circSLTM knockdown attenuates chondrocyte inflammation, apoptosis and ECM degradation in osteoarthritis by regulating the miR-515-5p/VAPB axis.

Osteoarthritis (OA) is a prevalent joint disorder characterized by cartilage degeneration. Circular RNAs (circRNAs) have emerged as pivotal players in OA progression, orchestrating various biological processes such as proliferation, apoptosis, inflammation, and extracellular matrix (ECM) reorganization. Among these circRNAs, circSLTM exhibits aberrant expression in OA, yet its precise regulatory mechanism remains elusive. This study aimed to elucidate the regulatory mechanisms of circSLTM in OA pathogenesis, with a focus on its role as a competing endogenous RNA (ceRNA). Human cartilage tissues were procured from both OA patients and non-OA individuals, while human chondrocyte cells were subjected to lipopolysaccharide (LPS) treatment to mimic OA-like conditions. Our findings revealed upregulation of circSLTM in OA patients and LPS-treated chondrocytes. Loss-of-function assays were conducted, demonstrating that silencing circSLTM via shRNAs mitigated LPS-induced effects on chondrocytes, as evidenced by enhanced proliferation, reduced apoptosis, and inflammatory factors, and altered expression of extracellular matrix proteins. Further exploration into the regulatory mechanism of circSLTM unveiled its interaction with microRNA-515-5p (miR-515-5p) to modulate vesicle-associated membrane protein (VAPB) expression in chondrocytes. VAPB, also upregulated in OA, was positively regulated by circSLTM. Rescue assays corroborated that VAPB overexpression reinstated the protective effects of circSLTM knockdown on LPS-treated chondrocytes. Moreover, concurrent knockdown of both circSLTM and VAPB demonstrated synergistic protection against LPS-induced chondrocyte injury. Additionally, we delineated that LPS triggered the activation of the NF-κB pathway in chondrocytes, which was counteracted by circSLTM silencing. To assess the effects of circSLTM on OA in vivo, anterior cruciate ligament transection (ACLT) mouse models were established, revealing that circSLTM deficiency ameliorated cartilage defects in vivo. In conclusion, circSLTM exacerbates osteoarthritis progression by orchestrating the miR-515-5p/VAPB axis and activating the NF-κB pathway, providing novel insights for targeted therapy in OA management.

[miR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes].

Objective: To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA). Methods: Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1ß (IL-1ß) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1ß for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay. Results: After the treatment of C28/I2 cells with IL-1ß, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G 2 phase decreased significantly, and the proportion of cells at G 1 phase increased significantly, suggesting that the cell cycle was blocked after IL-1ß treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1ß, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1ß, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1ß-induced apoptosis and inflammation of OA chondrocytes. Conclusion: miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.

Endogenous hsa-circ_0007113 binds hsa-miR-515-5p to regulate senescence in human embryonic lung fibroblasts.

BACKGROUND: Cellular senescence can lead to many diseases. However, the roles and regulation of circular RNAs (circRNAs) in senescence are poorly understood. OBJECTIVES: To investigate the altered expression pattern and mechanism of circRNA during cellular senescence and find potential targets to prevent senescence. MATERIAL AND METHODS: The Arraystar Human circRNA Array and bioinformatics were used to profile the differentially expressed circRNAs in human embryonic lung fibroblasts (IMR-90) between young cells and senescent cells and quantification in the clinical materials. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. The miRNA targets were predicted using TargetScan and miRanda. RESULTS: A total of 113 differentially expressed circRNAs were identified, including 109 upregulated and 4 downregulated circRNAs (fold change >2 and p-value <0.05). Real-time qualitative polymerase chain reaction (qPCR) showed that the expression levels of 4 circRNA were significantly increased in senescent cells, and that of hsa_circ_0007113 was significantly decreased, consistent with the microarray. siRNA against hsa_circ_0007113 increased p21 and p53 expression levels and ß-gal staining. The hsa_circ_0007113 has a binding site for miR-515-5p, which is involved in regulating the p53/p21 signaling pathway. The expression level of hsa_circ_0007113 was also decreased in aged people. CONCLUSIONS: The study showed an altered circRNA expression pattern in cellular senescence, which might play important roles in senescence-related physiological processes. These findings provide a new direction for studying the molecular mechanism underlying senescence and a new possibility for the treatment of senescence by modulating circRNAs.

Circ-UBR4 regulates the proliferation, migration, inflammation, and apoptosis in ox-LDL-induced vascular smooth muscle cells via miR-515-5p/IGF2 axis.

The aim of our study is to disclose the role and underlying molecular mechanisms of circular RNA ubiquitin protein ligase E3 component n-recognin 4 (circ-UBR4) in atherosclerosis (AS). Our data showed that circ-UBR4 expression was upregulated in AS patients and oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cells (VSMCs) compared with healthy volunteer and untreated VSMCs. In addition, ox-LDL stimulated proliferation, migration, and inflammation but decreased apoptosis in VSMCs, which were overturned by the inhibition of circ-UBR4. miR-515-5p was sponged by circ-UBR4, and its inhibitor reversed the inhibitory effect of circ-UBR4 knockdown on proliferation, migration, and inflammation in ox-LDL-induced VSMCs. Insulin-like growth factor2 (IGF2) was a functional target of miR-515-5p, and overexpression of IGF2 reversed the suppressive effect of miR-515-5p on ox-LDL-stimulated VSMCs proliferation, migration, and inflammation. Collectively, circ-UBR4 knockdown decreased proliferation, migration, and inflammation but stimulated apoptosis in ox-LDL-induced VSMCs by targeting the miR-515-5p/IGF2 axis.

[Effect of LncRNA GATA3-AS1 Targeting MiR-515-5p on Cell Proliferation and Apoptosis in Childhood Acute Lymphoblastic Leukemia Cells].

OBJECTIVE: To investigate the effects of long non-coding RNA (lncRNA) GATA3 antisense RNA 1 (GATA3-AS1) targeting miR-515-5p on the proliferation and apoptosis of childhood acute lymphoblastic leukemia (ALL) cells. METHODS: RT-qPCR was used to determine the expression of GATA3-AS1 and miR-515-5p in the plasma of controls and ALL children. Human ALL cells Jurkat were divided into si-GATA3-AS1, si-NC, miR-NC, miR-515-5p, si-GATA3-AS1+anti-miR-NC and si-GATA3-AS1+anti-miR-515-5p groups. CCK-8 assay was used to detect the cell proliferation, and flow cytometry was used to detect the cell apoptosis. The targeting relationship between GATA3-AS1 and miR-515-5p was determined by dual-luciferase reporter assay. RESULTS: The expression level of GATA3-AS1 in the plasma of ALL children was significantly higher than that of controls (P <0.001), while the expression level of miR-515-5p was significantly lower than that of controls (P <0.001). Compared with the si-NC group, the cell inhibition rate, apoptosis rate, and miR-515-5p expression level in si-GATA3-AS1 group were significantly increased (P <0.001). Compared with the miR-NC group, the cell inhibition rate and apoptosis rate in miR-515-5p group were significantly increased (P <0.001). GATA3-AS1 could directly and specifically bind to miR-515-5p. Compared with the si-GATA3-AS1+anti-miR-NC group, the cell inhibition rate and apoptosis rate in si-GATA3-AS1+anti-miR-515-5p group were significantly decreased (P <0.001). CONCLUSION: Down-regulation of GATA3-AS1 can inhibit proliferation and induce apoptosis of childhood ALL cells by targeting up-regulation of miR-515-5p expression.

MAPKAPK5-AS1/miR-515-5p/CAB39 Axis Contributes to Non-small Cell Lung Cancer Cell Proliferation and Migration.

Several studies have elucidated the pivotal function that long noncoding RNAs (lncRNAs) exerted on the initiation and development of various human carcinomas, encompassing non-small cell lung cancer (NSCLC). In spite of the fact that lncRNA MAPKAPK5 antisense RNA 1 (MAPKAPK5-AS1) has already been investigated by researchers and confirmed to play oncogenic roles in colorectal cancer, the underlying regulatory function of MAPKAPK5-AS1 in NSCLC cells still remain unclear. In our research, we found that MAPKAPK5-AS1 was expressed at high levels in NSCLC cells. Biological functional assays unclosed that downregulation of MAPKAPK5-AS1 repressed proliferative and migratory capacities whereas promoted apoptotic level in NSCLC cells. Molecular mechanism experiments confirmed that, in NSCLC cells, MAPKAPK5-AS1 combined with miR-515-5p and negatively modulated miR-515-5p expression level. Besides, calcium-binding protein 39 (CAB39) expression level was verified to be negatively modulated by miR-515-5p whereas positively modulated by MAPKAPK5-AS1 in NSCLC cells. Furthermore, rescued-function assays disclosed that inhibited miR-515-5p expression or overexpressed CAB39 could restore the suppressive influence of MAPKAPK5-AS1 silence on NSCLC progression. In summary, MAPKAPK5-AS1 upregulates CAB39 expression level to facilitate NSCLC progression by sequestering miR-515-5p, providing promising biomarkers for NSCLC treatment.

circ_0006089 Facilitates Gastric Cancer Progression via Decoying miR- 515-5p and Up-regulating CXCL6.

BACKGROUND: Gastric cancer (GC) is the most common cancer globally. Recent research has suggested that circular RNAs (circRNAs) play crucial roles in GC tumorigenesis and progression. The present study is performed to clarify the possible mechanism of circRNA has_circ_0006089 (circ_0006089) in GC. METHODS: The differentially expressed circRNAs were screened out by analyzing the dataset GSE83- 521. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect circ_0006089, miR-515-5p and CXCL6 expression levels in GC tissues and cell lines. CCK-8, BrdU and Transwell assays were adopted to examine the biological function of circ_0006089 in GC cells. The interaction between miR-515-5p and circ_0006089, as well as between CXCL6 and miR-515-5p, was confirmed through bioinformatics, RNA immunoprecipitation (RIP) assay, dual-luciferase reporter gene assay and RNA pull-down assay. RESULTS: Circ_0006089 was significantly upregulated in GC tissues and cells, and miR-515-5p was remarkably downregulated. After knocking down circ_0006089 or overexpressing miR-515-5p, the growth, migration and invasion of GC cells were markedly reduced. In terms of mechanism, miR-515- 5p was verified to be the target of circ_0006089, and CXCL6 was validated as miR-515-5p's downstream target gene. Inhibiting miR-515-5p reversed the inhibitory effect knocking down circ_0006089 had on GC cell proliferation, migration and invasion. CONCLUSION: Circ_0006089 facilitates the malignant biological behaviors of GC cells via the miR-515- 5p/CXCL6 axis. Circ_0006089 can probably act as one of the important biomarkers and therapeutic targets in GC treatment strategies.

[Hsa_circ_0000670 promoted the progression of gastric cancer through the miR-515-5p/SIX1 molecular axis].

Objective: To explore whether hsa_circ_0000670 promotes the progression of gastric cancer by regulating the miR-515-5p/SIX1 molecular axis. Methods: The gastric cancer and adjacent normal tissues of 35 gastric cancer patients admitted to Rugao Hospital Affiliated to Nantong University from 2014 to 2015 were collected. The expression levels of circ_0000670, miR-515-5p and Sine oculis homeobox 1 (SIX1) in gastric cancer tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The correlations between circ_0000670 and miR-515-5p, miR-515-5p and SIX1, circ_0000670 and SIX1 were analyzed by the Pearson method. Patients were divided into low circ_0000670 expression group (17 cases) and high circ_0000670 expression group (18 cases) based on the median of circ_0000670 expression level, and Kaplan-Meier was used to analyze the 5-year survival of patients. Cell proliferation was assessed via clone formation assay. Cell cycle and apoptosis were detected by flow cytometry. Wound healing and Transwell assays were used to detect cell migration and invasion ability. The targeting relationship between miR-515-5p and circ_0000670 or SIX1 was confirmed by the dual luciferase reporter assay. Nude mice were injected into HGC-27 cells transfected with sh-NC or sh-circ_0000670, and the volume and weight of the transplanted tumor were measured, also, the levels of circ_0000670, miR-515-5p and SIX1 in the transplanted tumor tissue were detected. Results: The expression levels of circ_0000670 and SIX1 in gastric cancer tissues and cell lines were significantly increased (P<0.05), while the expression levels of miR-515-5p were significantly decreased (P<0.05). The survival rate of patients in the low circ_0000670 expression group (82.4%) was significantly higher than that in the high circ_0000670 expression group (28.7%, P=0.034). Circ_0000670 was negatively correlated with miR-515-5p (r=-0.846, P<0.001), and miR-515-5p was negatively correlated with SIX1 (r=-0.615, P<0.001), but circ_0000670 was positively correlated with SIX1 (r=0.814, P<0.001). Transfection of si-circ_0000670 or miR-515-5p mimic could significantly reduce the number of clone-forming cells, migration distance, migration and invasion cells (P<0.05), and increase the ratio of G(0)/G(1) phase cells, apoptosis rate and the protein level of E-cadherin (P<0.05), decreased the proportion of S-phase cells and the protein level of Vimentin (P<0.05). The dual luciferase report assay confirmed that circ_0000670 could target miR-515-5p, and miR-515-5p could bind to SIX1. Co-transfection of si-circ_0000670 and miR-515-5p inhibitor could significantly attenuate the effects of si-circ_0000670 on cell proliferation, migration, invasion, cell cycle and apoptosis (P<0.05). Co-transfection of miR-515-5p mimic and pcDNA-SIX1 could significantly reduce the effects of miR-515-5p mimic on cell proliferation, migration, invasion, cell cycle and apoptosis (P<0.05). Compared with the sh-NC group [volume=(596.20±125.46) mm(3) and weight=(538.00±114.39) g], the volume and weight of transplanted tumors in the sh-circ_0000670 group [volume=(299.20±47.58) mm 3 and weight=(289.80±48.73 g)] were significantly reduced (P<0.05), the expression levels of circ_0000670 and SIX1 were significantly reduced (P<0.05), and the expression level of miR-515-5p was significantly increased (P<0.05). Conclusion: Knockdown of circ_0000670 could inhibit cell proliferation, migration, invasion of gastric cancer cells, induce cell cycle arrest in G(0)/G(1) phase and promote cell apoptosis by regulating the miR-515-5p/SIX1 axis.

Overexpression of FTO alleviates osteoarthritis by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis.

OBJECTIVE: Osteoarthritis (OA) is regarded as the most prevalent chronic joint disease. Fat-mass and obesity-associated gene (FTO) is involved in OA alleviation. This study elucidated the role of FTO in OA and the associated mechanism. METHODS: We established a cell injury model by stimulating human normal chondrocytes (C28/I2) with lipopolysaccharide (LPS), and measured cell viability, apoptosis, and inflammatory cytokines using CCK-8, flow cytometry, Western blot, and ELISA. TLR4, MyD88, p/t-p65, and p/t-IκBα levels, FTO, COX-2, and iNOS mRNA levels, and m6A methylation levels were measured by Western blot, RT-qPCR, and colorimetry. RNA immunoprecipitation and co-immunoprecipitation were conducted to confirm the interaction between FTO and DGCR8. pri-miR-515-5p process was regulated in an m6A-dependent manner. After predicting the presence of several binding sites between miR-515-5p and TLR4 on Targetscan, we further confirmed their relationship by dual-luciferase assay. OA rat models were established by monosodium iodoacetate injection. The pathological changes in knee joint were observed by HE staining. RESULTS: FTO was diminished in LPS-induced C28/I2 cells. With the increase of LPS concentration, cell viability was repressed, apoptosis rate was increased, and inflammatory markers were promoted, which were annulled by FTO overexpression. FTO interacted with DGCR8 and modulated the pri-miR-515-5p processing in an m6A-dependent manner. miR-515-5p silencing partially averted the inhibitory effect of FTO on LPS-induced cell injury. Given that TLR4 was a direct target of miR-515-5p, miR-515-5p inactivated the MyD88/NF-κB pathway by targeting TLR4. FTO overexpression improved cartilage structure in OA rats, reduced apoptosis, inhibited inflammation in synovial fluid, and repressed the TLR4/MyD88/NF-κB axis. CONCLUSION: FTO alleviated OA in an m6A-dependent manner via the miR-515-5p/TLR4/MyD88/NF-κB axis.

Depletion of circPDSS1 inhibits ITGA11 production to confer cisplatin sensitivity through miR-515-5p in gastric cancer.

Chemoresistance limits cisplatin (DDP)-mediated treatment for gastric cancer (GC). Circular RNA (circRNA) acts an important role in chemoresistance. However, the underlying mechanism of circPDSS1 regulating DDP sensitivity in GC remains unclear. The expression patterns of circPDSS1, miR-515-5p and integrin subunit alpha 11 (ITGA11) were analyzed by qRT-PCR. Protein expression was checked by Western blotting analysis. Cell viability was investigated by 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was evaluated by colony formation assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. The analysis of cell apoptosis, migration and invasion was performed by flow cytometry analysis and transwell assays. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted to identify the associations among circPDSS1, miR-515-5p and ITGA11. In vivo assay was implemented using a xenograft mouse model assay. CircPDSS1 and ITGA11 expression were significantly upregulated, whereas miR-515-5p was downregulated in DDP-resistant GC tissues and cells in comparison with controls. CircPDSS1 depletion reduced DDP resistance, cell proliferation, migration and invasion but induced cell apoptosis in DDP-resistant GC cells. CircPDSS1 directly bound to miR-515-5p. CircPDSS1-mediated actions were dependent on the regulation of miR-515-5p. Besides, miR-515-5p was associated with ITGA11, and circPDSS1 regulated ITGA11 expression by binding to miR-515-5p. Overexpression of miR-515-5p improved DDP sensitivity owing to the downregulation of ITGA11. Further, circPDSS1 mediated DDP sensitivity by regulating miR-515-5p and ITGA11 in vivo. CircPDSS1 conferred DDP resistance through the miR-515-5p/ITGA11 axis in GC cells.

miR-515-5p suppresses trophoblast cell invasion and proliferation through XIAP regulation in preeclampsia.

MicroRNAs (miRNAs) are non-coding small RNA molecules that can be secreted into the circulation and which exist in remarkably stable forms. Circulating miRNAs regulate numerous biological process and are aberrantly expressed in pathological conditions. Differentially expressed circulating miRNAs have received attention as potential biomarkers for many diseases. In this study, we revealed that miR-515-5p was significantly upregulated in maternal serum from preeclampsia patients in comparison to normal pregnant women. Bioinformatics prediction and a dual-luciferase reporter gene assay revealed that miR-515-5p directly targets the X-linked inhibitor of apoptosis protein (XIAP) 3'-untranslated region. In addition, the overexpression of miR-515-5p inhibited the proliferation and invasion of HTR-8/SVneo trophoblast cells. The decreased XIAP expression and reduced epithelial-mesenchymal transition (EMT) were observed in the preeclamptic placenta. Collectively, miR-515-5p may play critical roles in the pathogenesis of preeclampsia through suppression of XIAP, and serum miR-515-5p may act as a potential biomarker for preeclampsia.

Plasmacytoma variant translocation 1 inhibits miR-515-5p function and modulates high mobility group B3 to promote the growth of prostate cancer cells.

AIM: This study is performed to analyze the role of long non-coding RNA plasmacytoma variant translocation 1 in prostate cancer. METHODS AND MATERIALS: Plasmacytoma variant translocation 1, miR-515-5p, and high mobility group B3 mRNA expressions were examined using quantitative real-time polymerase chain reaction and immunohistochemistry. After gain-of-function and loss-of-function models were established, the changes in cell proliferation, migration, and invasion were evaluated using cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, and Transwell experiments. Validation of the targeting relationships between plasmacytoma variant translocation 1 and miR-515-5p, and between miR-515-5p and high mobility group B3 was conducted using bioinformatics prediction, a dual-luciferase reporter assay, and an RNA immunoprecipitation experiment. Moreover, the effects of plasmacytoma variant translocation 1 and miR-515-5p on high mobility group B3 protein expression were examined using Western blot. RESULTS: Plasmacytoma variant translocation 1 expression and high mobility group B3 expression were up-regulated in prostate cancer tissues and cell lines while miR-515-5p expression was down-regulated. Plasmacytoma variant translocation 1 knockdown restrained the proliferation, migration, and invasion of LNCaP and DU145 cells in vitro, and the transfection with miR-515-5p inhibitors reversed these effects. Mechanistically, plasmacytoma variant translocation 1 could repress the function of miR-515; high mobility group B3 was proved to be a target gene of miR-515-5p, and its expression could be indirectly positively modulated by plasmacytoma variant translocation 1. CONCLUSION: Plasmacytoma variant translocation 1 accelerates prostate cancer progression by repressing miR-515-5p's function to upregulate high mobility group B3 expression.

Hsa_circ_0000670 promoted the progression of gastric cancer through the miR-515-5p/SIX1 molecular axis / 中华肿瘤杂志

Objective: To explore whether hsa_circ_0000670 promotes the progression of gastric cancer by regulating the miR-515-5p/SIX1 molecular axis. Methods: The gastric cancer and adjacent normal tissues of 35 gastric cancer patients admitted to Rugao Hospital Affiliated to Nantong University from 2014 to 2015 were collected. The expression levels of circ_0000670, miR-515-5p and Sine oculis homeobox 1 (SIX1) in gastric cancer tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The correlations between circ_0000670 and miR-515-5p, miR-515-5p and SIX1, circ_0000670 and SIX1 were analyzed by the Pearson method. Patients were divided into low circ_0000670 expression group (17 cases) and high circ_0000670 expression group (18 cases) based on the median of circ_0000670 expression level, and Kaplan-Meier was used to analyze the 5-year survival of patients. Cell proliferation was assessed via clone formation assay. Cell cycle and apoptosis were detected by flow cytometry. Wound healing and Transwell assays were used to detect cell migration and invasion ability. The targeting relationship between miR-515-5p and circ_0000670 or SIX1 was confirmed by the dual luciferase reporter assay. Nude mice were injected into HGC-27 cells transfected with sh-NC or sh-circ_0000670, and the volume and weight of the transplanted tumor were measured, also, the levels of circ_0000670, miR-515-5p and SIX1 in the transplanted tumor tissue were detected. Results: The expression levels of circ_0000670 and SIX1 in gastric cancer tissues and cell lines were significantly increased (P<0.05), while the expression levels of miR-515-5p were significantly decreased (P<0.05). The survival rate of patients in the low circ_0000670 expression group (82.4%) was significantly higher than that in the high circ_0000670 expression group (28.7%, P=0.034). Circ_0000670 was negatively correlated with miR-515-5p (r=-0.846, P<0.001), and miR-515-5p was negatively correlated with SIX1 (r=-0.615, P<0.001), but circ_0000670 was positively correlated with SIX1 (r=0.814, P<0.001). Transfection of si-circ_0000670 or miR-515-5p mimic could significantly reduce the number of clone-forming cells, migration distance, migration and invasion cells (P<0.05), and increase the ratio of G(0)/G(1) phase cells, apoptosis rate and the protein level of E-cadherin (P<0.05), decreased the proportion of S-phase cells and the protein level of Vimentin (P<0.05). The dual luciferase report assay confirmed that circ_0000670 could target miR-515-5p, and miR-515-5p could bind to SIX1. Co-transfection of si-circ_0000670 and miR-515-5p inhibitor could significantly attenuate the effects of si-circ_0000670 on cell proliferation, migration, invasion, cell cycle and apoptosis (P<0.05). Co-transfection of miR-515-5p mimic and pcDNA-SIX1 could significantly reduce the effects of miR-515-5p mimic on cell proliferation, migration, invasion, cell cycle and apoptosis (P<0.05). Compared with the sh-NC group [volume=(596.20±125.46) mm(3) and weight=(538.00±114.39) g], the volume and weight of transplanted tumors in the sh-circ_0000670 group [volume=(299.20±47.58) mm 3 and weight=(289.80±48.73 g)] were significantly reduced (P<0.05), the expression levels of circ_0000670 and SIX1 were significantly reduced (P<0.05), and the expression level of miR-515-5p was significantly increased (P<0.05). Conclusion: Knockdown of circ_0000670 could inhibit cell proliferation, migration, invasion of gastric cancer cells, induce cell cycle arrest in G(0)/G(1) phase and promote cell apoptosis by regulating the miR-515-5p/SIX1 axis.

Circ_0040929 Serves as Promising Biomarker and Potential Target for Chronic Obstructive Pulmonary Disease.

Background: Circular RNAs (circRNAs) can act as essential regulators in many diseases, including chronic obstructive pulmonary disease (COPD). We aimed to explore the role and underlying mechanism of circ_0040929 in COPD. Methods: A cellular model of COPD was constructed by treating human bronchial epithelial cells (16HBE) with cigarette smoke extract (CSE). The levels of circ_0040929, microRNA-515-5p (miR-515-5p) and insulin-like growth factor-binding protein 3 (IGFBP3) were measured by quantitative real-time PCR. Cell proliferation was assessed by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine assays. Cell apoptosis was evaluated by flow cytometry. Protein expression was measured using Western blot assay. The levels of inflammatory factors and airway remodeling were assayed via enzyme-linked immunosorbent assay. The interaction between miR-515-5p and circ_0040929/IGFBP3 was confirmed by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. Exosomes were detected using transmission electron microscopy. Results: Circ_0040929 expression and IGFBP3 expression were upregulated in the serum of smokers (n = 22) compared to non-smokers (n = 22) and more significantly upregulated in the serum of COPD patients (n = 22). However, miR-515-5p expression was decreased in the serum of smokers compared to non-smokers and further reduced in the serum of COPD. Circ_0040929 knockdown attenuated CSE-induced cell injury by increasing proliferation and reducing apoptosis, inflammation, and airway remodeling in 16HBE cells. MiR-515-5p was a direct target of circ_0040929, and miR-515-5p inhibition reversed the effect of circ_0040929 knockdown in CSE-treated 16HBE cells. IGFBP3 was a direct target of miR-515-5p, and miR-515-5p overexpression alleviated CSE-induced cell injury via targeting IGFBP3. Moreover, circ_0040929 regulated IGFBP3 expression by targeting miR-515-5p. Importantly, circ_0040929 was upregulated in serum exosomes from COPD patients. Conclusion: Circ_0040929 played a promoting role in CSE-induced COPD by regulating miR-515-5p/IGFBP3 axis, suggesting that it might be a novel potential target for COPD treatment.

LncRNA HOXA11-AS promotes vascular endothelial cell injury in atherosclerosis by regulating the miR-515-5p/ROCK1 axis.

AIMS: Long non-coding RNA HOXA11-AS participated in heart disease. In this study, we aim to evaluate the potential roles of HOXA11-AS in atherosclerosis and its underlying mechanisms. METHODS AND RESULTS: The expression levels of HOXA11-AS in ox-LDL-treated HUVECs and arch tissues of high-fat diet-fed ApoE-/- mice (n = 10) were assessed by qRT-PCR. The effects of HOXA11-AS knockdown on the development of atherosclerosis were evaluated using in vitro and in vivo models. Luciferase reporter and RNA immunoprecipitation (RIP) assays verified the potential relationships between HOXA11-AS or ROCK1 and miR-515-5p. The interactive roles between HOXA11-AS and miR-515-5p and between miR-515-5p and ROCK1 were further characterized in ox-LDL-treated HUVECs. Our data showed that HOXA11-AS was significantly up-regulated (P < 0.001), whereas miR-515-5p was dramatically down-regulated in AS mice tissues (P < 0.001) and ox-LDL-treated HUVECs (P < 0.01). Ox-LDL could induce endothelial injuries by inhibiting cell proliferation (P < 0.001) and SOD synthesis (P < 0.001), promoting apoptosis (P < 0.01), ROS (P < 0.001), and MDA production (P < 0.001), increasing Bax (P < 0.001) and cleaved Caspase-3 (P < 0.001), and decreasing Bcl-2 (P < 0.001) and phosphorylated eNOS (P < 0.01). HOXA11-AS knockdown attenuated endothelial injuries via increasing eNOS phosphorylation. Luciferase assay and RIP results confirmed that miR-515-5p is directly bound to HOXA11-AS and ROCK1. HOXA11-AS promoted ox-LDL-induced HUVECs injury by directly inhibiting miR-515-5p from increasing ROCK1 expression and subsequently decreasing the expression and phosphorylation of eNOS. MiR-515-5p mimics could partially reverse the effects of HOXA11-AS knockdown. CONCLUSIONS: HOXA11-AS contributed to atherosclerotic injuries by directly regulating the miR-515-5p/ROCK1 axis. This study provided new evidence that HOXA11-AS might be a candidate for atherosclerosis therapy.

Identification of the regulatory role of the circ_0004788/miR-515-5p/FGF2 network in nasopharyngeal carcinoma development.

BACKGROUND: CircularRNAs (circRNAs) played vital roles in nasopharyngeal carcinoma (NPC). However, the impacts of circ_0004788 on the development of NPC have not been explored. METHODS: Cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU) and colony formation assays were applied to determine cell proliferation. Wound healing, transwell invasion assay, tube formation assay, and flow cytometry were employed for the detection of cell migration, invasion, angiogenesis, and apoptosis, respectively. Xenograft tumor experiment was used to explore the biological role of circ_0004788 in NPC in vivo. RESULTS: Circ_0004788 and fibroblast growth factor 2 (FGF2) were significantly elevated, and microRNA-515-5p (miR-515-5p) was dramatically decreased in NPC tissues and cells. The impacts of circ_0004788 knockdown on cell progression in NPC cells were reversed by miR-515-5p inhibitor, and FGF2 overexpression could block the suppressive effect of miR-515-5p on cell progression in NPC cells. CONCLUSION: Circ_0004788 knockdown restrained the progression of NPC via the miR-515-5p/FGF2 axis.

Long non-coding RNA CATIP antisense RNA 1 (lncRNA CATIP-AS1) downregulation contributes to the progression and metastasis of thyroid cancer via epithelial-mesenchymal transition (EMT) pathway.

Thyroid cancer (THCA) is the most common cancer of the endocrine system across the globe. To date, the mechanism of development of THCA remains scarcely known. In this study, we aim to elucidate the long non-coding RNA CATIP antisense RNA 1 (lncRNA CATIP-AS1/CATIP-AS1) role in the pathogenesis of THCA and its regulatory mechanism. The result shows that the CATIP-AS1 was significantly downregulated in THCA tissues and cells and was associated with a poor prognosis of patients diagnosed with THCA. The overexpression of CATIP-AS1 significantly inhibited THCA cell proliferation, migration, and epithelial-mesenchymal transition (EMT) but increased the THCA cell apoptosis. We found that CATIP-AS1 endogenously sponges miR-515-5p and its overexpression could inhibit miR-515-5p regulatory effect. Moreover, the overexpression of miR-515-5p repressed the Smad4 expression level, consequently reversed the inhibiting effect of overexpressed CATIP-AS1 on the proliferation, and migration of THCA cell. It also reversed the increased THCA cell apoptosis and the downregulated-CATIP-AS1-induced cell EMT inhibition. Summarily, we demonstrated that the CATIP-AS1 promotes the progression and metastasis of THCA via EMT pathway partly through regulating the miR-515-5p and Smad4 expression in THCA cell. The CATIP-AS1 could be a promising biomarker for early THCA detection and prognosis and a possible therapeutic target for its treatment.

Targeting of MAD2L1 by miR-515-5p involves the regulation of cell cycle arrest and apoptosis of colorectal cancer cells.

Although many previous studies have found that the mitotic arrest deficient 2-like 1 (MAD2L1) protein contributes to the proliferation of colorectal cancer (CRC) cells, but the upstream mechanism of MAD2L1 is still largely elusive. This study aimed to explore the microRNAs (miRNAs) upstream of MAD2L1 to improve our understanding of the mechanism of the MAD2L1 gene in CRC. The upstream target miRNAs (miR-515-5p) of MAD2L1 were predicted by the online databases miRWalk, miRDIP, and TargetScan. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of miR-515-5p in human CRC tissues. The targeting relationship between miR-515-5p and MAD2L1 was tested by dual luciferase reporter gene assays. The effects of miR-515-5p on the biological behaviors of CRC cells by regulating MAD2L1 expression were verified by qRT-PCR, western blot, Cell Counting Kit-8, and flow cytometry. The results showed that miR-515-5p was a highly reliable upstream miRNA of the MAD2L1 gene. As an upstream target miRNA of MAD2L1, miR-515-5p was lowly expression in CRC tissues. The overexpression of miR-515-5p could inhibit the proliferation of CRC cells and induce cell cycle arrest at the G1 phase leading to cell apoptosis. However, MAD2L1 gene overexpression could reverse the effects of miR-515-5p overexpression on the biological behaviors of CRC cells above. This study illustrated that miR-515-5p can inhibit proliferation and induce G1 phase arrest leading to apoptosis in CRC cells. The mechanism underlying this phenomenon may be related to the negative targeted regulation of MAD2L1.

Circular RNA circ-LARP1B contributes to cutaneous squamous cell carcinoma progression by targeting microRNA-515-5p/TPX2 microtubule nucleation factor axis.

Circular RNAs (circRNAs) have shown pivotal regulatory roles in tumorigenesis and progression. Our purpose was to analyze the role of circRNA La ribonucleoprotein 1B (circ-LARP1B; hsa_circ_0070934) in cutaneous squamous cell carcinoma (CSCC) progression and its associated mechanism. Cell viability, colony formation ability, migration, and invasion were analyzed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay, colony formation assay, wound healing assay, and transwell invasion assay. Flow cytometry was performed to analyze cell apoptosis and cell cycle progression. Cell glycolytic metabolism was analyzed using Glucose Uptake Colorimetric Assay kit, Lactate Assay Kit II, and ATP colorimetric Assay kit. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were performed to verify the interaction between microRNA-515-5p (miR-515-5p) and circ-LARP1B or TPX2 microtubule nucleation factor (TPX2). Circ-LARP1B expression was up-regulated in CSCC tissues and cell lines. Circ-LARP1B knockdown suppressed cell viability, colony formation ability, migration, invasion, cell cycle progression, and glycolysis and triggered cell apoptosis in CSCC cells. miR-515-5p was a direct target of circ-LARP1B in CSCC cells, and circ-LARP1B silencing-mediated anti-tumor effects were largely counteracted by miR-515-5p knockdown. miR-515-5p directly interacted with the 3' untranslated region (3'UTR) of TPX2. TPX2 overexpression largely overturned miR-515-5p-mediated anti-tumor effects in CSCC cells. Circ-LARP1B could up-regulate TPX2 expression by sponging miR-515-5p in CSCC cells. Circ-LARP1B knockdown suppressed tumor growth in vivo. In conclusion, circ-LARP1B contributed to CSCC progression by targeting miR-515-5p/TPX2 axis. The circ-LARP1B/miR-515-5p/TPX2 axis might provide novel therapeutic targets for CSCC patients.

The effects of circFBXL5 on the malignant biological behaviors of bladder cancer T24 cells by targeting miR-515-5p and its molecular mechanisms / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨circFBXL5通过靶向miR-515-5p影响膀胱癌T24细胞的增殖、迁移、侵袭及其分子机制。方法： 收集2020年4月至2020年6月间在苏州市中西医结合医院手术切除的41例膀胱癌组织及其癌旁组织，采用qPCR法检测circFBXL5、miR-515-5p的表达；双荧光素酶报告实验验证circFBXL5与miR-515-5p之间的靶向关系，体外培养人膀胱癌T24细胞，实验分为si-NC组、si-circFBXL5组、anti-miR-NC+si-circFBXL5组和si-circFBXL5+anti-miR-515-5p组；MTT法、细胞克隆形成实验、FCM、Transwell实验和WB法分别检测转染后T24细胞的增殖、细胞克隆形成、迁移、侵袭和凋亡及BAX、Bcl-2蛋白水平。结果：膀胱癌组织中circFBXL5呈高表达，miR-515-5p呈低表达（均P<0.05）；circFBXL5靶向且负向调控miR-515-5p的表达；敲减circFBXL5后T24细胞的增殖抑制率、凋亡率和BAX蛋白水平均显著增高（均P<0.05），细胞克隆形成数和迁移、侵袭细胞数均显著减少（均P<0.05），Bcl-2蛋白水平显著降低（P<0.05）；同时敲减circFBXL5和miR-515-5p可部分逆转敲减circFBXL5对T24细胞增殖的抑制作用。结论：circFBXL5通过调控miR-515-5p表达影响膀胱癌T24细胞的增殖、迁移、侵袭，circFBXL5和miR-515-5p可能膀胱癌治疗的潜在分子靶标。