MiR-4729 regulates TIE1 mRNA m6A modification and angiogenesis in hemorrhoids by targeting METTL14
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Hemorrhoids are a frequently-occurring disease of the anorectal system that is often accompanied by vascular hyperplasia and edema. A METTL14-mediated RNA N-6 methyladenosine (m6A) modification can improve mRNA stability and increase its transcriptional and translational activities, closely related to the occurrence of many diseases.Western blot, qPCR, and immunofluorescence staining were used to detect the levels of gene and protein expression. Haematoxylin and eosin staining was used for histopathological examination. RNA immunoprecipitation-PCR and RNA dot blotting were used to detect mRNA m6A modification.Obvious signs of angiogenesis (CD31+/vWF+) were identified in the hemorrhoids. High levels of METTL14 expression on vascular endothelial cells (CD31+) suggested that angiogenesis was accompanied by differential modification of m6A RNA. It was subsequently found that the level of miR-4729 expression was significantly decreased in hemorrhoid tissues. The luciferase reporter enzyme assay results suggested that miR-4729 silenced its expression by targeting the 3'UTR of METTL14 mRNA. MiR-4729 overexpression in human umbilical vein endothelial cells (HUVECs) inhibited the proliferation and migration of HUVECs in vitro and vascular structure formation in the outer matrix. MiR-4729 overexpression significantly inhibited endogenous METTL14 expression in HUVECs and reduced the entire m6A RNA modification, especially the level of m6A methylation at the specific site of the 3' UTR of TIE1 mRNA. Moreover, miR-4729 overexpression significantly inhibited the molecular loop of the TIE1/VEGFA signaling pathway in HUVECs.Our findings confirmed that the down-regulation of miR-4729 in hemorrhoid vascular endothelial cells was one of the main reasons for vascular proliferation. The overexpression of miR-4729 in vascular endothelial cells decreased the global mRNA methylation and TIE1 mRNA 3'UTR-specific site methylation by silencing METTL14 expression, reducing TIE1 mRNA stability, down-regulating the TIE1/VEGFA signal molecular loop expression, and weakening angiogenesis ability.Keywords:
CD31
Low physical activity correlates with increased cancer risk in various cancer types, including colorectal cancer (CRC). However, the ways in which swimming can benefit CRC remain largely unknown. In this study, mice bearing tumors derived from CT-26 cells were randomly divided into the control and swimming groups. Mice in the swimming group were subjected to physical training (swimming) for 3 weeks. Compared with the control group, swimming clearly attenuated tumor volume and tumor weight in CT-26 tumor-bearing mice. RNA sequencing (RNA-seq) identified 715 upregulated and 629 downregulated transcripts (including VEGFA) in tumor tissues of mice in the swimming group. KEGG pathway analysis based on differentially expressed transcripts identified multiple enriched signaling pathways, including angiogenesis, hypoxia, and vascular endothelial growth factor (VEGF) pathways. Consistently, IHC analysis revealed that swimming significantly downregulated CD31, HIF-1α, VEGFA, and VEGFR2 protein expression in tumor tissues. In conclusion, swimming significantly attenuates tumor growth in CT-26 tumor-bearing mice by inhibiting tumor angiogenesis via the suppression of the HIF-1α/VEGFA pathway.
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Angiogenesis as a prognostic indicator in tumours has been extensively studied, demonstrating a positive correlation for various malignant tumours. Yet, in osteosarcomas (OSAs), its role remains a topic of debate. VEGFA is considered the most important pro-angiogenic factor involved in the development of the vasculature. In order to investigate the possibility that intra-tumoral microvessel density (MVD) expression may be related to VEGFA and may provide useful prognostic information in canine OSA, 12 histological specimens of primary canine appendicular OSA were immunostained using an endothelial marker CD31 and VEGFA antibody. No significant differences in mean MVD were found when comparing with various clinicopathological features, development of pulmonary metastasis and patient prognosis. Notably, the number of vessels counted in VEGFA-High expression specimens was significantly greater than those in the VEGFA-Low expression (p <0.05). In this study, we were able to demonstrate that canine appendicular OSA is a relatively vascular tumour and that the local MVD in primary canine appendicular OSA is significantly correlated with VEGFA immunostaining expression in the tumour tissue. These observations suggest that VEGFA secreted by canine appendicular OSA cells elicits angiogenesis. However, the degree of MVD does not provide prognostic information. It is likely that angiogenesis plays a key role in the tumorigenesis of canine appendicular OSA and may be a potential target for novel anti-angiogenic therapies.
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Rapid growth of residual tumors can occur as a result of their recurrence and progression. The present study aimed to investigate the expression of hypoxia inducible factor‑2 subunit α (HIF‑2α), vascular endothelial growth factor A (VEGFA), erythropoietin-producing hepatocellular A2 (EphA2) and angiogenesis in residual hepatocellular carcinoma (HCC), following treatment with high‑intensity focused ultrasound (HIFU) ablation, in order to investigate the association between protein expression and tumor recurrence and growth. Athymic BALB/c (nu/nu) mice were subcutaneously inoculated with the HCC cell line HepG2, in order to create xenograft tumors. Approximately 30 days post‑inoculation, eight mice were treated with HIFU, whereas eight mice received no treatment and acted as the control group. Residual tumor tissues were obtained from the experimental groups after one month. Levels of HIF‑2α, VEGFA, EphA2 and cluster of differentiation 31 (CD31) expression was measured by immunohistochemical staining. CD31‑positive vascular endothelial cells were counted to calculate microvascular density (MVD), and western blot analysis was performed to determine levels of HIF‑2α, VEGFA, and EphA2 protein. It was found that the expression levels of HIF‑2α, VEGFA, EphA2, and MVD proteins in residual HCC tissues were significantly higher than in the control group tissues (P<0.05). Tumor MVD was strongly correlated with VEGFA (R=0.957, P<0.01) and EphA2 (R=0.993, P<0.01) protein expression levels. Furthermore, there was a significant positive correlation between HIF‑2α and EphA2 expression (R=0.991, P<0.01). The correlation between VEGFA and EphA2 expression was also positive (R=0.985, P<0.01). These data suggest that overexpression of HIF‑2α, VEGFA and EphA2 is related to angiogenesis in residual HCC following HIFU ablation, potentially via their association with key mediators of recurrence.
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Abstract MALAT1 is one of the most hopeful members implicated in angiogenesis in a variety of non-malignant diseases. In multiple myeloma (MM), MALAT1 is recognized as the most highly expressed long non-coding RNA. However, the functional roles of MALAT1 in angiogenesis and the responsible mechanisms have not yet been explored. Herein, we discovered a novel regulatory network dependent on MALAT1 in relation to MM tumorigenesis and angiogenesis. We observed that MALAT1 was upregulated in MM and significantly associated with poor overall survival. MALAT1 knockdown suppressed MM cell proliferation and promoted apoptosis, while restricting endothelial cells angiogenesis. Moreover, MALAT1 directly targeted microRNA-15a/16, and microRNA-15a/16 suppression partly reverted the effects of MALAT1 deletion on MM cells in vitro as well as tumor growth and angiogenesis in vivo. In addition, further study indicated that MALAT1 functioned as a competing endogenous RNA for microRNA-15a/16 to regulate vascular endothelial growth factor A (VEGFA) expression. Our results suggest that MALAT1 plays an important role in the regulatory axis of microRNA-15a/16–VEGFA to promote tumorigenicity and angiogenesis in MM. Consequently, MALAT1 could serve as a novel promising biomarker and a potential antiangiogenic target against MM.
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Angiogenesis plays a vital role in the development of bladder cancer (BC). The Y-box-binding protein 1 (YB-1) is a well-known oncoprotein which is closely related to angiogenesis of tumors, but the relationship and mechanism of YB-1 and angiogenesis in BC remain unclear. Based on 56 clinical BC specimens, this study found that high expression of YB-1 samples demonstrated a higher expression of vascular endothelial growth factor A (VEGFA) than those of YB-1 low expression. Subsequently, the expression of YB-1 and miR-29b-3p was regulated in the BC cell lines where we noted that YB-1 promoted VEGFA expression by downregulating the expression of miR- 29b-3p. The ability of BC cells to induce angiogenesis decreased after YB-1 was knocked down. Moreover, the in vivo study further confirmed that YB-1 promotes angiogenesis in BC. Our findings enhance the understanding of how YB-1 promotes angiogenesis in BC and provide evidence for YB-1 as a therapeutic target of BC. Moreover, this may provide new inspiration for miRNAs replacement therapies.
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Objective To study the isolation,culture and identification of human umbilical vein endothelial cells(HUVECs) in vitro,and analyze antigens expression.Methods Cultured cells were isolated from umbilical cord by enzyme digestion and were cultured in vitro.Cultured cells were subjected to flow cytometer to analyze the expression of CD31,CD34,VWF,KDR and CD133.Immunofluorescence was performed to detect VWF and dual binding of ac-LDL and UEA-1.Results Cultured HUVECs were like slabstones,which were positively stained for CD31,CD34,VWF,KDR,but negative for CD133.Cultured HUVECs could endocytose ac-LDL and UEA-1.Conclusions HUVECs with the characters of endothelial cells can be cultured abundantly by this method.Purity of HUVECs can be assayed by analyzing antigens expression,which can be used to establish the model of endothelial cells.
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The profile of microRNAs (miRNAs) altered following middle cerebral artery occlusion (MCAO) and miRNAs are involved in angiogenesis following cerebral ischemia. miR‑376b‑5p was decreased following MCAO, however, whether miR‑376b‑5p is important in angiogenesis remains to be elucidated. The present study was designed to identify whether miR‑376b‑5p is involved in angiogenesis following cerebral ischemia and to elucidate the underlying mechanisms. A rat MCAO model was established and quantitative polymerase chain reaction was performed to analyze the mRNA expression level of miR‑376b‑5p for 1 to 7 days. In addition, the density of microvessels and the relative mRNA and protein levels of hypoxia‑inducible factor‑1 α (HIF‑1α), vascular endothelial growth factor A (VEGFA) and Notch1 were measured. The miR‑376b‑5p mimic or the miR‑376b‑5p inhibitor were transfected into hypoxic human umbilical vein endothelial cells (HUVECs), and the proliferation, migration and tube formation were measured. To further examine the underlying mechanisms, shRNA was transfected into cells to knock down HIF‑1α, and angiogenesis and the expression of associated molecules, including HIF‑1α, VEGFA and Notch1 were compared between each group. Our results demonstrated that miR‑376b‑5p repressed angiogenesis in vivo and in vitro, and miR‑376b‑5p inhibited angiogenesis in HUVECs by targeting the HIF‑1α‑mediated VEGFA/Notch1 signaling pathway. These findings provide new insights into angiogenesis therapy for cerebral ischemia.
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To investigate the effect of recombinant adenovirus-mediated HIF-1 alpha (HIF-1α) on the expression of vascular endothelial growth factor (VEGFA) and HIF-1α in hypoxic brain microvascular endothelial cells (BMEC) in rats.Primary cultured rat BMEC in vitro were treated without or with either recombinant adenovirus-mediated hypoxia-inducible factor-1 alpha (AdHIF-1α) or recombinant adenovirus empty vector (Ad) in the presence of CoCl2 (simulating hypoxia conditions), or were grown under normoxia conditions. The expression of VEGFA and HIF-1α was analyzed at 12h, 24h, 48h and 72h incubation time, respectively. We also accessed a GEO dataset of stroke to analyze in vivo the alteration of HIF-1α and VEGFA expression, and the correlations between HIF-1α, VEGFA and CD31 mRNA levels in vascular vessels after stroke.VEGFA and HIF-1α expression were significantly higher in at each time point in the AdHIF-1α than other groups (p<0.05), whereas the Ad group and hypoxia group, showed no statistically significant difference (p>0.05). Moreover, VEGFA and HIF-1α levels were significantly higher in BMEC under hypoxia conditions than normoxia conditions (p <0.05). Both HIF-1α and VEGFA expression significantly increased after stroke in vivo with 1.30 and 1.57 fold-change in log2, respectively. There were significantly positive associations between HIF-1α, VEGFA and CD31 mRNA levels in vivo after stroke.Hypoxia-induced HIF-1α and VEGFA expression in vascular vessels, and recombinant AdHIF-1α could up-regulate VEGFA, and enhance HIF-1ααlevels in BMEC in vitro, which may play an important role in the recovery of stroke.
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