Cell Migration Capacity Research Articles

UCA1 executes an oncogenic role in pancreatic cancer by regulating miR-582-5p/BRCC3.

As a fatal disease, the mechanism of pancreatic cancer is unclear. Urothelial carcinoma antigen 1(UCA1), a long noncoding RNA (lncRNA) that was first reported in bladder cancer, acts as an oncogene. However, the regulatory role and mechanism of UCA1 in pancreatic cancer remain unknown. This study aims to investigate the expression level and prognostic value of UCA1 in pancreatic cancer tissues, the effects and mechanism of UCA1 in regulating cell proliferation, apoptosis and metastasis. UCA1 expression levels in tissues were detected by in situ hybridization (ISH) and the prognostic value was evaluated by univariate and multivariate survival analysis. For in vitro experiments, proliferation was evaluated by a cell count kit assay, Edu experiments, and a clone formation assay. Apoptosis was evaluated by fluorescence-activated cell sorting flow-cytometry. Cell migration and invasion capacities were detected by wound healing and transwell assays. Western blots were performed to detect apoptotic associated molecules and epithelial-mesenchymal transition (EMT) markers. For the in vivo experiment, subcutaneous transplantation models of pancreatic cancer in nude mice were established to observe the tumor growth. The regulatory mechanism of UCA1 was explored by proteomics, bioinformatic analysis, luciferase reporter assays, and rescue experiments. ISH staining revealed that UCA1 levels between cancer tissues (n=94) and tumor-adjacent tissues (n=73) did not show significant differences. Survival analysis indicated that high expression of UCA1 was an unfavorable prognosis factor for pancreatic cancer. Downregulation of UCA1 by siRNA significantly inhibited cell proliferation, decreased the capacities of cell migration and invasion, induced cell apoptosis, and inhibited EMT. Furthermore, we demonstrated that UCA1 positively regulated the expression of BRCC3 by inhibiting miR-582-5p. Rescue experiments indicated that either inhibiting the expression of miR-582-5p or enhancing expression of BRCC3 could partly attenuate the antitumor effects of downregulation of UCA1. UCA1 acted as an oncogene in pancreatic cancer by partly regulating miR-582-5p/BRCC3, which could be a new therapeutic target for pancreatic cancer.

The CellProfiler pipeline analysis of cell migration

We demonstrate herein a refined method to evaluate the migration capacity of monolayer cells using the CellProfiler pipeline. We used MDA-MB-231 cells, a triple-negative breast cancer cell line, as a model to perform the wound healing assay and proceeded with the pipeline analysis. In order to see a contrast in our analysis of cell migration, we treated the cells with 10 µM kartogenin for 48 h and compared the result to the control cells treated with 0.1 % dimethyl sulfoxide (DMSO). The migration rate of MDA-MB-231 cells could be measured precisely using this method whereby in the presence of 10 µM kartogenin, the cells migrated at 6.3 ± 1.7µmh−1 whilst the vehicle control migrated at 9.1 ± 3.2 µmh−1 (p < 0.05). The small changes in the rate of migration could be significantly differentiated and we believe that this method is accurate in analyzing data of scratch assays as it is of high precision and therefore can be used for high-throughput screenings.

Backscatter from therapeutic doses of ionizing irradiation does not impair cell migration on titanium implants in vitro

ObjectiveThe influence of radiation backscatter from titanium on DNA damage and migration capacity of human osteoblasts (OBs) and mesenchymal stem cells (MSCs) may be critical for the osseointegration of dental implants placed prior to radiotherapy. In order to evaluate effects of radiation backscatter, the immediate DNA damage and migration capacity of OBs and MSCs cultured on titanium or plastic were compared after exposure to ionizing irradiation. Materials and methodsHuman OBs and MSCs were seeded on machined titanium, moderately rough fluoride-modified titanium, or tissue culture polystyrene, and irradiated with nominal doses of 2, 6, 10, or 14 Gy. Comet assay was performed immediately after irradiation, while a scratch wound healing assay was initiated 24 h post-irradiation. Fluorescent live cell imaging documented the migration.ResultsDNA damage increased with higher dose and with backscatter from titanium, and MSCs were significantly more affected than OBs. All doses of radiation accelerated the cell migration on plastic, while only the highest dose of 10 Gy inhibited the migration of both cell types on titanium.ConclusionsHigh doses (10 Gy) of radiation inhibited the migration capacity of both cell types on titanium, whereas lower doses (2 and 6 Gy) did not affect the migration of either OBs or MSCs.Clinical relevanceFractionated doses of 2 Gy/day, as distributed in conventional radiotherapy, appear not to cause severe DNA damage or disturb the migration of OBs or MSCs during osseointegration of dental implants.

Low-Temperature Plasma-Activated Medium Inhibits the Migration of Non-Small Cell Lung Cancer Cells via the Wnt/β-Catenin Pathway.

This study explored the molecular mechanism of the plasma activation medium (PAM) inhibiting the migration ability of NSCLC (non-small cell lung cancer) cells. The effect of PAM incubation on the cell viability of NSCLC was detected through a cell viability experiment. Transwell cells and microfluidic chips were used to investigate the effects of PAM on the migration capacity of NSCLC cells, and the latter was used for the first time to observe the changes in the migration capacity of cancer cells treated with PAM. Moreover, the molecular mechanisms of PAM affecting the migration ability of NSCLC cells were investigated through intracellular and extracellular ROS detection, mitochondrial membrane potential, and Western blot experiments. The results showed that after long-term treatment with PAM, the high level of ROS produced by PAM reduced the level of the mitochondrial membrane potential of cells and blocked the cell division cycle in the G2/M phase. At the same time, the EMT process was reversed by inhibiting the Wnt/β-catenin signaling pathway. These results suggested that the high ROS levels generated by the PAM treatment reversed the EMT process by inhibiting the WNT/β-catenin pathway in NSCLC cells and thus inhibited the migration of NSCLC cells. Therefore, these results provide good theoretical support for the clinical treatment of NSCLC with PAM.

The effect of ubiquitin-specific peptidase 21 on proliferation, migration, and invasion in DU145 cells

Abstract Objectives Although ubiquitin-specific peptidase 21 (USP21) has been shown to promote the development of various cancers, its role in prostate cancer has been poorly reported. Therefore, this study attempted to investigate the involvement of USP21 in prostate adenocarcinoma (PRAD) progression. Methods Information from public databases was used to evaluate the pattern of USP21 expression in PRAD tissues and its effect on patient prognosis. Subsequently, we either upregulated or knocked down USP21 expression in the human PRAD cell line DU145 to assess cell growth, migration, invasiveness, and apoptosis. Results The transcript levels of USP21 in PRAD tissues were low, indicating a poor prognosis. In DU145 cells, USP21 silencing impaired cell proliferation, colony formation, cell cycle progression, migratory capacity, and invasiveness, while it increased rates of apoptosis. Furthermore, cell proliferation, migration, and invasion were all induced by upregulating USP21. In addition, gene enrichment analysis revealed that USP21 had the potential to regulate cell adhesion and the cell cycle. This observation was further validated by the detection of expression of related genes in cells with either knockdown or increased USP21 expression levels. The expression and copy numbers of USP21 were significantly correlated with the infiltration levels of immune cells. Conclusions Expression level of USP21 is associated with PRAD progression and poor prognosis, and may have a role in potential therapeutic strategies for patients with PRAD.

Disulfiram-Loaded Niosomes Reduces Cancerous Phenotypes in Oral Squamous Cell Carcinoma Cells.

Surgery and chemotherapy are the most common therapeutic strategies proposed for oral squamous cell carcinoma (OSCC). However, some of the disadvantages associated with the current methods like unwanted side effects and poor drug response lead the scientist to seek for novel modalities and delivery approaches to enhance the efficacy of treatments. The study aimed to assess the effectiveness of disulfiram (DSF)-loaded Niosomes on cancerous phenotypes of the OSCC cells. In this experimental study, an optimum formulation of DSF-loaded Niosomes was developed for the treatment of OSCC cells to reduce drug doses and improve the poor stability of DSF in the OSCC environment. The design expert software was utilized to optimize the particles in terms of size, polydispersity index (PDI), and entrapment efficacy (EE). Acidic pH increased the release rate of DSF from these formulations. The size, PDI, and EE of Niosomes were more stable at 4°C compared to 25°C. The results indicated that DSF-loaded Niosomes could induce apoptosis (P=0.019) in the OSCC cells compared to the control group. Moreover, it could reduce colony formation ability (P=0.0046) and also migration capacity of OSCC cells (P=0.0015). Our findings indicated that the application of proper dose of DSF-loaded Niosomes (12.5 μg/ml) increases apoptosis, decreases colony formation capacity and declines the migration ability of OSCC cells.

Mechanistic insights into genomic structure and functions of a novel oncogene YEATS4.

As a novel oncogene, the role of YEATS domain-containing protein 4 (YEATS4) in the occurrence, development, and treatment of tumors is now beginning to be appreciated. YEATS4 plays an important role in regulating DNA repair during replication. The upregulation of YEAST4 promotes DNA damage repair and prevents cell death, whereas its downregulation inhibits DNA replication and induces apoptosis. Additionally, accumulating evidence indicates that the aberrant activation of YEATS4 leads to changes in drug resistance, epithelial-mesenchymal transition and also in the migration and invasion capacity of tumor cells. Therefore, specific inhibition of the expression or activity of YEATS4 protein may be an effective strategy for inhibiting the proliferation, motility, differentiation, and/or survival of tumor cells. Taken together, YEATS4 has emerged as a potential target for multiple cancers and is an attractive protein for the development of small-molecule inhibitors. However, research on YEAST4 in tumor-related fields is limited and its biological functions, metabolism, and the regulatory mechanism of YEATS4 in numerous cancers remain undetermined. This review comprehensively and extensively summarizes the functions, structure and oncogenic roles of YEATS4 in cancer progression and aims to further contribute to the study of its underlying molecular mechanism and targeted drugs.

LncRNA MAGI2-AS3-Encoded Polypeptide Restrains the Proliferation and Migration of Breast Cancer Cells.

Accumulating articles have reported the coding potential of long non-coding RNAs (lncRNAs). However, only a few lncRNAs-encoded peptides have been studied. Breast cancer (BRCA) progression-related gene modules were determined by weighted gene co-expression network analysis (WGCNA). Cell viability, proliferation, and migration capacities were assessed by Cell counting kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), and transwell assays. Immunofluorescence (IF) assay was implemented to observe protein expression. Co-immunoprecipitation (Co-IP) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were employed to analyze MAGI2 antisense RNA 3 (MAGI2-AS3)-ORF5-interacted proteins. WGCNA identified that MEpurple and MEblack modules were significantly negatively correlated with T stage in BRCA patients. MAGI2-AS3 was screened as one of the differentially expressed (DE) lncRNAs with translational potential in MEblack and MEpurple modules in BRCA. The data in The Cancer Genome Atlas (TCGA) uncovered that MAGI2-AS3 abundance was significantly decreased in invasive BRCA patients, and it had high diagnostic and prognostic values. MAGI2-AS3-ORF5 notably restrained BRCA cell viability, proliferation, and migration. Mechanically, MAGI2-AS3-ORF5 might affect the progression of BRCA cells by binding to extracellular matrix (ECM)-related proteins. MAGI2-AS3-ORF5 played an anti-tumor role by inhibiting BRCA cell viability, proliferation, and migration. MAGI2-AS3-ORF5 might modulate BRCA cell migration through ECM-associated proteins.

MicroRNA-378a-3p plays a regulatory role in trophoblast cell function in preeclampsia by targeting CMTM3

BackgroundPreeclampsia (PE) is a potential multisystemic disease in the middle and late pregnancy. Although its precise etiology and pathogenesis remain unknown, it is a significant cause of morbidity and mortality in both pregnant women and newborns. This study explored the effects of the miR-378a-3p/CKLF-like MARVEL transmembrane domain containing 3 (CMTM3) upon the trophoblast biological functions in PE. MethodsThe placental pathology of PE were identified by hematoxylin-eosin (HE) staining, and miR-378a-3p expression in placental tissues of PE was verified by RT-qPCR. Trophoblast cells (HTR-8/SVneo and JEG-3) were treated with lipopolysaccharide (LPS), and cell counting kit-8 (CCK-8) assay, flow cytometry, scratch assay, and Transwell assay were carried out to measure cell viability, apoptosis, migratory and invasive capacities, respectively. Western blot was performed to determine the expression levels of the cell migration-related proteins. The binding of miR-378a-3p to CMTM3 was verified through a dual-luciferase reporter gene assay. ResultsmiR-378a-3p expression levels were down-regulated in placental tissues and primary trophoblast cells from women with PE compared to the control group. The overexpression of miR-378a-3p promoted the capabilities of LPS-treated trophoblast cells to proliferate, migrate and invade. In contrast, it impeded cell apoptosis, promoted matrix metallopeptidase (MMP)-2 and MMP-9 expression and inhibiting TIMP metallopeptidase inhibitor (TIMP)-1 and TIMP-2 expression. Regarding the molecular mechanism, miR-378a-3p was chosen as the target to modulate the expression level of CMTM3. CMTM3 expression was increased in placental tissues and primary trophoblast cells from women with PE compared to the control group. CMTM3 overexpression could partially neutralize the effects of the overexpressed miR-378a-3p on trophoblast cell function and the expression levels of migration-associated proteins. ConclusionOur study provides a foundation for miRNA-targeted therapy for preeclampsia by establishing for the first time a potential role for the miR-378a-3p/CMTM3 axis in regulating trophoblast cell activities by altering the expression of migration-related proteins.

METTL3-mediated m6A RNA modification promotes corneal neovascularization by upregulating the canonical Wnt pathway during HSV-1 infection

BackgroundCorneal neovascularization (CNV) is a symptom of herpes simplex keratitis (HSK), which can result in blindness. The corneal angiogenesis brought on by herpes simplex virus type 1 (HSV-1) is strongly affected by vascular endothelial growth factor A (VEGFA). The N6-methyladenosine (m6A) modification catalyzed by methyltransferase-like 3 (METTL3) is a crucial epigenetic regulatory process for angiogenic properties. However, the roles of METTL3 and m6A in HSK-induced CNV remain unknown. Here, we investigated these roles in vitro and in vivo. MethodsA PCR array in HSV-1-infected human umbilical vein endothelial cells (HUVECs) was used to screen for METTL3 among the epitranscriptomic genes. Tube formation and scratch assays were conducted to investigate cell migration capacity. The global mRNA m6A abundance was evaluated using a dot blot assay. Gene expression was assessed by RT-qPCR, western blotting, and fluorescence immunostaining. In addition, bioinformatic analysis was conducted to identify the downstream molecules of METTL3 in HUVECs. METTL3 knockdown and STM2457 treatment clarified the specific underlying molecular mechanisms affecting HSV-1-induced angiogenesis in vitro. An acute HSK mouse model was established to examine the effects of METTL3 knockdown or inhibition using STM2457 on pathological angiogenic development in vivo. ResultsMETTL3 was highly upregulated in HSV-1-infected HUVECs and led to increased m6A levels. METTL3 knockdown or inhibition by STM2457 further reduced m6A levels and VEGFA expression and impaired migration and tube formation capacity in HUVECs after HSV-1 infection. Mechanistically, METTL3 regulated LRP6 expression through post-transcriptional mRNA modification in an m6A-dependent manner, increasing its stability, upregulating VEGFA expression, and promoting angiogenesis in HSV-1-infected HUVECs. Furthermore, METTL3 knockdown or inhibition by STM2457 reduced CNV in vivo. ConclusionOur findings revealed that METTL3 promotes pathological angiogenesis through canonical Wnt and VEGF signaling in vitro and in vivo, providing potential pharmacological targets for preventing the progression of CNV in HSK.

P-346 Migration-associated microRNAs are dysregulated in endometriosis: potential diagnostic biomarkers

Abstract Study question What is the role of dysregulated endometrial microRNAs (miRNAs) in the development of endometriosis? Summary answer Dysregulated miRNAs in the endometrium of women with endometriosis can also be found in endometriomas and may affect the migratory ability of endometriotic cells. What is known already The molecular mechanisms underlying the pathogenesis of endometriosis are poorly understood. One of the hypotheses is that changes in cell properties observed in endometriotic lesions could be initiated already in the endometrium. Dysregulation of miRNAs in the endometrium of women with endometriosis has been reported in previous microarray-based studies. However, little overlap has been seen between published miRNA expression data. Moreover, the potential role of dysregulated miRNAs in the endometrium of women with endometriosis and whether these changes are also present in the endometrioma are largely unknown. Study design, size, duration In this experimental case-control study miRNA gene expression in proliferative phase endometrium was compared between 15 women with laparoscopically confirmed endometriosis (stage III–IV) and 17 age-matched controls who were laparoscopically confirmed to be free of endometriosis. Selected miRNAs were further compared between paired proliferative-phase endometrium and endometrioma from a new cohort of women with endometriosis (stage III–IV) and studied in vitro to understand their effect on cell migration. Participants/materials, setting, methods Samples were collected during laparoscopic operations performed at Tartu University Hospital, Estonia. Dysregulated endometrial miRNAs were detected with small RNA sequencing and differential gene expression analysis. Target genes and biological pathways were predicted to understand their potential role in the disease. Selected miRNAs were further studied for their expression in endometriomas using qRT-PCR and in-vitro for their proliferation and migration ability using a transwell-migration assay after miRNA mimic transfection of the 12Z endometriotic cell line. Main results and the role of chance In total, we identified 9 upregulated and 5 downregulated (-2 &amp;lt;fold change &amp;gt; 2, FDR &amp;lt; 0.05) miRNAs in the endometrium of women with endometriosis compared to controls. In-silico analyses showed that predicted target genes of the dysregulated miRNAs were significantly enriched in migration-related KEGG pathways such as adherens junctions, focal adhesion, MAPK-, PI3-AKT-, and TGF-beta signaling. The most down-regulated miRNA, miR-193b-5p, and the most up-regulated miRNA, miR-374b-5p, were selected for further characterization. Validation of their expression in endometriomas showed a significant up-regulation of both miRNAs compared to paired endometrium (Fold change &amp;gt; 2, FDR &amp;lt; 0.05). Since it has been reported that altered cell migratory ability could be involved in the pathogenesis of endometriosis and our dysregulated miRNA were associated with migration-related pathways, we explored if miR-193b-5p mimic transfection affects the migration capacity of 12Z cells. A 2-fold decrease in cell migration (p-value 0.0021) was observed after 12Z cell mimic transfection. No change in proliferation was demonstrated. Limitations, reasons for caution Although our findings both in-silico and in-vitro suggest a link between dysregulated miRNAs and cell migration, further in-vitro studies in primary cells and in-vivo studies in animal models are needed to reveal the specific pathways that these miRNAs regulate to explain the observed functional changes in the context of endometriosis. Wider implications of the findings This study gives molecular insight into the pathogenesis of endometriosis, a poorly understood disease, by demonstrating changes in miRNA expression in both the endometrium and endometrioma that potentially can be linked to a changed cell migratory ability. Furthermore, identified miRNAs could be further evaluated as diagnostic biomarkers in larger studies. Trial registration number Not applicable

Circ-PDZD8 promotes cell growth and glutamine metabolism in non-small cell lung cancer by enriching LARP1 via sequestering miR-330-5p.

The deregulation of circular RNA (circRNA) is widely reported in carcinogenesis. The purpose of this study was to investigate the role of circRNA-PDZ domain containing 8 (circ-PDZD8) in non-small cell lung cancer (NSCLC) progression. The histological structure of tissues was identified by hematoxylin-eosin (HE) staining analysis. The expression levels of circ-PDZD8, miR-330-5p and la ribonucleoprotein 1 (LARP1) mRNA were ascertained by qPCR. Cell counting kit-8, colony formation, flow cytometry, and transwell assays were employed for functional analysis. Glutamine metabolism was monitored by glutamine consumption, alpha ketoglutarate (α-KG) level and adenosine triphosphate (ATP) level. A xenograft model was established to ascertain the role of circ-PDZD8 in vivo. The putative binding relationships were verified by dual-luciferase and RIP studies. Circ-PDZD8 expression was highly increased in NSCLC. Circ-PDZD8 knockdown inhibited cell growth, migratory capacity, invasiveness and glutamine metabolism but enhanced cell apoptosis in NSCLC cells. Circ-PDZD8 blocked miR-330-5p expression, and miR-330-5p inhibition overturned the effects of circ-PDZD8 absence. LARP1 targeted by miR-330-5p, and miR-330-5p upregulation-impaired cell growth, motility and glutamine metabolism were recovered by LARP1 overexpression. Circ-PDZD8 knockdown was also shown to impede solid tumor growth. Circ-PDZD8 promotes NSCLC cell growth and glutamine metabolism by increasing LARP1 via competitively targeting miR-330-5p.

PITX2 in pancreatic stellate cells promotes EMT in pancreatic cancer cells via the Wnt/β-catenin pathway.

Since the prognosis of patients with pancreatic cancer is very poor and there is a lack of treatment methods, this study is performed to investigate the function of PITX2 in pancreatic stellate cells (PSCs) in the progression of pancreatic cancer. Scientific hypotheses are proposed according to bioinformatics analysis and tissue microarray analysis. Stable knockdown of PITX2 in PSCs is achieved through lentiviral infection. The relative expressions of PITX2, α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are measured in wild-type PSCs and PITX2-knockdown PSCs. Proliferative capacity is measured by EdU assay. After coculture with PSCs, the proliferation, invasion and migration capacity of pancreatic cancer cells are tested. EMT and Wnt/β-catenin downstream genes of pancreatic cancer cells are investigated to reveal the potential mechanism. Bioinformatics analysis reveals that the PITX2 gene is highly expressed in stromal cells in pancreatic cancer and is correlated with squamous-type PDAC. Analysis of PDAC tissue microarray further demonstrates that high PITX2 level in stromal cells is correlated with poor prognosis in PDAC. After stable knockdown of PITX2 in PSCs, the relative protein levels of α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are decreased, and the proliferative capacity of PSCs is also decreased. After coculture with PSCs, in which PITX2 expression is downregulated, the proliferation, invasion and migration capacities of pancreatic cancer cells are inhibited. Thus, our results show that PITX2-silenced PSCs inhibit the growth, migration and invasion of pancreatic cancer cells via reduced EMT and Wnt/β-catenin signaling.

Oridonin promotes endoplasmic reticulum stress via TP53-repressed TCF4 transactivation in colorectal cancer

BackgroundThe incidence of colorectal cancer and cancer death rate are increasing every year, and the affected population is becoming younger. Traditional Chinese medicine therapy has a unique effect in prolonging survival time and improving the prognosis of patients with colorectal cancer. Oridonin has been reported to have anti-cancer effects in a variety of tumors, but the exact mechanism remains to be investigated.MethodsCell Counting Kit-8 assay (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) staining assay, Tranwell, and Wound healing assays were performed to measure cell proliferation, invasion, and migration capacities, respectively. The protein and mRNA expression levels of various molecules were reflected by Western blot and Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR). Transcription Factor 4 (TCF4) and its target genes were analyzed by Position Weight Matrices (PWMs) software and the Gene Expression Omnibus (GEO) database. Immunofluorescence (IF) was performed to visualize the expression and position of Endoplasmic Reticulum (ER) stress biomarkers. The morphology of the ER was demonstrated by the ER tracker-red. Reactive Oxygen Species (ROS) levels were measured using a flow cytometer (FCM) or fluorescent staining. Calcium ion (Ca2+) concentration was quantified by Fluo-3 AM staining. Athymic nude mice were modeled with subcutaneous xenografts.ResultsOridonin inhibited the proliferation, invasion, and migration of colorectal cancer, and this effect was weakened in a concentration-dependent manner by ER stress inhibitors. In addition, oridonin-induced colorectal tumor cells showed increased expression of ER stress biomarkers, loose morphology of ER, increased vesicles, and irregular shape. TCF4 was identified as a regulator of ER stress by PWMs software and GEO survival analysis. In vitro and in vivo experiments confirmed that TCF4 inhibited ER stress, reduced ROS production, and maintained Ca2+ homeostasis. In addition, oridonin also activated TP53 and inhibited TCF4 transactivation, further exacerbating the elevated ROS levels and calcium ion release in tumor cells and inhibiting tumorigenesis in colorectal cancer cells in vivo.ConclusionsOridonin upregulated TP53, inhibited TCF4 transactivation, and induced ER stress dysregulation in tumor cells, promoting colorectal cancer cell death. Therefore, TCF4 may be one of the important nodes for tumor cells to regulate ER stress and maintain protein synthesis homeostasis. And the inhibition of the TP53/TCF4 axis plays a key role in the anti-cancer effects of oridonin.

#4976 DIFFERENTIAL ROLES OF REGNASE3 IN RESIDENT MACROPHAGE VERSUS TUBULAR EPITHELIAL CELLS IN THE HEALTHY OR POSTISCHEMIC KIDNEY

Abstract Background and Aims Acute kidney injury (AKI) is a prevalent yet severe condition that occurs in clinical settings. The prognosis for AKI is poor, with many patients progressing to AKD, CKD, and even ESKD especially in those who were admitted to the ICU. RNA-binding proteins (RBPs) are a class of proteins that play a vital role in regulating gene expression and have been implicated in a wide range of diseases. Regnase3 is a member of the Regnase RBPs family and has been shown to promote inflammation by increasing TNF in macrophages. Despite this knowledge, the full extent of the role played by Regnase3 in AKI remains unknown. Therefore, we hypothesized that Regnase3 plays a role in both macrophages and tubular epithelial cells, influencing inflammation and tubular repair in AKI. Method In order to investigate the role of Regnase3 in kidney injury, a series of genetically-modified mice were developed on the C57BL/6J genetic background. These mice included the paired box gene 8 (Pax8)-reverse tetracycline transactivator (rTtA, Pax8rTtA), tetracycline resistance protein (TetO)-Cre (TetOCre), transgenic receptor activator of nuclear factor kappa-Β (Rank)-Cre (RankCre), and Regnase3 floxed (Regnase-3fl/fl) mice. Using these genetically modified mice, we applied a range of experimental animal models to study the effects of Regnase3 on kidney injury, including unilateral kidney ischemia-reperfusion surgery with or without nephrectomy and nephrocalcinosis-related kidney injury. Additionally, we employed in vitro models utilizing primary cells to study Regnase3 on the cellular level. Furthermore, we utilized scRNA-seq and RNA-seq to investigate the underlying mechanisms of Regnase3’s function. Results We conducted a thorough investigation and discovered that the Regnase3 is highly expressed in macrophages located within the kidney after AKI. The scRNA-seq showed the expression Regnase3 positively correlated with the phagocytosis, chemokines, and monocyte maturation. Furthermore, Rank-Regnase3 mice suffered from more inflammation and kidney injury after AKI, characterized by an increase in CCR2 positive leukocyte infiltration. Additionally, in vitro experiments revealed that Regnase3 is involved in modulating macrophage behavior, acting as a regulator of macrophage polarization towards an M1 and M2 phenotype and influencing the capacity of cell migration. These findings indicate that Regnase3 is essential in the migration of macrophages after AKI, which contribute to both early phase inflammation and progression towards AKI- CKD. Next, we aimed to examine the role of Regnase3 in the context of renal tubule injury. We observed that Regnase3 is highly expressed in healthy renal tubules, but its expression is reduced following injury. Through in silico, in vivo and in vitro experiments, we found that Regnase3 controls early apoptosis, cell death, proliferation, and wound healing capability of tubular epithelial cells. These effects contribute to kidney injury in postischemic kidneys. Additionally, we discovered that the deletion of Regnase3 provides protection against ischemic AKI (Figure 1). Mechanistically, Regnase3 targets pre-RNA and modulates alternative splicing by increasing the proportion of targeted skipped exon events and decreasing the probability of targeted retained intron events. Overall, our findings indicate that Regnase3 plays a vital role in tubular epithelial cells in the response to kidney injury by regulating alternative splicing. Conclusion In summary, our findings indicate that Regnase3 contributes to kidney injury in postischemic kidneys. However, the impact of Regnase3 is contingent upon the specific cell lineage in question. The Rank-Regnase3 leads to an exacerbation of kidney injury by increasing macrophage recruitment, whereas the Pax8-Regnase3 leads to an improvement in kidney injury through its effects on cell death and wound healing capability of tubular epithelial cells. Therefore, the role of Regnase3 in kidney injury is dependent on the cell lineage, which ultimately determines the detrimental or beneficial impact on kidney injury.

Kaempferol and Fisetin-Related Signaling Pathways Induce Apoptosis in Head and Neck Cancer Cells.

Despite the relative effectiveness of standard cancer treatment strategies, head and neck cancer (HNC) is still considered one of the leading causes of mortality and morbidity. While selected bioactive compounds of plant origin reveal a pro-apoptotic effect, kaempferol and fisetin flavonols have been reported as potential anti-cancer agents against malignant neoplasms. To date, their exact role in signaling pathways of head and neck cancer cells is largely unknown. Based on the various methods of cytotoxicity testing, we elucidated that kaempferol and fisetin inhibit proliferation, reduce the capacity of cell migration, and induce apoptosis in SCC-9, SCC-25, and A-253 HNC cells in a dose-dependent manner in vitro (p < 0.05, fisetin IC50 values of 38.85 µM, 62.34 µM, and 49.21 µM, and 45.03 µM, 49.90 µM, and 47.49 µM for kaempferol-SCC-9, SCC-25, and A-253, respectively). The obtained results showed that exposure to kaempferol and fisetin reduces Bcl-2 protein expression, simultaneously leading to the arrest in the G2/M and S phases of the cell cycle. Kaempferol and fisetin inhibit cell proliferation by interfering with the cell cycle, which is strongly associated with the induction of G2/M arrest, and induce apoptosis by activating caspase-3 and releasing cytochrome c in human HNC cells. In addition, investigating flavonols, by inhibiting anti-apoptotic proteins from the Bcl-2 family and damaging the mitochondrial transmembrane potential, increased the level of cytochrome c. While flavonols selectively induce apoptosis of head and neck cancer cells, they may support oncological therapy as promising agents. The discovery of new derivatives may be a breakthrough in the search for effective chemotherapeutic agents with less toxicity and thus fewer side effects.

Esomeprazole Alleviates Cisplatin Resistance by Inhibiting the AKT/mTOR Pathway in Ovarian Cancer Cells.

Ovarian cancer is the most lethal malignancy in gynecology. Due to limited treatment strategies and platinum resistance, newer drugs and therapeutic options are needed. Esomeprazole (ESO) has been reported to have multiple anticancer activities in preclinical and clinical research. Therefore, this study aimed to explore the anticancer effects of esomeprazole on ovarian cancer and its underlying molecular mechanisms. CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect cell viability and proliferation. The Transwell assay was used to evaluate cell migration and invasion capacity. Flow cytometry was used to detect cell apoptosis. Western blotting and immunofluorescence were used to detect protein expression. ESO effectively inhibited the cell viability, proliferation, invasion, migration, and induced apoptosis of ovarian cancer cells in a concentration-dependent manner. Treatment with ESO decreased the expression of c-MYC, SKP2, E2F1, N-cadherin, vimentin, and matrix metalloproteinase 2 (MMP2), while it increased E-cadherin, caspase3, p53, BAX, and cleaved poly (ADP-ribose) polymerase (PARP) expression, and downregulated the PI3K/AKT/mTOR signaling pathway. Furthermore, ESO combined with cisplatin showed synergistic effects in inhibiting proliferation, invasion, and migration of cisplatin-resistant ovarian cancer cells. The mechanism may be related to the increased inhibition of c-MYC, epithelial-mesenchymal transition (EMT), and the AKT/mTOR signaling pathway and enhanced the upregulation of the pro-apoptotic protein BAX and cleaved PARP levels. Moreover, ESO combined with cisplatin synergistically upregulated the expression of the DNA damage marker γH2A.X. ESO exerts multiple anticancer activities and has a synergistic effect in combination with cisplatin on cisplatin-resistant ovarian cancer cells. This study provides a promising strategy to improve chemosensitivity and overcome resistance to cisplatin in ovarian cancer.

Exosomes from Bacteria (Streptococcus parauberis) challenged olive flounder (Paralichthys olivaceus): Isolation, molecular characterization, wound healing, and regeneration activities.

Exosomes are a group of extracellular vesicles carrying membrane proteins, lipids, RNAs, and, cytosolic proteins, which play key role in intercellular communication and homeostasis. This study describes the isolation, physicochemical, morphological and molecular characterization, toxicity, wound healing, and regeneration properties of plasma derived exosomes from naive (phosphate-buffered saline [PBS]-injected; PBS-Exo) and Streptococcus parauberis-challenged (Sp-Exo) olive flounder (Paralichthys olivaceus). The average diameters of PBS-Exo and Sp-Exo were 120.5 ± 6.1 and 113.1 ± 9.3 nm, respectively, and they presented unique cup shape morphologies. Both exosomes exhibited classical tetraspanin surface markers (CD81, CD9, and CD63) and were enriched with acetylcholinesterase. High-throughput miRNA profiling revealed differentially expressed miRNAs (log2 fold change >1; P < 0.05), including 14 known and 22 novel miRNAs, in Sp-Exo. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that the target genes of the miRNAs contribute towards various physiological and immunological functions, including wound healing and fin regeneration. Sp-Exo exhibited a rapid wound healing (cell migration) capacity in human fibroblast cells, and its mRNA and protein expression patterns corroborated its activity. Higher larval fin regeneration was more prevalent in Sp-Exo than in PBS-Exo, which further confirmed its functional significance. Our study provides the first basic physiochemical, morphometric, molecular (miRNA profiling), and wound healing evidences of Sp-Exo in olive flounder and highlights important miRNA cargoes in exosomes that may be potential therapeutic candidates in wound healing.

LINC01614 Promotes Colorectal Cancer Cell Growth and Migration by Regulating miR-217-5p/HMGA1 Axis.

Colorectal cancer (CRC) substantially contributes to cancer-related deaths worldwide. Recently, a long non-coding RNA (lncRNA), LINC01614, has emerged as a vital gene regulator in cancer progression. Yet, how LINC01614 affects CRC progression remains enigmatic. Here, we defined LINC01614 expression in CRC, investigated the performance of CRC cells lacking LINC01614, and elucidated the underpinning mechanism. We observed that LINC01614 was upregulated in both CRC tissues and cell lines. LINC01614 knockdown repressed the proliferation and metastasis capacity of CRC cell lines. Consistently, an in vivo LINC01614 deficiency model exhibited slow tumor growth rate. Moreover, luciferase reporter assay, RNA pull-down, and immunoprecipitation confirmed that LINC01614 targeted miR-217-5p. LINC01614 knockdown reduced the expression of HMGA1 and N-cadherin, while increasing that of E-cadherin, resulting in decreased viability, proliferation, migration, and invasion capacity of CRC cells. Our results demonstrate that LINC01614 regulates CRC progression by modulating the miR-217-5p/HMGA1 axis, thus holding great potential as a prognostic biomarker for CRC diagnosis and treatment.

RPA3 promotes the proliferation, migration, and invasion of gliomas by activating the PI3K-AKT-mTOR pathway.

Gliomas are the most common primary malignant brain tumors, with a poor prognosis and high mortality, and there is no effective treatment regimen. A number of studies have shown that replication protein A3 (RPA3) can regulate DNA replication and that the abnormal expression of RPA3 can lead to genomic instability and induce the development of a variety of tumors. However, the relationship between RPA3 and gliomas and the mechanism of action remains unclear. In this study, we investigated the role of RPA3 in the development of gliomas and the possible mechanism. The Chinese Glioma Genome Atlas (CGGA), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) databases were used to analyze the expression level of RPA3 and its correlation with clinical prognosis. A univariate Cox regression model was established to predict the prognosis of glioma patients and analyze the correlation between RPA3 and immune cell infiltration and activation. Immunohistochemistry, RT-PCR, and Western blot (WB) were used to detect the expression of RPA3 in glioma specimens. After knocking down and overexpressing RPA3 with plasmids, effects on glioma cell proliferation, migration and invasive capacity were investigated in vitro. The possible molecular mechanisms were analyzed using WB. Results showed that the expression of RPA3 in glioma tissue and cells was significantly higher than that in normal glial cells and was positively correlated with the poor prognosis of patients with gliomas. The overexpression of RPA3 expression activated the phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of the rapamycin (mTOR) pathway by promoting the phosphorylation of PI3K, AKT, and mTOR, thereby promoting the proliferation, migration and invasion of glioma cells. In conclusion, RPA3 is highly expressed in gliomas and promotes the proliferation, migration and invasion of gliomas by activating the PI3K-AKT-mTOR pathway. Therefore, RPA3 may be a prognostic biomarker and therapeutic target for gliomas.