an important role in cancer cell proliferation, migration and invasion by inhibiting the NF-κB signalling pathway in cervical cancer.

CircZNF609/miR-134-5p/BTG-2 axis regulates proliferation and migration of glioma cell.

MicroRNAs are abundant in eukaryotic cells and play key roles in cancers. Circular RNAs (CircRNAs) served as the competing endogenous RNAs (ceRNAs) in mediating multiple cell processes. This study aims to define the role of CircRNA CircZNF609/miR-134-5p in glioma as well as the underlying regulating mechanism. Relative expression of miR-134-5p, CircZNF609 and BTG-2 mRNA was determined by quantitative real-time PCR. Cell proliferation was analysed by CCK-8 assay. Cell migration was assessed by cell wound scratch assay. The direct regulatory of miR-134-5p on BTG-2 and CircZNF609 was verified by luciferase report gene assay. MiR-134-5p was significantly upregulated in glioma cells. The overexpression of miR-134-5p inhibited cell proliferation and migration of glioma cell U251 and U87. Reversely, knock-down of miR-134-5p enhanced cell proliferation and migration. Both BTG-2 and CircZNF609 are the direct targets of miR-134-5p, and their expression could be negatively regulated by miR-134-5p. CircZNF609 was significantly upregulated in U251 and U87 cells and acted as an oncogene to promote cell proliferation and cell migration of glioma cell U251 and U87. These data proved that CircZNF609 served as a competing RNA to bind miR-134-5p that promoted BTG-2 expression leading to reduced proliferation and migration of glioma cell.

PD-L1 Expression Is Regulated By NF-κB During EMT Signaling In Gastric Carcinoma.

PurposeThe aim of this study was to investigate the influence of epithelial-mesenchymal transition (EMT) occurring in gastric carcinoma cells and the involvement of programmed death ligand 1 (PD-L1) expression in tumor cells that undergo EMT. The mechanisms underlying PD-L1 expression during EMT in gastric carcinoma cells were also explored.MethodsThe capacities of migration and invasion were tested by cell scratch-wound assay and transwell chamber assay. PD-L1 expression by SGC7901 cell line and related mechanism were measured by Western blot and QRT-PCR.ResultsTreating with TGF-β1 promotes the motility of SGC7901 and PD-L1 expression in vitro, while activating the NF-κB signal pathway.ConclusionEMT increases the capacities of migration and invasion in gastric cancer cells, which resulted in up-regulation of PD-L1 expression via a mechanism that is dependent on NF-κB activation.

Ethanol extract of Ligustrum lucidum Ait. leaves suppressed hepatocellular carcinoma in vitro and in vivo

BackgroundThe present study investigated the pharmacological activity and mechanism of ethanol extract of Ligustrum lucidum Ait. leaves (EEL) on HCC.MethodsCell viability was determined using cell counting kit-8 (CCK-8) assay. The effects of EEL on cellular biological activities were analyzed by flow cytometry (FCM), cell wound scratch assay and transwell assay. The expression levels of related mRNA and protein were determined by performing quantitative real-time PCR (qRT-PCR), Western blotting assay and immunocytochemistry. Methylation-specific PCR (MSP) was carried out to investigate the possible mechanism underlying the DNA methylation of PTEN.ResultsEEL showed cytotoxicity to both Bel-7402 and Huh-7 cell lines. We also found that EEL enhanced the apoptosis of Bel-7402 and Huh-7 cells by regulating the expressions of Bcl-2 associated X (Bax), B cell lymphoma 2 (Bcl-2) and Cytochrome-C and the activity of caspase-3 and therefore promoted cell cycle arrest. Moreover, EEL also suppressed cell migration and invasion. EEL increased the expression of tissue inhibitor of metalloproteinases 2 (TIMP2) but decreased the expressions of matrix metalloproteinase2 (MMP2) and MMP9. Furthermore, EEL inhibited the phosphorylation of PI3K/Akt pathway. MSP results showed that EEL promoted the demethylation of PTEN, suggesting that the inactivation of PI3K/Akt may be related to DNA de-methylation of PTEN. In addition, EEL inhibited the tumor growth of HCC in vivo.ConclusionsEEL exerted anti-tumor effect on HCC in vitro and in vivo. EEL mediated by the inhibition of PI3K/Akt may be closely related to DNA de-methylation of PTEN. Thus, EEL could be used as a potential anti-cancer therapeutic agent of HCC.