Ethynyl Deoxyuridine Research Articles

Exosome-transported circ_0001955 as a potent driver of breast cancer by regulating the miR-708-5p/PGK1 axis.

Increasing evidence shows that exosome-mediated delivery of circular RNA (circRNA) is implicated in breast cancer progression. This study aimed to elucidate the role of exosome-transported circ_0001955 in breast cancer. The expression of circ_0001955, miR-708-5p, and phosphoglycerate kinase 1 (PGK1) messenger RNA (mRNA) was detected by quantitative real-time polymerase chain reaction (qRT-PCR); the protein levels of PGK1 and hexokinase 2 (HK2) were detected by western blot (WB). 5'-Ethynyl-2'-deoxyuridine (EdU) and colony formation assay were used to determine cell proliferation. Glycolytic metabolism was analyzed by corresponding kits to detect the associated indicators. The role of circ_0001955 in vivo was studied by establishing animal models. The potential binding relationship between miR-708-5p and circ_0001955 or PGK1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Circ_0001955 was highly expressed in breast cancer tissues and cell lines, as well as in exosomes from breast cancer cell lines. The deficiency of circ_0001955 blocked proliferation, decreased the IC50 value of paclitaxel (PTX), and blocked glycolysis in MCF-7 and MDA-MB-231 cells. Circ_0001955 knockdown also inhibited tumor growth in vivo. Circ_0001955 directly combined with miR-708-5p, and the miR-708-5p inhibitor reversed the effects of sh-circ_0001955. PGK1 was a target of miR-708-5p, and circ_0001955 indirectly promoted PGK1 expression by binding to miR-708-5p. PGK1 overexpression abolished the function of miR-708-5p in breast cancer. Exosomal circ_0001955 excreted from breast cancer cells facilitated proliferation and glycolysis and enhanced the IC50 value of PTX in breast cancer cells by sponging miR-708-5p to upregulate PGK1.

Intramammary Labeling of Epithelial Cell Division

Thymidine analogs such as ethynyl deoxyuridine (EdU) or bromodeoxyuridine (BrdU) can be used to label mitosis of mammary epithelial cells (MEC) and to quantify their proliferation. However, labeling cells in larger animals requires considerable amounts of chemical that can be costly and hazardous. We developed a strategy to infuse EdU into the mammary glands of ewes to directly label mitotic MEC. First, each udder half of nulliparous ewes (n = 2) received an intramammary infusion of one of four different concentrations of EdU (0, 0.1, 1.0 or 10 mM) which was compared to BrdU IV (5 mg/kg) 24 h later. Tissues were analyzed by immunofluorescent histochemistry to detect EdU, BrdU, and total MEC. Of the EdU doses tested, 10 mM EdU yielded the greatest labeling index, while a proportion of MEC were labeled by both EdU and BrdU. We next sought to establish whether intramammary labeling could detect the induction of mitosis after exposure to exogenous estrogen and progesterone (E + P). We first infused EdU (10 mM) into the right udder half of ewes (n = 6) at t 0, followed by thymidine (100 mM) 24 h later to prevent further labeling. Three ewes were then administered E + P for 5 d, while n = 3 ewes served as controls. On d 5, EdU was infused into the left udder half of all mammary glands alongside BrdU IV (5 mg/kg). By the time of necropsy 24 h later an average MEC labeling index of 2.9% resulted from EdU delivered at t 0. In the left half of the udder on d 5, CON glands had a final EdU labeling index of 3.4% while glands exposed to E + P had a labeling index of 4.6% (p = 0.05). The corresponding degree of labeling with BrdU was 5.6% in CON glands, and 12% following E + P (p < 0.001). Our findings reveal that intramammary labeling is an efficient and cost-effective method for single- and dual-labeling of cell division in the mammary glands.

Overexpression of NOP58 Facilitates Proliferation, Migration, Invasion, and Stemness of Non-small Cell Lung Cancer by Stabilizing hsa_circ_0001550.

NOP58 ribonucleoprotein (NOP58) is associated with the recurrence of lung adenocarcinoma. Few investigations concentrate on the role of NOP58 in non-small cell lung cancer (NSCLC), which is the focus of our current study. Following transfection, the proliferation, migration, and invasion of NSCLC cells were assessed by 5- ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assays. The percentage of CD9+ cells was evaluated by flow cytometry assay. Based on target genes and binding sites predicted through bioinformatics analysis, a dual-luciferase reporter assay was performed to verify the targeting relationship between hsa_circ_0001550 and NOP58. The effect of NOP58 overexpression on hsa_circ_0001550 stability was gauged using Actinomycin D. The hsa_circ_0001550 and NOP58 expression levels, as well as protein expressions of CD44, CD133, OCT4, and SOX2 in NSCLC cells were determined by quantitative real-time PCR and Western blot, respectively. Hsa_circ_0001550 was remarkably up-regulated in NSCLC cell lines A549 and PC9, silencing of which weakened cell abilities to proliferate, migrate and invade, decreased CD9+ cell ratio, and diminished protein expressions of CD44, CD133, OCT4, and SOX2. NOP58 could bind to hsa_circ_0001550 and stabilize its expression, and NOP58 overexpression partially abrogated hsa_circ_0001550 knockdown-inhibited NSCLC cell proliferation, migration, invasion and stemness. Overexpression of NOP58 facilitates proliferation, migration, invasion, and stemness of NSCLC cells by stabilizing hsa_circ_0001550, hinting that NOP58 is a novel molecular target for NSCLC therapy.

Long non-coding RNA MAGEA4-AS1 binding to p53 enhances MK2 signaling pathway and promotes the proliferation and metastasis of oral squamous cell carcinoma

Long non-coding RNAs (lncRNAs) regulate the occurrence, development and progression of oral squamous cell carcinoma (OSCC). We elucidated the expression features of MAGEA4-AS1 in patients with OSCC and its activity as an OSCC biomarker. Furthermore, the impact of up-regulation of MAGEA4-AS1 on the cellular behaviors (proliferation, migration and invasion) of OSCC cells and intrinsic signal mechanisms were evaluated. Firstly, we analyzed MAGEA4-AS1 expression data in The Cancer Genome Atlas (TCGA) OSCC using a bioinformatics approach and in 45 pairs of OSCC tissues using qPCR. Then CCK-8, ethynyl deoxyuridine, colony formation, transwell and wound healing assays were conducted to assess changes in the cell proliferation, migration and invasion protential of shMAGEA4-AS1 HSC3 and CAL27 cells. The RNA sequence of MAGEA4-AS1 was identified using the rapid amplification of cDNA ends (RACE) assay. And whole-transcriptome sequencing was used to identify MAGEA4-AS1 affected genes. Additionally, dual-luciferase reporter system, RNA-binding protein immunoprecipitation (RIP), and rescue experiments were performed to clarify the role of the MAGEA4-AS1-p53-MK2 signaling pathway. As results, we found MAGEA4-AS1 was up-regulated in OSCC tissues. We identified a 418 nucleotides length of the MAGEA4-AS1 transcript and it primarily located in the cell nucleus. MAGEA4-AS1 stable knockdown weakened the proliferation, migration and invasion abilities of OSCC cells. Mechanistically, p53 protein was capable to activate MK2 gene transcription. RIP assay revealed an interaction between p53 and MAGEA4-AS1. MK2 up-regulation in MAGEA4-AS1 down-regulated OSCC cells restored MK2 and epithelial-to-mesenchymal transition related proteins’ expression levels. In conclusion, MAGEA4-AS1-p53 complexes bind to MK2 promoter, enhancing the transcription of MK2 and activating the downstream signaling pathways, consequently promoting the proliferation and metastasis of OSCC cells. MAGEA4-AS1 may serve as a diagnostic marker and therapeutic target for OSCC patients.

Secretion of GPNMB from Neural Stem Cells Induced by ET-1 Contributes to Angiogenesis after Spinal Cord Injury.

The feeble plasticity of spinal cord microvascular endothelial cells (SCMECs) after trauma is one of the major causes of spinal cord injury (SCI). Neural stem cells (NSCs) play an important role in nerve repair. Glycoprotein nonmetastatic B (GPNMB) has neuroprotective effects and can be stimulated by endothelin 1 (ET-1), and its expression is upregulated in SCI. Here, we aim to investigate whether elevated ET-1 levels stimulate NSCs to secrete GPNMB, thereby further promoting angiogenesis. Mouse SCMECs and NSCs were isolated, cultured, and identified by flow cytometry and immunofluorescence staining. NSCs were treated with ET-1, while SCMECs were cocultured with NSCs, followed by treatment with ET-1. NCS and SCMEC viability were evaluated using cell counting kit 8 (CCK-8) assay, while cell proliferation, migration, invasion, and angiogenesis were examined using 5'-Ethynyl-2'-Deoxyuridine (EdU) staining, wound healing assay, Transwell assay, and tube formation assay. GPNMB expression in NCSs and SCMECs was quantified by western blot assay, quantitative Real-Time polymerase chain reaction (qRT-PCR), or enzyme-linked immunosorbent assay (ELISA). Mouse SCMECs and NSCs were successfully isolated and cultured. ET-1 promoted NSC viability and proliferation and upregulated GPNMB expression. NSCs and ET-1-treated NSCs promoted the viability, migration, invasion, angiogenesis, and GPNMB expression in SCMECs compared with control group cells, while GPNMB antibody reversed the above effects of ET-1 on the SCMECs. ET-1 promotes SCMEC migration and invasion, along with angiogenesis, by enhancing NSC-mediated GPNMB secretion, so ET-1 may be a novel therapeutic target for SCI.

TRIM47 promotes the Warburg effect and reduces ferroptosis in prostate cancer by FBP1 and FOXO1.

Prostate cancer (PC), a malignant tumor occurring in the male prostate tissue, has a high incidence rate. In this study, we explored the role of tripartite motif 47 (TRIM47) in the progression of PC and its underlying mechanism. PC and paracancerous tissues were collected from Shenzhen Peoples's Hospital. The following methods were employed in this experiment: quantitative polymerase chain reaction (qPCR), immunofluorescent staining, cell counting kit-8 (CCK-8), ethynyl deoxyuridine (EdU), and Western blot. The expression levels of TRIM47 were up-regulated in patients with PC. TRIM47 was found to promote cell growth and induce the Warburg effect, while also reducing ferroptosis in PC cells. Conversely, the knockdown of TRIM47 [small interfering RNA, (si)-TRIM47] decreased cell growth and the Warburg effect, while promoting ferroptosis in PC cells. Additionally, TRIM47 was observed to induce the protein expression levels of fructose-1,6-bisphosphatase 1 (FBP1) and forkhead box protein O1 (FOXO1) in PC cells. Further, TRIM47 protein was found to interact with both the FBP1 and FOXO1 proteins in the PC cells. The inhibition of FBP1 attenuated the effects of TRIM47 on the Warburg effect in PC cells, while the inhibition of FOXO1 diminished the effects of TRIM47 on ferroptosis in PC cells. Our findings suggest that TRIM47 promotes the Warburg effect of PC by inducing FBP1 and FOXO1. Thus, our findings suggest that targeting TRIM47 could serve as a viable therapeutic strategy for the treatment of PC.

Loureirin B ameliorates cholestatic liver fibrosis via AKT/mTOR/ATG7-mediated autophagy of hepatic stellate cells

Aim of the studyChronic cholestasis leads to liver fibrosis, which lacks effective treatment. In this study, we investigated the role and mechanisms of action of loureirin B (LB) in cholestatic liver fibrosis. Materials and methodsBile duct ligation (BDL)-induced hepatic fibrosis mice were used as in vivo models. Transforming growth factor-β1 (TGF-β1)-pretreated HSC-T6 cells were used to explore the mechanism by which LB attenuates liver fibrosis in vitro. RNA sequencing, quantitative PCR (qPCR), western blotting, immunohistochemistry and immunofluorescence were performed to detect the fibrosis markers and measure autophagy levels. Flow cytometry, cell counting kit-8 (CCK-8) assay, and 5′-ethynyl-2′-deoxyuridine (EdU) assay were conducted to detect cell proliferation and viability. GFP-RFP-LC3 adenovirus, autophagy-related protein 7 (ATG7) siRNA, and bafilomycin A1 (BafA1) were used to verify autophagic flux. ResultsOur results showed that LB ameliorates liver injury, inhibits collagen deposition, and decreases the expressions of fibrosis-related markers in BDL-induced mouse livers. In vitro, we found that LB inhibited proliferation and migration, promoted apoptosis, and inhibited the activation of HSC-T6 cells pretreated with TGF-β1. RNA sequencing analysis of HSC-T6 cells showed that LB treatment predominantly targeted autophagy-related pathways. Further protein analysis indicated that LB downregulated the expression of phosphorylated AKT (p-AKT) and phosphorylated mTOR (p-mTOR), and upregulated LC3-II, p62, and ATG7 both in vivo and in vitro. Intriguingly, ATG7 inactivation reversed the antifibrotic effects of LB on HSC-T6 cells. ConclusionsLB can improve BDL-induced liver fibrosis by inhibiting the activation and proliferation of HSCs and is expected to be a promising antifibrotic drug.

Anticancer potential of grifolin in lung cancer treatment through PI3K/AKT pathway inhibition

ObjectiveGrifolin is a natural secondary metabolite isolated from edible fruiting bodies of the mushroom Albatrellus confluens. Grifolin has antitumor activities in several types of cancer. We aimed to determine the effects of grifolin on lung cancer. MethodsWe determined the proliferation, migration, invasion, and apoptosis of lung cancer cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Ethynyl deoxyuridine, colony formation, wound scratch, transwell, flow cytometry, and xenograft mouse assays. Molecular docking evaluated the binding relation between grifolin and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA). The levels of PIK3CA, AKT, and p-AKT were measured by western blot. ResultsGrifolin (10, 20, or 40 μM) inhibited the proliferation, migration, and invasion of lung cancer cells, and induced cell cycle arrest and apoptosis. Grifolin also decreased CDK4, CDK6, and CyclinD1 expression and significantly decreased PIK3CA and p-AKT expression in lung cancer cells. These anticancer effects were abolished by 740Y-P. ConclusionsGrifolin regulates the PI3K/AKT pathway, thus inhibiting lung cancer progression.

Circ_0020460 drives tumorigenesis in cervical cancer through miR-485-3p sponging

Deregulation of circular RNAs (circRNAs) is widely recognized in cancer progression. Our study aims to investigate the role of circ_0020460 in the development of cervical cancer (CC) and its potential mechanism of action. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot assays were used to detect the expression levels of circ_0020460, miR-485-3p and C-X-C motif chemokine ligand 1 (CXCL1). The roles of circ_0020460 on cell proliferation, cell migration, cell invasion, cell apoptosis, and angiogenesis were investigated using cell counting kit-8 (CCK-8) and Ethynyl deoxyuridine (Edu) assay, wound healing assay, transwell assay, flow cytometry assay, and tube formation assay, respectively. The putative relationship predicted by bioinformatics analysis was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Xenograft models were constructed to explore the role of circ_0020460 in vivo. The expression of circ_0020460 and CXCL1 expression were increased, while miR-485-3p expression was declined in CC tissues and cells. Circ_0020460 knockdown suppressed CC cell proliferation, cell migration, cell invasion, angiogenesis, and promoted cell apoptosis. Circ_0020460 functioned as a miR-485-3p sponge to inhibit miR-485-3p level, and the anti-cancer effects mediated by circ_0020460 knockdown were reversed by miR-485-3p inhibitor. MiR-485-3p bound to CXCL1 3ʹ untranslated region (3ʹUTR) to degrade CXCL1 expression, and the anti-cancer effects of miR-485-3p restoration were impaired by CXCL1 overexpression. Circ_0020460 downregulation inhibited CC xenograft tumor growth. These results suggest that circ_0020460 promoted the malignant behavior of CC cells by modulating the miR-485-3p/CXCL1 axis.

Inhibition of ZDHHC16 promoted osteogenic differentiation and reduced ferroptosis of dental pulp stem cells by CREB

BackgroundThe repair of bone defects caused by periodontal diseases is a difficult challenge in clinical treatment. Dental pulp stem cells (DPSCs) are widely studied for alveolar bone repair. The current investigation aimed to examine the specific mechanisms underlying the role of Zinc finger DHHC-type palmitoyl transferases 16 (ZDHHC16) in the process of osteogenic differentiation (OD) of DPSCs.MethodsThe lentiviral vectors ZDHHC16 or si-ZDHHC16 were introduced in the DPSCs and then the cells were induced by an odontogenic medium for 21 days. Subsequently, Quantitate Polymerase Chain Reaction (PCR), immunofluorescent staining, proliferation assay, ethynyl deoxyuridine (EdU) staining, and western blot analysis were used to investigate the specific details of ZDHHC16 contribution in OD of DPSCs.ResultsOur findings indicate that ZDHHC16 exhibited a suppressive effect on cellular proliferation and oxidative phosphorylation, while concurrently inducing ferroptosis in DPSCs. Moreover, the inhibition of ZDHHC16 promoted cell development and OD and reduced ferroptosis of DPSCs. The expression of p-CREB was suppressed by ZDHHC16, and immunoprecipitation (IP) analysis revealed that ZDHHC16 protein exhibited interconnection with cAMP-response element binding protein (CREB) of DPSCs. The CREB suppression reduced the impacts of ZDHHC16 on OD and ferroptosis of DPSCs. The activation of CREB also reduced the influences of si-ZDHHC16 on OD and ferroptosis of DPSCs.ConclusionsThese findings provide evidences to support a negative association between ZDHHC16 and OD of DPSCs, which might be mediated by ferroptosis of DPSCs via CREB.

Cell cycle dynamics of food-entrapping cells of sponges: an experimental approach.

Sponges (phylum Porifera) exhibit surprisingly complex tissue dynamics, maintaining constant cell turnover and migration, rearranging internal structures, and regenerating after severe injuries. Such tissue plasticity relies on the activity of proliferating cells represented primarily by the food-entrapping cells, choanocytes. Although there are plenty of studies regarding the dynamics of regeneration and tissue rearrangement in sponges, cell cycle kinetics of choanocytes in intact tissues remains a controversial issue. This study is devoted to the comparative description of choanocyte cell cycle dynamics in intact tissues of two sponges, Halisarca dujardinii (class Demospongiae) and Leucosolenia corallorrhiza (class Calcarea). We have identified populations of proliferating cells and synchronized them in the S-phase to estimate the growth fraction of cycling cells. Using continuous exposure to labeled thymidine analog ethynyl deoxyuridine (EdU), we calculated choanocyte cell cycle duration and the length of the S phase. We also applied double labeling with EdU and antibodies against phosphorylated histone 3 to estimate the lengths of choanocyte M and G2 phases. Finally, flow-cytometry-based quantitative analysis of DNA content provided us with the lengths of G2 and G1 phases. We found that tissue growth and renewal in the studied sponges are generally maintained by a relatively large population of slowly cycling choanocytes with a total cell cycle duration of 40 h in H. dujardinii and 60 h in L. corallorrhiza. In both species, choanocytes are characterized by an extremely short M-phase and heterogeneity in the duration of the G2 phase.

CircCDYL Contributes to Apoptosis, Ferroptosis, and Oxidative Stress of Ang II-Induced Vascular Smooth Muscle Cells in Thoracic Aortic Aneurysm.

Circular RNAs (circRNAs) have important regulation in thoracic aortic aneurysm (TAA). The function and mechanism of circCDYL (circ_0008285) was explored in TAA here. Angiotensin II (Ang II) was used to construct a TAA model. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed for the detection of circCDYL, miR-1270, and a disintegrin and metalloproteinase 10 (ADAM10). Cell viability was examined via cell counting kit-8 (CCK-8) assay and proliferation was analyzed using Ethynyl-2'-deoxyuridine (EdU) assay. Apoptosis rate was assessed via flow cytometry. Western blot was used for protein detection. Oxidative stress was evaluated by commercial kits. CircCDYL was upregulated in TAA tissues and Ang II-induced circCDYL upregulation in vascular smooth muscle cells (VSMCs). Knockdown of circCDYL weakened Ang II-aroused inhibition of viability, proliferation, and promotion of apoptosis, ferroptosis, and oxidative stress in VSMCs. CircCDYL served as a miR-1270 sponge. The mitigated regulation of circCDYL knockdown for Ang II-induced injury was restored after miR-1270 downregulation. CircCDYL positively regulated ADAM10 through interacting with miR-1270. Overexpression of miR-1270 abated Ang II-induced injury by downregulating ADAM10. In conclusion, circCDYL was involved in the Ang II-induced VSMC injury in TAA via the miR-1270/ADAM10 axis.

Measurement of Myonuclear Accretion In Vitro and In Vivo.

Adult skeletal muscle stem cells (MuSC) are the regenerative precursors of myofibers and also have an important role in myofiber growth, adaptation, and maintenance by fusing to the myofibers-a process referred to as "myonuclear accretion." Due to a focus on MuSC function during regeneration, myofibers remain a largely overlooked component of the MuSC niche influencing MuSC fate. Here, we describe a method to directly measure the rate of myonuclear accretion in vitro and in vivo using ethynyl-2'-deoxyuridine (EdU)-based tracing of MuSC progeny. This method supports the dissection of MuSC intrinsic and myofiber-derived factors influencing myonuclear accretion as an alternative fate of MuSCs supporting myofiber homeostasis and plasticity.

CD73-positive pediatric urethral mesenchymal stem-like cell-derived small extracellular vesicles stimulate angiogenesis

IntroductionAngiogenesis plays an important role in the repair of urethral injury, and stem cells and their secretomes can promote angiogenesis. We obtained pediatric urethral mesenchymal stem-like cells (PU-MSLCs) in an earlier study. This project studied the pro-angiogenic effect of PU-MSLC-derived small extracellular vesicles (PUMSLC-sEVs) and the underlying mechanisms. Materials and methodsPUMSLCs and PUMSLC-sEVs were cultivated and identified. Then, biological methods such as the ethynyl deoxyuridine (EdU) incorporation assay, Cell Counting Kit-8 (CCK-8) assay, scratch wound assay, Transwell assay, and tube formation assay were used to study the effect of PUMSLC-sEVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). We explored whether the proangiogenic effect of PUMSLC-sEVs is related to CD73 and whether adenosine (ADO, a CD73 metabolite) promoted angiogenesis. GraphPad Prism 8 software was used for data analysis. ResultsWe observed that PUMSLC-sEVs significantly promoted the proliferation, migration, and tube-forming abilities of HUVECs. PUMSLC-sEVs delivered CD73 molecules to HUVECs to promote angiogenesis. The angiogenic ability of HUVECs was enhanced after treatment with extracellular ADO produced by CD73, and PUMSLC-sEVs further promoted angiogenesis by activating Adenosine Receptor A2A (A2AR). ConclusionsThese observations suggest that PUMSLC-sEVs promote angiogenesis, possibly through activation of the CD73/ADO/A2AR signaling axis.

Combining MTI-31 with RAD001 inhibits tumor growth and invasion of kidney cancer by activating autophagy.

At most of the times, patients who are diagnosed with kidney cancer should be provided with systemic treatment as drug resistance is a challenging issue in the treatment of this disease. The progression of the cancer can be inhibited with the help of mTOR inhibitors namely RAD001 (everolimus) and MTI-31. In literature, it has been revealed that these mTOR inhibitors have the potential to stimulate autophagy. This degradation pathway boosts the survival rate of the cancerous cells that are subjected to anti-cancer therapy. In this study, CCK8, colony formation assays, and ethynyl deoxyuridine (EdU) analysis were conducted to detect cell proliferation. Furthermore, Transwell assays were also conducted for cell migration analysis. In addition to these, the researchers also performed the flow cytometry process to identify the cells that are undergoing apoptosis. In vivo, experiments were conducted to measure the growth of tumors and metastasis. In this study, the treatment provided through a combination of MTI-31 and RAD001 significantly inhibited the kidney cancer cells' proliferation and tumor growth. Furthermore, there was a notable reduction in the migration and invasion of kidney cancer cells upon the neighboring cells. The outcomes from the mechanistic studies infer that the combination of MTI-31 and RAD001 increases the LC3 levels, which in turn translates into the activation of autophagy. To conclude, the combination of MTI-31 and RAD001 improves the anti-cancerous impact produced by RAD001 in vivo through the promotion of autophagy.

CircPDSS1 accelerates malignant progression of renal cell carcinoma through sponging of miR-182-5p

Purpose: To investigate the biological function and mechanisms of circPDSS1 in triggering malignant progression of renal cell carcinoma (RCC).&#x0D; Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to determine circPDSS1 levels in 50 pairs of RCC and para-cancerous tissues. The relationship between circPDSS1 level and pathological indices in RCC patients was analyzed, while the in vitro effect of circPDSS1 in regulating RCC proliferation was assessed using cell counting kit-8 (CCK-8), colony formation and 5- ethynyl-2’- deoxyuridine (EdU) assay. The sponge effect of circPDSS1 on miR-182-5p was examined by bioinformatics analysis and dual- luciferase reporter assay, while their involvement in mediating malignant progression of RCC was analyzed using rescue experiments. In vivo, the influence of circPDSS1 on RCC growth was determined by establishing a xenograft model in nude mice. Thereafter, RCC tissues were harvested from mice to assess relative levels of miR-182-5p and Ki-67.&#x0D; Results: CircPDSS1 was highly expressed in RCC tissues (p &lt; 0.05). A high level of circPDSS1 correlated with advanced tumor staging and low overall survival. Knockdown of circPDSS1 inhibited RCC cell proliferation, and CircPDSS1 sponged and negatively regulated miR-182-5p (p &lt; 0.05). MiR182-5p was able to abolish regulatory effect of circPDSS1 on malignant proliferative potential in RCC cells. In nude mice bearing RCC, in vivo knockdown of circPDSS1 slowed down tumor growth and decreased positive expression of Ki-67 in tumor tissues (p &lt; 0.05).&#x0D; Conclusion: CircPDSS1 predicts tumor stage and prognosis in RCC patients. It triggers malignant progression of RCC through sponging of miR-182-5p.

Bifidobacterium bifidum BGN4 fractions ameliorate palmitic acid-induced hepatocyte ferroptosis by inhibiting SREBP1-CYP2E1 pathway.

Metabolic dysfunction-associated fatty liver disease (MAFLD) is strongly associated with disturbances in the intestinal microbiota. Herein, the biological effects and mechanism of Bifidobacterium bifidum BGN4 fractions in regulating hepatocyte ferroptosis during MAFLD progression were investigated. To establish an in vitro model of MAFLD, LO2 cells were subjected to palmitic acid (PA). The mRNA and protein expressions were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. LO2 cell proliferation was examined using 5-diphenyltetrazolium bromide (MTT) and ethynyl-2'-deoxyuridine (EdU) assays, whereas its apoptosis was evaluated by flow cytometry. Furthermore, level of reactive oxygen species (ROS) was measured using 2', 7,-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Additionally, the levels of Fe2+, malondialdehyde (MDA), and glutathione (GSH), as well as the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were detected using corresponding kits. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were performed to analyze the interaction between sterol-regulatory element binding protein 1 (SREBP1) and cytochrome P450-2E1 (CYP2E1) promoter. Our results revealed that Bifidobacterium bifidum BGN4 fractions effectively ameliorated PA-induced hepatocyte injury, oxidative stress, and ferroptosis. However, these beneficial effects of BGN4 fractions on PA-induced hepatocyte were dramatically reversed by SREBP1 overexpression, suggesting that BGN4 attenuated MAFLD by acting on SREBP1. Moreover, we observed that BGN4 fractions inhibited CYP2E1 transcription by suppressing SREBP1 nuclear translocation. In addition, CYP2E1 overexpression eliminated the inhibitory effect of BGN4 fractions on PA-induced hepatocyte oxidative stress and ferroptosis. These findings collectively indicated that BGN4 fractions reduced CYP2E1 expression by inhibiting SREBP1 nuclear translocation, thereby suppressing hepatocyte oxidative stress and ferroptosis during the development of MAFLD.

Circ_0003575 knockdown alleviates ox-LDL-induced human aortic endothelial cell dysfunction in atherosclerosis by miR-637/TRAF6 axis.

Circular RNAs (circRNAs) are involved in the progression of atherosclerosis (AS). The present study aimed to determine the functions and mechanism of circ_0003575 in AS. Oxidized low-density lipoprotein (ox-LDL) was used to induce human aortic endothelial cells (HAECs) to establish an AS cell model. Cell Counting Kit-8 (CCK-8) assay and 5'-ethynyl-2'-deoxyuridine (EdU) assay were conducted to assess cell proliferation. Flow cytometry analysis was utilized to quantify cell apoptosis. Tube formation assay was performed to analyze angiogenesis ability. Enzyme linked immunosorbent assay (ELISA) was used to examine the concentrations of inflammatory factors. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were manipulated for the expression of circ_0003575, microRNA-637 (miR-637) and TNF receptor associated factor 6 (TRAF6). Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were adopted to estimate the downstream targets of circ_0003575. Ox-LDL treatment repressed the proliferation and angiogenesis and promoted the apoptosis and inflammation in HAECs. Circ_0003575 knockdown ameliorated ox-LDL-induced injury of HAECs. Circ_0003575 interacted with mi-R-637, which directly targeted TRAF6. Inhibition of miR-637 reversed the impacts of circ_0003575 knockdown on HAEC injury. Moreover, miR-637 overexpression promoted cell proliferation and angiogenesis and inhibited cell apoptosis and inflammation by targeting TRAF6 in ox-LDL-treated HAECs. Further, circ_0003575 silencing inhibited the activation of NF-κB pathway. Circ_0003575 knockdown alleviated ox-LDL-induced HAEC damage by regulating miR-637/TRAF6 and NF-κB pathways.

Cathepsin D Attenuates the Proliferation of Vascular Smooth Muscle Cells Induced by the AGE/RAGE Pathway by Suppressing the ERK Signal.

In this study, we aimed to clarify the role and mechanism by which Cathepsin D (CTSD) mediates the advanced glycation end products (AGEs)-induced proliferation of vascular smooth muscle cells (VSMCs). We conducted a Western blotting assay and co-immunoprecipitation assay to detect the expression of target proteins and the interaction between different proteins. Cell Counting Kit-8 (CCK-8) assay and 5- ethynyl-2'-deoxyuridine (EdU) were used to evaluate the proliferation. AGEs significantly promoted phenotypic switching and proliferation of VSMCs in a concentration-dependent manner. This effect of AGEs was accompanied by inhibition of CTSD. Both the proliferation of VSMCs and inhibition of CTSD induced by AGEs could be attenuated by the specific inhibitor of the receptor for advanced glycation end products (RAGE), FPS-ZM1. Overexpression of CTSD significantly alleviated these effects of AGEs on VSMCs. The mechanism of CTSD action in VSMCs was also explored. Overexpression of CTSD reduced the activation of p-ERK caused by AGEs. By contrast, the knockdown of CTSD, elicited using a plasmid containing short hairpin RNA (shRNA) against CTSD, further increased the activation of p-ERK compared to AGEs alone. Additionally, co-immunoprecipitation studies revealed an endogenous interaction between CTSD, a protease, and p-ERK, its potential substrate. It has been demonstrated that CTSD downregulates the level of phosphorylated ERK by degrading its target, and this interaction plays a critical role in the proliferation of VSMCs induced by the AGE/RAGE axis. These results provide a novel insight into the prevention and treatment of vascular complications in diabetes.

A Novel Circ_Arf3/miR-452-5p/Mbnl1 Axis Regulates Proliferation and Expression of Fibrosis-Related Proteins of Mouse Mesangial Cells Under High Glucose.

Diabetic nephropathy (DN) is a serious microvascular complication of diabetes that may lead to chronic renal failure and end-stage renal disease. Circular RNAs (circRNAs) play important roles in DN progression. However, the action of circRNA ADP ribosylation factor 3 (circ_Arf3) in high glucose (HG)-induced change is still unclear. Mouse mesangial cells (MCs) were treated with 30 mM HG as a DN cell model in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to examine the expression levels of circ_Arf3, microRNA (miR)-452-5p and muscleblind like splicing regulator 1 (Mbnl1). The proliferation of HG-treated MCs was assessed using 5 Ethynyl 2' deoxyuridine (EdU) and cell counting kit-8 (CCK-8) assays, and the levels of proliferation and fibrosis-related proteins and Mbnl1 were detected by Western blot. Dual-luciferase reporter and RNA pull-down assays were utilized to determine the relationship between miR-452-5p and circ_Arf3 or Mbnl1. Our results discovered that circ_Arf3 and Mbnl1 were lowly expressed in HG-treated MCs, while miR-452-5p expression was up-regulated. Moreover, circ_Arf3 was mainly located in the cytoplasm and had a ring-like stable structure. Functional assays demonstrated that overexpression of circ_Arf3 prevented cell proliferation and fibrous formation in HG-treated MCs. Circ_Arf3 could sponge miR-452-5p, and the effect of circ_Arf3 overexpression was reversed by enhanced expression of miR-452-5p. Mbnl1 was a direct target of miR-452-5p. Knockdown of Mbnl1 abolished the suppressive effects of miR-452-5p inhibitor on proliferation and fibrosis-related protein expression in HG-treated MCs. Moreover, circ_Arf3 regulated Mbnl1 through miR-452-5p. Overexpression of circ_Arf3 prevents cell proliferation and fibrous formation in HG-treated MCs by regulating the expression of Mbnl1 via miR-452-5p.