VEGFA Axis Research Articles

Ursodeoxycholic Acid Platinum(IV) Conjugates as Antiproliferative and Antimetastatic Agents: Remodel the Tumor Microenvironment through Suppressing JAK2/STAT3 Signaling.

Tumor microenvironment (TME) is a pivotal factor driving the tumor metastasis and leading to the failure of tumor therapy. Here, a series of ursodeoxycholic acid platinum(IV) conjugates with potency in remodeling the TME through suppressing JAK2/STAT3 signaling was developed. A candidate was screened out, which displayed potent antiproliferative and antimetastatic performance both in vitro and in vivo. It displayed superior pharmacokinetic properties compared to cisplatin. Serious DNA injury was induced, and then mitochondria-mediated apoptosis was initiated through the Bcl-2/Bax/Caspase3 pathway. The JAK2/STAT3 and TGF-β1 signaling pathways were remarkably inhibited, and pro-death autophagy was subsequently promoted. The inflammatory and hypoxic TME was suppressed by downregulating COX-2, MMP9, and HIF-1α, which resulted in inhibited angiogenesis in tumors by inhibiting the HIF-1α/VEGFA axis. Additionally, the immunosuppressive TME was reversed by blocking the immune checkpoint PD-L1, further improving the density of CD3+ and CD8+ tumor-infiltrating lymphocytes, and promoting macrophage polarization from M2- to M1-type.

CircUQCRC2 promotes asthma progression in children by activating the VEGFA/NF-κB pathway by targeting miR-381-3p.

This study targeted to explore circUQCRC2's role and mechanism in childhood asthma. A mouse model of ovalbumin-induced asthma was established to evaluate the effects of circUQCRC2 on childhood asthma in terms of oxidative stress, inflammation, and collagen deposition. The effects of circUQCRC2 on platelet-derived growth factor-BB (PDGF-BB)-induced smooth muscle cells (SMCs) were evaluated, the downstream mRNA of miRNA and its associated pathways were predicted and validated, and their effects on asthmatic mice were evaluated. circUQCRC2 levels were upregulated in bronchoalveolar lavage fluid of asthmatic mice and PDGF-BB-treated SMCs. Depleting circUQCRC2 alleviated tissue damage in asthmatic mice, improved inflammatory levels and oxidative stress in asthmatic mice and PDGF-BB-treated SMC, inhibited malignant proliferation and migration of SMCs, and improved airway remodeling. Mechanistically, circUQCRC2 regulated VEGFA expression through miR-381-3p and activated the NF-κB cascade. circUQCRC2 knockdown inactivated the NF-κB cascade by modulating the miR-381-3p/VEGFA axis. Promoting circUQCRC2 stimulates asthma development by activating the miR-381-3p/VEGFA/NF-κB cascade. Therefore, knocking down circUQCRC2 or overexpressing miR-381-3p offers a new approach to treating childhood asthma.

Comprehensive analysis of circRNA-miRNA-mRNA network related to angiogenesis in recurrent implantation failure

BackgroundAbnormal endometrial blood flow causes a decrease in endometrial receptivity and is considered a relatively independent risk factor for recurrent implantation failure (RIF). This study aimed to explore the potentially functional circRNA-miRNA-mRNA network in RIF, and further explore its mechanism.MethodsDatasets were downloaded from the GEO database to identify differentially expressed circRNAs, miRNAs and mRNAs. The circRNA–miRNA–mRNA and PPI networks were constructed using Cytoscape 3.6.0 and the STRING database, the hub genes were identified with the cytoHubba plug-in, and a circRNA–miRNA–hub mRNA regulatory sub-network was constructed. Then, GO and KEGG pathway enrichment analyses of the hub genes were performed to comprehensively analyze the mechanism of hub mRNAs in RIF. Due to the results of circRNAs-miRNAs-hub mRNAs regulatory network, we verified the expression of circRNA_0001721, circRNA_0000714, miR-17-5p, miR-29b-3p, HIF1A and VEGFA in the RIF mouse model by qRT‒PCR and western blotting.ResultsWe initially identified 175 DEmRNAs, 48 DEmiRNAs and 56 DEcircRNAs in RIF associated with angiogenesis and constructed a circRNA-miRNA‒mRNA network and PPI network. We further identified six hub genes in the acquired network. Based on these genes, functional enrichment analysis revealed that the HIF-1 signaling pathway plays a vital role in endometrial angiogenesis in RIF. In addition, the interaction networks of circRNA_0001721/miR-17-5p/HIF1A and the circRNA_0000714/miR-29b-3p/VEGFA axis were predicted. In the RIF mouse model, circRNA_0001721, circRNA_0000714, HIF1A and VEGFA were down-regulated, whereas miR-17-5p and miR-29b-3p were up-regulated according to qRT‒PCR and western blotting.ConclusionThis study revealed that the HIF-1 signaling pathway plays a vital role in endometrial angiogenesis in RIF. The circRNA_0001721/miR-17-5p/HIF1A and circRNA_0000714/miR-29b-3p/VEGFA axes might play a role in the pathogenesis of endometrial angiogenesis in RIF.

EIF4A3-Induced Circ_0059914 Promoted Angiogenesis and EMT of Glioma via the miR-1249/VEGFA Pathway.

Gliomas are common brain tumors. Despite extensive research, the 5-year survival rate of glioma remains low. Many studies have reported that circular RNAs (circRNAs) play a role in promoting the malignant progression of glioma; however, the role of circ_0059914 in this process remains unclear. In this study, we aimed to investigate the function and underlying mechanism of circ_0059914 in glioma. Western blotting and qRT-PCR were used to determine the levels of circ_0059914, miR-1249, VEGFA, N-cadherin, vimentin, Snail, and EIF4A3. EDU and colony formation assays were conducted to evaluate cell proliferation. Transwell assays were used to explore cell migration and invasion and tube formation assays were used to analyze angiogenesis. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were used to explore the relationship between EIF4A3, circ_0059914, miR-1249, and VEGFA. A xenograft tumor assay was performed to determine the role of circ_0059914 in vivo. Circ_0059914 expression was upregulated in gliomas. Knockdown of gliomal circ_0059914 expression reduced the proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, and growth of glioma cells in vivo. Circ_0059914 sponged miR-1249, and miR-1249 inhibition reversed the circ_0059914 knockdown-mediated effects in glioma cells. VEGFA was found to be a target gene of miR1249; overexpression of VEGFA reversed the effect of miR-1249 up-regulation in glioma. Finally, EIF4A3 increased the expression of circ_0059914. EIF4A3-induced circ_0059914 expression plays a role in promoting glioma via the miR-1249/VEGFA axis.

Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology.

Angiogenesis, as a tumor hallmark, plays an important role in the growth and development of the tumor vasculature system. There is a huge amount of evidence suggesting that the vascular endothelial growth factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2 signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In this study, we employ phage display technology and solution-phase biopanning (SPB) to isolate specific single-chain variable fragments (scFvs) against VEGFR-2 and report on the receptor binding characteristics of the candidate scFvs A semisynthetic phage antibody library to isolate anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression and purification, the specificity of the selected scFv clones was further analyzed by enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoblotting. The competition assay was undertaken to identify the VEGFR-2 receptor-blocking properties of the scFvs. Furthermore, the molecular binding characteristics of candidate scFvs were extensively studied by peptide-protein docking. Polyclonal ELISA analysis subsequent to four rounds of biopanning showed a significant enrichment of VEGFR-2-specific phage clones by increasing positive signals from the first round toward the fourth round of selection. The individual VEGFR-2-reactive scFv phage clones were identified by monoclonal phage ELISA. The sequence analysis and complementarity-determining region alignment identified the four unique anti-VEGFR-2-scFv clones. The soluble and purified scFvs displayed binding activity against soluble and cell-associated forms of VEGFR-2 protein in the ELISA and flow cytometry assays. Based on the inference from the molecular docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and 3 on the VEGFR-2 protein and displayed competition with VEGF-A for binding to VEGFR-2. The competition assay confirmed that scFvs H1 and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.

Metformin Regulates the miR-205/VEGFA Axis in Renal Cell Carcinoma Cells: Exploring a Clinical Synergism with Tyrosine Kinase Inhibitors

Introduction: Metformin (MF) intake could be associated with a favorable outcome in sunitinib (SUT)- and axitinib (AX)-treated clear cell renal cell carcinoma (ccRCC) patients. Functionally, MF induces miR-205, a microRNA serving as a tumor suppressor in several cancers. Methods: Real-time quantitative PCR, viability assays, and Western blotting analyzed MF and SUT/AX effects in RCC4 and 786-O cells. A tetracycline-inducible overexpression model was used to study the role of miR-205 and its known target gene, VEGFA. We analyzed miR-205 and VEGFA within a public and an in-house ccRCC cohort. Human umbilical vein endothelial cell (HUVEC) sprouting assays examined miR-205 effects on angiogenesis initiation. To determine the influence of the von Hippel-Lindau tumor suppressor (VHL), we examined VHL^wt reexpressing RCC4 and 786-O cells. Results: Viability assays confirmed a sensitizing effect of MF toward SUT/AX in RCC4 and 786-O cells. Overexpression of miR-205 diminished VEGFA expression – as did treatment with MF. Tumor tissue displayed a downregulation of miR-205 and an upregulation of VEGFA. Accordingly, miR-205 caused less and shorter vessel sprouts in HUVEC assays. Finally, VHL^wt-expressing RCC4 and 786-O cells displayed higher miR-205 and lower VEGFA levels. Conclusion: Our results support the protective role of MF in ccRCC and offer functional insights into the clinical synergism with tyrosine kinase inhibitors.

The microRNA-4766/VEGFA axis mediates macrophage M2-type polarization to inhibit colorectal cancer proliferation and migration

ObjectiveTo investigate the miR-4766/VEGFA axis in regulating M2-type macrophage polarization under hypoxia and its effect on the proliferation and migration of colorectal cancer (CRC) cells. MethodsThe differentially expressed genes (DEGs) in macrophages before and after hypoxia treatment in the dataset GSE154427 were analyzed. microRNA (miR)-4766 and VEGFA were selected as the research objects and then detected for mRNA expression and protein level using qRT-PCR and Western blot, respectively. The expression levels of M2 macrophage markers such as CD206, CD163, and ARG1 were detected to determine the M2-type macrophage polarization. The targeted binding of miR-4766 to VEGFA was verified using Dual-luciferase reporter gene assay. CCK-8 and Transwell assays were performed, respectively, to detect the capacity of cells to proliferate and migrate. IL-10 and TGF-β levels in the conditioned medium were detected using ELISA. ResultsmiR-4766 was upregulated, and VEGFA was downregulated in hypoxia-treated macrophages. miR-4766 inhibited, while VEGFA promoted the polarization of M2-type macrophages. miR-4766 targeted and negatively regulated VEGFA. miR-4766 inhibited the polarization of M2-type macrophages and then suppressed CRC cell proliferation and migration via targeting VEGFA. ConclusionRestoring miR-4766 expression to inhibit VEGFA expression promised to be a potential strategy to suppress CRC development.

Quinacrine inhibits HIF-1α/VEGF-A mediated angiogenesis by disrupting the interaction between cMET and ABCG2 in patient-derived breast cancer stem cells

BackgroundBreast cancer stem cells (BCSCs) have a critical role in progression of breast cancer by inducing angiogenesis. Several therapeutic strategies have been designed for the treatment of breast cancer by specifically preventing angiogenesis. But there is a dearth of study regarding the treatment procedure which can specifically target and kill the BCSCs and cause lesser harm to healthy cells of the body. A plant-based bioactive compound Quinacrine (QC) specifically kills cancer stem cells (CSCs) without harming healthy cells and also inhibits cancer angiogenesis but the detailed mechanistic study of its anti-CSCs and anti-angiogenic activity is yet to explore. HypothesisEarlier report showed that both cMET and ABCG2 play an essential role in cancer angiogenesis. Both are present on the cell surface of CSCs and share an identical ATP-binding domain. Interestingly, QC a plant based and bioactive compound which was found to inhibit the function of CSCs marker cMET and ABCG2. These relevant evidence led us to hypothesize that cMET and ABCG2 may interact with each other and induce the production of angiogenic factors, resulting in activation of cancer angiogenesis and QC might disrupt the interaction between them to stop this phenomena. MethodsCo-immunoprecipitation assay, immunofluorescence assay, and western blotting were performed by using ex vivo patient-derived breast cancer-stem-cells (PDBCSCs) and human umbilical vein endothelial cells (HUVECs). In silico study was carried out to check the interaction between cMET and ABCG2 in presence or absence of QC. Tube formation assay using HUVECs and in ovo Chorioallantoic membrane (CAM) assay using chick fertilized eggs were performed to monitor angiogenesis. In vivo patient-derived xenograft (PDX) mice model was used to validate in silico and ex vivo results. ResultsData revealed that in a hypoxic tumor microenvironment (TME), cMET and ABCG2 interact with each other and upregulate HIF-1α/VEGF-A axis to induce breast cancer angiogenesis. In silico and ex vivo study showed that QC disrupted the interaction between cMET and ABCG2 to inhibit the angiogenic response in endothelial cells by reducing the secretion of VEGF-A from PDBCSCs within the TME. Knockdown of cMET, ABCG2 or both, significantly downregulated the expression of HIF-1α and reduced the secretion of pro-angiogenic factor VEGF-A in the TME of PDBCSCs. Additionally, when PDBCSCs were treated with QC, similar experimental results were obtained. ConclusionIn silico, in ovo, ex vivo and in vivo data confirmed that QC inhibited the HIF-1α/VEGF-A mediated angiogenesis in breast cancer by disrupting the interaction between cMET and ABCG2.

Exosomes with overexpressed miR 147a suppress angiogenesis and infammatory injury in an experimental model of atopic dermatitis

Atopic dermatitis is defined as an intensely systemic inflammation among skin diseases. Exosomes derived from adipose-derived stem cells may be a novel cell-free therapeutic strategy for atopic dermatitis treatment. This study aims to elucidate the possible underlying mechanism of adipose-derived stem cells-exosomes harboring microRNA-147a in atopic dermatitis pathogenesis. BALB/c mice treated with Dermatophagoides farinae extract/2,4-dinitrochlorobenzene were defined as a mouse model of atopic dermatitis, either with inflamed HaCaT cells and HUVECs exposed with TNF-α/IFN-γ stimulation were applied for a cell model of atopic dermatitis. The concentrations of IL-1β and TNF-α in the supernatants were examined by ELISA. Cell viability and migration were assessed by MTT and Transwell assay. The apoptosis was examined using flow cytometry and TUNEL staining. The tube formation assay was employed to analyzed angiogenesis. The molecular regulations among miR-147a, MEF2A, TSLP and VEGFA were confirmed using luciferase reporter assay, either with ChIP. microRNA-147a was markedly downregulated in the serum and skin samples of atopic dermatitis mice, of which overexpression remarkably promoted HaCaT cell proliferation, meanwhile inhibiting inflammatory response and cell apoptosis. microRNA-147a in adipose-derived stem cells was subsequently overexpressed, and exosomes (Exos-miR-147a mimics) were collected. Functionally, exos-microRNA-147a mimics attenuated TNF-α/IFN-γ-induced HaCaT cell inflammatory response and apoptosis, and suppressed HUVECs angiogenesis. Encouraging, molecular interaction experiments revealed that exosomal microRNA-147a suppressed TNF-α/IFN-γ-induced HUVECs angiogenesis by targeting VEGFA, and exosomal microRNA-147a repressed HaCaT cells inflammatory injury through the MEF2A-TSLP axis. Mechanistically, exosomal microRNA-147a repressed pathological angiogenesis and inflammatory injury during atopic dermatitis progression by targeting VEGFA and MEF2A-TSLP axis. microRNA-147a-overexpressing adipose-derived stem cells-derived exosomes suppressed pathological angiogenesis and inflammatory injury in atopic dermatitis by targeting VEGFA and MEF2A-TSLP axis.

LINC00460 promotes angiogenesis by enhancing NF-κB-mediated VEGFA expression in cervical cancer cells

Angiogenesis is a characteristic of tumor development and is key for tumor growth and metastasis. LINC00460 is a long non-coding RNA that plays important yet complex roles in cancer development and progression. Here, we explored the functional mechanism of action of LINC00460 in cervical cancer (CC) angiogenesis for the first time. We found that conditioned medium (CM) from LINC00460-knockdown CC cells attenuated human umbilical vein endothelial cell (HUVEC) migration, invasion, and tube formation, whereas LINC00460 upregulation had the opposite effects. Mechanistically, LINC00460 stimulated VEGFA transcription. Suppressing VEGF-A reversed the effects of CM from LINC00460-overexpressing CC cells on HUVEC angiogenesis. Recombinant VEGFA eliminated the suppressive effects of CM from LINC00460-knockdown CC cells. Furthermore, LINC00460 enhanced VEGFA expression and promoted angiogenesis by activating the NF-κB pathway. Our data illustrate that LINC00460 can promote angiogenesis by activating the NF-κB–VEGFA axis, suggesting that the axis is a promising target for blocking tumor angiogenesis.

Vascular Endothelial Growth Factor A Contributes to Increased Mammalian Respiratory Epithelial Permeability Induced by Pasteurella multocida Infection.

Pasteurella multocida infection can cause significant zoonotic respiratory problems in both humans and animals, but little is known about the mechanisms used by P. multocida to invade and cross the mammalian respiratory barrier. In this study, we investigated the influence of P. multocida infection on the dysfunction of the respiratory epithelial barrier. In vivo tests in mouse infection models demonstrated that P. multocida infection significantly increased epithelial permeability and increased the expression of vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) in murine tracheae and lungs. In murine lung epithelial cell (MLE-12) models, P. multocida infection decreased the expression of tight junctions (ZO-1) and adherens junctions (β-catenin and E-cadherin) proteins but induced the activation of hypoxia-inducible factor 1α (HIF-1α) and VEGFA signaling. When the expression of HIF-1α is suppressed, the induction of VEGFA and ZO-1 expression by P. multocida infection is decreased. We also found that intervention of HIF-1α and VEGFA signaling affected infection outcomes caused by respiratory bacteria in mouse models. Most importantly, we demonstrate that P. multocida infection increases the permeability of human respiratory epithelial cells and that this process is associated with the activation of HIF-1α and VEGFA signaling and likely contributes to the pathogenesis of P. multocida infection in humans. IMPORTANCE The mammalian respiratory epithelium forms the first line of defense against infections with P. multocida, an important zoonotic respiratory pathogen. In this study, we found that P. multocida infection increased respiratory epithelial permeability and promoted the induction of the HIF-1α-VEGFA axis in both mouse and murine cell models. Similar findings were also demonstrated in human respiratory epithelial cells. The results from this study provide important knowledge about the pathogenesis of P. multocida causing infections in both animals and humans.

Exosomes derived from Nr-CWS pretreated MSCs facilitate diabetic wound healing by promoting angiogenesis via the circIARS1/miR-4782-5p/VEGFA axis.

Mesenchymal stem cell (MSC)-derived exosomes (Exos) were reported to a prospective candidate in accelerating diabetic wound healing due to their pro-angiogenic effect. MSCs pretreated with chemistry or biology factors were reported to advance the biological activities of MSC-derived exosomes. Hence, this study was designed to explore whether exosomes derived from human umbilical cord MSCs (hucMSCs) preconditioned with Nocardia rubra cell wall skeleton (Nr-CWS) exhibited superior proangiogenic effect on diabetic wound repair and its underlying molecular mechanisms. The results showed that Nr-CWS-Exos facilitated the proliferation, migration and tube formation of endothelial cells in vitro. In vivo, Nr-CWS-Exos exerted great effect on advancing wound healing by facilitating the angiogenesis of wound tissues compared with Exos. Furthermore, the expression of circIARS1 increased after HUVECs were treated with Nr-CWS-Exos. CircIARS1 promoted the pro-angiogenic effects of Nr-CWS-Exos on endothelial cellsvia the miR-4782-5p/VEGFA axis. Taken together, those data reveal that exosomes derived from Nr-CWS-pretreated MSCs might serve as an underlying strategy for diabetic wound treatment through advancing the biological function of endothelial cells via the circIARS1/miR-4782-5p/VEGFA axis.

Prolonged administration of a secretin receptor antagonist inhibits biliary senescence and liver fibrosis in Mdr2 -/- mice.

Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5-27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2 -/- [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5-27 treatment in Mdr2 -/- mice. In vivo studies were performed in male wild-type and Mdr2 -/- mice treated with saline or SCT 5-27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2 -/- mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-β1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2 -/- mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2 -/- compared with wild-type mice, which was reversed by long-term SCT 5-27 treatment. In vitro , SCT 5-27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-β1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5-27-induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. Modulating the SCT/SR axis may be critical for managing PSC.

Stathmin1 promotes lymph node metastasis in hypopharyngeal squamous cell carcinoma via regulation of HIF‑1α/VEGF‑A axis and MTA1 expression.

Extensive neck lymph node metastasis (LNM) is an important clinical feature of hypopharyngeal squamous cell carcinoma (HSCC). Stathmin1 (STMN1) is closely associated with LNM in numerous human cancers. In the present study, the association between STMN1 and neck LNM in HSCC and the underlying molecular mechanisms were explored. First, postoperative samples of HSCC were screened and the association between STMN1 and neck LNM in HSCC was analyzed. Then, cell functional experiments were performed to assess the potential of STMN1 to promote invasion and migration. Subsequently, the potential target genes and pathways of STMN1 were predicted using bioinformatics analysis. Finally, the obtained target genes and pathways of STMN1 were validated by reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses to confirm the potential mechanisms by which STMN1 promotes LNM in HSCC. As a result, a total of 117 postoperative samples of HSCC were screened, and STMN1 was proven to be associated with neck LNM in HSCC. Further, cell functional experiments established that high expression of STMN1 could actually promote FaDu cell invasion and metastasis. Bioinformatics analysis revealed that high expression of STMN1 was associated with the activation of hypoxia inducible factor-1alpha (HIF-1α) pathway and increased expression of metastasis-associated protein 1 (MTA1). Finally, RT-qPCR and western blot analyses confirmed that STMN1 promotes the expression levels of HIF-1α/vascular endothelial growth factor (VEGF)-A and MTA1 in FaDu cell lines. In conclusion, it was found that high expression of STMN1 promoted neck LNM in HSCC and the potential mechanisms may be via regulation of the HIF-1α/VEGF-A axis and MTA1 expression.

Construction of ceRNA network and key gene screening in cervical squamous intraepithelial lesions.

This study aimed to construct an endogenous competition network for cervical squamous intraepithelial lesions using differential gene screening. GSE149763 was used to screen differentially expressed long non-coding RNAs (lncRNAs) and mRNAs to predict correlated microRNAs (miRNAs). The correlated miRNAs and GSE105409 were used to screen differentially expressed miRNAs for differential co-expression analysis, and the co-expressed differentially expressed miRNAs were used to predict correlated mRNAs. Differentially expressed mRNAs, miRNAs, and lncRNAs were visualized, and differential gene screening, enrichment, and pathway analysis were performed. The ceRNA network of cervical squamous intraepithelial was successfully established and a potential differentially expressed network was identified. The key genes were VEGFA and FOS, and the key pathway was the MAPK signaling pathway. The differential expression and potential effects of the lncRNA BACH1-IT1/miR-140-5p/VEGFA axis, key genes, VEGFA and FOS, and MAPK signaling in CIN were clarified, and the occurrence and potential effects of CIN were further clarified. The underlying molecular mechanism provides a certain degree of reference for subsequent treatments and experimental research.

MiR-590-3p affects the function of adipose-derived stem cells (ADSCs) on the survival of skin flaps by targeting VEGFA.

IntroductionPartial necrosis of skin flaps is still a substantial problem in plastic and reconstructive surgery. In this study, the role of miR-590-3p in adipose-derived stem cells (ADSCs) transplantation in improving the survival of skin flap in a mouse model was delved into.MethodAn abdominal perforator flap model was established in mice. The histopathological examination of mice skin tissues after ADSCs transplantation was implemented using Hematoxylin & eosin (H&E) staining. Immunohistochemistry (IHC) or immunofluorescence (IF) staining was utilized to assess the PCNA or CD31 levels. The concentrations of VEGFA in the culture medium were quantified using a VEGFA ELISA kit.ResultThe damage of tissue in the skin flap was dramatically relieved by ADSCs transplantation. MiR-590-3p overexpression notably suppressed, while miR-590-3p knockdown facilitated skin flap survival by regulating PCNA, VCAM-1, and VEGFA levels. MiR-590-3p targeted VEGFA to regulate its expression. The knockdown of VEGFA significantly inhibited, while overexpression of VEGFA notably promoted the survival of skin flap.ConclusionADSCs transplantation promotes skin flap survival by boosting angiogenesis. The miR-590-3p/VEGFA axis modulates skin flap angiogenesis and survival in ADSCs. These results reveal that interfering with miR-590-3p in ADSCs could potentially be a novel therapeutic target for the improvement of skin flap survival.

DZW-310, a novel phosphoinositide 3-kinase inhibitor, attenuates the angiogenesis and growth of hepatocellular carcinoma cells via PI3K/AKT/mTOR axis

Hepatocellular carcinoma (HCC) is one of the most malignant tumors worldwide with high lethality and prevalence. The deregulated phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway plays an indispensable role in mediating the progression of HCC. Among them, PI3K acts as the most pivotal initiator, contributing to multiple malignant biological processes, like proliferation, apoptosis and angiogenesis. Many PI3K inhibitors (PI3Kis) have been proved or proceeded into clinical as antineoplastic drugs. Nevertheless, the application of PI3Kis for the treatment of HCC remains a blank. Accordingly, our study identified a novel PI3Ki (DZW-310) with strong anti-HCC activity in vitro and in vivo. This study aimed to evaluate its anti-HCC effect and elucidate its potential mechanism. Our current results revealed that DZW-310 significantly attenuated HCC cell growth through promoting intrinsic apoptosis and G0/G1 phase cell arrest. Moreover, DZW-310 suppressed angiogenesis by regulating the HIF-1α/VEGFA axis. Further mechanistic investigation demonstrated that DZW-310, functioned as a PI3Ki, exerted strong anti-HCC activity by acting on PI3Kα (a major subtype of PI3K) and ulteriorly deactivating the PI3K/AKT/mTOR pathway. Collectively, our studies identified that DZW-310 is expected to become a promising HCC therapeutic agent and broaden clinical application of PI3Ki in HCC chemotherapy.

Characterization of a Novel LUCAT1/miR-4316/VEGF-A Axis in Metastasis and Glycolysis of Lung Adenocarcinoma.

Objective: Accumulating literatures suggested that long non-coding RNAs (lncRNAs) were involved in tumorigenesis and cancer progression in lung adenocarcinoma (LUAD). However, the precise regulatory mechanism of lncRNA Lung cancer-associated transcript 1 (LUCAT1) in LUAD is not well defined. In this study, we aimed to investigate the biological function and mechanism of lncRNA LUCAT1 in regulating tumor migration and glycolysis of LUAD. Methods: High throughput sequencing was performed to identify differentially expressed lncRNAs between LUAD patients and healthy controls. The expression levels of LUCAT1 in LUAD clinical specimens or cell lines were evaluated by In situ hybridization (ISH) and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Functional experiments, including wound-healing, transwell invasion assays, glucose absorption, lactate metabolism and tumor xenograft experiments were conducted to identify the biological functions of LUCAT1 in LUAD. Silencing of LUCAT1, over-expression of LUCAT1 and miR-4316 were generated in LUAD cell lines to verify the regulatory mode of LUCAT1-mir-4316-VEGFA axis. Results: Our findings revealed that lncRNA LUCAT1 was significantly up-regulated in LUAD serum exosomes, tumor tissues, and LUAD cells in comparison with corresponding controls. Receiver operating characteristic curve (ROC) analysis indicated that the area under the curve (AUC) value of serum exosomal LUCAT1 reached 0.852 in distinguishing LUAD patients from healthy individuals. High expression of LUCAT1 in LUAD patient tissues was associated with enhanced Lymph Node Metastasis (LNM), advanced Tumor Node Metastasis (TNM) stage and poorer clinical outcome in LUAD patients. Knockdown of LUCAT1 inhibited LUAD cell metastasis and glycolysis in vitro as well as tumor metastasis in vivo, while overexpression of LUCAT1 induced a promoted LUAD metastasis and glycolysis. Furthermore, mechanistic investigations revealed that LUCAT1 elevated LUAD cell metastasis and glycolysis by sponging miR-4316, which further led to the upregulation of VEGFA. Finally, the regulatory axis LUCAT1-miR-4316-VEGFA was verified in LUAD. Conclusion: Our present research suggested that LUCAT1 facilitate LUAD cell metastasis and glycolysis via serving as a competing endogenous RNA to regulate miR-4316/VEGFA axis, which provided a novel diagnostic marker and therapeutic target for LUAD patients.

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR Tcells.

Unlimited generation of chimeric antigen receptor (CAR) Tcells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR Tcell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ Tcell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory Tcells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR Tcell characteristics. Expanded iPSC CD19-CAR Tcells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR Tcells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR Tcells also mediated potent antitumor activity invivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR Tcells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

LncRNA H19 Promotes Ox-LDL-Induced Dysfunction of Human Aortic Endothelial Cells through the miR-152/VEGFA Axis.

Objective lncRNA H19 (H19) elevation is related to the risk of coronary artery disease. DIANA-lncBase database analysis suggested that microRNA-152 (miR-152) and H19 have binding sites. Here, the effect and mechanism of H19 and miR-152 in the oxidized low-density lipoprotein (ox-LDL)-induced human aortic endothelial cells (HAECs) were explored. Methods The expression of H19, miR-152, and vascular endothelial growth factor (VEGF)-A in the HAECs treated with 5 μg/mL ox-LDL was detected by qRT-PCR. MTT, wound-healing assay, and tube formation assay were analyzed to evaluate the angiogenic activity of H19 and miR-152 in the HAECs cells knocked down H19. Dual-luciferase assay was performed to verify the targeting relationship of miR-152 to either H19 or VEGFA, respectively. Western blot was used to detect the expression of epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin and vimentin) and VEGFA protein in the cells. Results After ox-LDL treatment, the expression of H19 and VEGFA was significantly increased, miR-152 expression was remarkably decreased. H19 was mainly expressed in the cytoplasm of HAECs. Knocking down H19 or overexpression of miR-152 significantly inhibited the cellular proliferation, migration, tube formation, and EMT trend of the HAECs. On the contrary, miR-152 interference reversed H19 silencing-mediated effects in the ox-LDL-induced HAECs. The dual-luciferase assay showed that miR-152 had a targeting relationship with H19 and VEGFA. MiR-152 was negatively corrected with the VEGFA expression. Conclusion Ox-LDL negatively regulates miR-152 via H19, promotes the expression of VEGFA, and induces the dysfunction of HAECs.