Circ-Ntrk2 acts as a miR-296-5p sponge to activate the TGF-ß1/p38 MAPK pathway and promote pulmonary hypertension and vascular remodelling.

BACKGROUND: Circular RNAs (circRNAs), a novel class of non-coding RNAs, play an important regulatory role in pulmonary arterial hypertension (PAH); however, the specific mechanism is rarely studied. In this study, we aimed to discover functional circRNAs and investigate their effects and mechanisms in hypoxia-induced pulmonary vascular remodelling, a core pathological change in PAH. METHODS: RNA sequencing was used to illustrate the expression profile of circRNAs in hypoxic PAH. Bioinformatics, Sanger sequencing, and quantitative real-time PCR were used to identify the ring-forming characteristics of RNA and analyse its expression. Then, we established a hypoxia-induced PAH mouse model to evaluate circRNA function in PAH by echocardiography and hemodynamic measurements. Moreover, microRNA target gene database screening, fluorescence in situ hybridisation, luciferase reporter gene detection, and western blotting were used to explore the mechanism of circRNAs. RESULTS: RNA sequencing identified 432 differentially expressed circRNAs in mouse hypoxic lung tissues. Our results indicated that circ-Ntrk2 is a stable cytoplasmic circRNA derived from Ntrk2 mRNA and frequently upregulated in hypoxic lung tissue. We further found that circ-Ntrk2 sponges miR-296-5p and miR-296-5p can bind to the 3'-untranslated region of transforming growth factor-ß1 (TGF-ß1) mRNA, thereby attenuating TGF-ß1 translation. Through gene knockout or exogenous expression, we demonstrated that circ-Ntrk2 could promote PAH and vascular remodelling. Moreover, we verified that miR-296-5p overexpression alleviated pulmonary vascular remodelling and improved PAH through the TGF-ß1/p38 MAPK pathway. CONCLUSIONS: We identified a new circRNA (circ-Ntrk2) and explored its function and mechanism in PAH, thereby establishing potential targets for the diagnosis and treatment of PAH. Furthermore, our study contributes to the understanding of circRNA in relation to PAH.

Circ_0071589 contributes to growth, angiogenesis, and metastasis of colorectal cancer through regulating miR-296-5p/EN2 axis.

To explore the function and regulation mechanism of circ_0071589 in colorectal cancer (CRC). The expression levels of circ_0071589, microRNA-296-5p (miR-296-5p), and Engrailed-2 (EN2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was performed to check the protein levels of EN2 and apoptosis-related proteins. Cell colony formation and 5-Ethynyl-29-deoxyuridine (EdU) assay were used to exhibit cell proliferation. Cell apoptosis was shown by flow cytometry. Tube formation assay manifested the angiogenesis ability of CRC cells. Transwell assay demonstrated cell migration and invasion. The interaction between miR-296-5p and circ_0071589 or EN2 was identified by dual-luciferase reporter assay. The effect of circ_0071589 on tumor formation was demonstrated by in vivo tumor formation experiments. Immunohistochemical (IHC) assay was used to detect the positive cell rate of Ki67 in tumor tissue. Circ_0071589 was upregulated in CRC tissue and cells. Circ_0071589 knockdown repressed CRC cells proliferation, angiogenesis, migration, invasion, and promoted cell apoptosis. MiR-296-5p was downregulated in CRC tissue and cells. And miR-296-5p inhibitor could reverse the malignant phenotypes and angiogenesis inhibition of CRC cells caused by circ_0071589 knockdown. Additionally, miR-296-5p decreased CRC cell colony formation, EdU-positive cells, angiogenesis, and increased cell apoptosis through reducing the expression level of EN2. Finally, circ_0071589 silencing inhibited tumor formation in vivo. Circ_0071589 upregulated EN2 expression through sponging miR-296-5p, thereby promoting the malignant phenotype and angiogenesis of CRC cells, which provided a new target for the treatment of CRC.

miR-296-5p promotes autophagy in mouse LS8 cells under excessive fluoride via AMPK/ULK1 pathways.

Numerous microRNAs participate in regulating the pathological process of autophagy. We have found miR-296-5p is one of the most significantly down-regulated microRNAs in a high concentration of sodium fluoride. However, it is not clear whether miR-296-5p augments autophagy in dental fluorosis. Our purpose is to explore the function of miR-296-5p in regulating autophagy of excessive fluoride development. Thus, the cell line of ameloblasts LS8 was exposed to a 1.5 mM dose of NaF and miR-296-5p-mimics, Real-time qPCR, CCK-8 assays, Fluorescence imaging and Western blot analysis were performed. Autophagy was observed. As our results indicated, miR-296-5p overexpression in mouse LS8 cells significantly accelerated autophagy. The autophagy inhibition effect of miR-296-5p underexpression was consistent with the effect of the AMPK inhibitor. And we found that the expression of LC3II was decreased via down-regulation of AMPK. The change of ULK1 by miR-296-5p may be accomplished through AMPK. Thus, miR-296-5p may improve the secretion of autophagic mediators by activating AMPK/ULK1 expression in fluorosis, suggesting that miR-296-5p, AMPK/ULK1 may be potential therapeutic targets under the higher fluoride stimulation.

SP1-induced lncRNA FOXD3-AS1 contributes to tumorigenesis of cervical cancer by modulating the miR-296-5p/HMGA1 pathway.

Long noncoding RNAs (lncRNAs) have drawn growing attention due to their regulatory roles in various diseases, including tumors. Recently, lncRNA FOXD3 antisense RNA 1 (FOXD3-AS1) was shown to be overexpressed in colon adenocarcinoma and glioma, exerting oncogenic functions. However, its expression and effects in cervical cancer (CC) remained unknown. In this research, our group first reported that the levels of FOXD3-AS1 were distinctly elevated in CC samples and cell lines. The distinct upregulation of FOXD3-AS1 was associated with lymphatic invasion, distant metastasis, and International Federation of Gynecology and Obstetrics stage, and also predicted poor clinical results of CC patients. Next, transcription factor SP1 was demonstrated to resulting in the upregulation of FOXD3-AS1 in CC. Functional assays indicated that knockdown of FOXD3-AS1 distinctly suppressed CC progression via affecting cell proliferation, cell apoptosis, and metastasis. Moreover, mechanistic studies suggested that FOXD3-AS1 acted as an endogenous sponge by directly binding miR-296-5p, resulting in the suppression of miR-296-5p. In addition, we also reported that high mobility group A, a direct target of miR-296-5p, could mediate the tumor-promotive effects that FOXD3-AS1 displayed. Overall, our present study might help to lead a better understanding of the pathogenesis of CC, provide a novel possible tumor biomarker, and probe the feasibility of lncRNA-directed treatments for CC.

Circular RNA hsa_circ_001895 serves as a sponge of microRNA-296-5p to promote clear cell renal cell carcinoma progression by regulating SOX12.

There is an urgent need to find novel potential therapeutic targets for the diagnosis and treatment of clear cell renal cell carcinoma (ccRCC) due to its highly invasive ability as a common urological malignant tumor. Circular RNAs (circRNAs) have been indicated as potentially critical mediators in various types of tumor progression. We first used qRT-PCR analysis to find dysregulated circRNAs in ccRCC. A novel circRNA, hsa_circ_001895, was upregulated in ccRCC specimens and associated with metastatic properties of ccRCC. However, the tumorigenic mechanism of hsa_circ_001895 on ccRCC is yet to be found. We first indicated that hsa_circ_001895 predicted a poor prognosis in ccRCC patients. Additionally, overexpression of hsa_circ_001895 not only promoted cell proliferation, invasion and migration of ccRCC, but also inhibited cell apoptosis, whereas hsa_circ_001895 knockdown reversed the effect on ccRCC progression. In vivo s.c. xenotransplanted tumor model also showed that silencing hsa_circ_001895 could suppress in vivo ccRCC growth. Mechanistically, hsa_circ_001895 directly binds with microRNA (miR)-296-5p and inhibits its expression. Moreover, sex determining region Y (SRY)-box 12 (SOX12) was identified as a target of miR-296-5p, the expression of which was suppressed by miR-296-5p. Notably, the inhibitory effect of hsa_circ_001895 on ccRCC progression was reversed by miR-296-5p inhibitor. In general, our findings indicated that hsa_circ_001895 may sponge miR-296-5p and promote SOX12 expression, which is the underlying mechanism of hsa_circ_001895-induced ccRCC progression.

MicroRNA-296-5p inhibits cell metastasis and invasion in nasopharyngeal carcinoma by reversing transforming growth factor-ß-induced epithelial-mesenchymal transition.

AIM: To explore the effect of miR-296-5p on the metastasis of nasopharyngeal carcinoma (NPC) cells and investigate the underlying mechanism. METHODS: The expressions of miR-296-5p in NPC tissues and cells were determined using GSE32920 database analysis and real-time PCR and miRNA microarray assays. An miR-296-5p mimic and inhibitor were transfected into NPC cells. Then, immunofluorescence imaging, scratch wound-healing, transwell migration and invasion assays were used to observe the effects of miR-296-5p on cell metastasis and invasion. Real-time PCR and western blotting were carried out to detect the expressions of genes and proteins related to epithelial-mesenchymal transition (EMT). A dual luciferase reporter assay was used to identify whether TGF-ß is the target gene of miR-296-5p. Finally, TGF-ß expression plasmids were transfected into NPC cells to verify the role of TGF-ß in the miR-296-5p-mediated inhibition of nasopharyngeal carcinoma cell metastasis. RESULTS: Our results show that miR-296-5p inhibits the migratory and invasive capacities of NPC cells by targeting TGF-ß, which suppresses EMT. Importantly, the miR-296-5p level was significantly lower in human NPC tissues than in adjacent normal tissues. It also negatively correlated with TGF-ß and was significantly associated with the lymph node metastasis of patients with NPC. CONCLUSIONS: Our findings show that miR-296-5p represses the EMT-related metastasis of NPC by targeting TGF-ß. This provides new insight into the role of miR-296-5p in regulating NPC metastasis and invasiveness.

CircPSMC3 suppresses the proliferation and metastasis of gastric cancer by acting as a competitive endogenous RNA through sponging miR-296-5p.

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs with a loop structure, but its functions remain largely unknown. Growing evidence has revealed that circRNAs play a striking role as functional RNAs in the progression of malignant disease. However, the precise role of circRNAs in gastric cancer (GC) remains unclear. METHODS: CircRNAs were determined by human circRNA array analysis and quantitative reverse transcription polymerase reaction. Luciferase reporter, RNA pull down, and fluorescence in situ hybridization assays were employed to test the interaction between circPSMC3 and miR-296-5p. Ectopic over-expression and siRNA-mediated knockdown of circPSMC3, proliferation, migration and invasion in vitro, and in vivo experiment of metastasis were used to evaluate the function of circPSMC3. RESULTS: CircPSMC3 rather than liner PSMC3 mRNA was down-regulated in GC tissues, corresponding plasmas from GC patients as well as GC cell lines compared to normal controls. Lower circPSMC3 expression in GC patients was correlated with higher TNM stage and shorter overall survival. Over-expression of circPSMC3 and miR-296-5p inhibitor could inhibit the tumorigenesis of gastric cancer cells in vivo and vitro whereas co-transfection of circPSMC3 and miRNA-296-5p could counteract this effect. Importantly, we demonstrated that circPSMC3 could act as a sponge of miR-296-5p to regulate the expression of Phosphatase and Tensin Homolog (PTEN), and further suppress the tumorigenesis of gastric cancer cells. CONCLUSION: Our study reveals that circPSMC3 can serve as a novel potential circulating biomarker for detection of GC. CircPSMC3 participates in progression of gastric cancer by sponging miRNA-296-5p with PTEN, providing a new insight into the treatment of gastric cancer.

MicroRNA-296-5p promotes healing of diabetic wound by targeting sodium-glucose transporter 2 (SGLT2).

BACKGROUND: Diabetic wounds are refractory and very difficult to heal. We aimed to use miRNA to identify novel and specific molecular markers for diabetes mellitus (DM) diagnosis and treatment. METHODS: The expression level of miR-296-5p was determined in tissue samples of 12 DM patients. The effect of miR-296-5p on proliferation of ß-cells was examined using Cell Counting Kit-8 (CCK-8) and colony formation assay. The effect of miR-296-5p on cell cycle progression was analysed using flow cytometry. The target gene was verified using luciferase reporter assay. A rat diabetes model was used to assess the effect of miR-296-5p in vivo. RESULTS: Overexpression of miR-296-5p suppressed cell proliferation, arrested cell cycle progression, and increased the healing rate of diabetic wounds both in vivo and in vitro. TargetScan analysis results showed that miR-296-5p is a direct regulator of SGLT2. CONCLUSIONS: miR-296-5p can increase the healing rate of diabetic wounds and may be an effective molecular tool in DM diagnosis and therapy.

Targeting of multiple oncogenic signaling pathways by Hsp90 inhibitor alone or in combination with berberine for treatment of colorectal cancer.

There is a wide range of drugs and combinations under investigation and/or approved over the last decade to treat colorectal cancer (CRC), but the 5-year survival rate remains poor at stages II-IV. Therefore, new, more-efficient drugs still need to be developed that will hopefully be included in first-line therapy or overcome resistance when it appears, as part of second- or third-line treatments in the near future. In this study, we revealed that heat shock protein 90 (Hsp90) inhibitors have high therapeutic potential in CRC according to combinative analysis of NCBI's Gene Expression Omnibus (GEO) repository and chemical genomic database of Connectivity Map (CMap). We found that second generation Hsp90 inhibitor, NVP-AUY922, significantly downregulated the activities of a broad spectrum of kinases involved in regulating cell growth arrest and death of NVP-AUY922-sensitive CRC cells. To overcome NVP-AUY922-induced upregulation of survivin expression which causes drug insensitivity, we found that combining berberine (BBR), a herbal medicine with potency in inhibiting survivin expression, with NVP-AUY922 resulted in synergistic antiproliferative effects for NVP-AUY922-sensitive and -insensitive CRC cells. Furthermore, we demonstrated that treatment of NVP-AUY922-insensitive CRC cells with the combination of NVP-AUY922 and BBR caused cell growth arrest through inhibiting CDK4 expression and induction of microRNA-296-5p (miR-296-5p)-mediated suppression of Pin1-ß-catenin-cyclin D1 signaling pathway. Finally, we found that the expression level of Hsp90 in tumor tissues of CRC was positively correlated with CDK4 and Pin1 expression levels. Taken together, these results indicate that combination of NVP-AUY922 and BBR therapy can inhibit multiple oncogenic signaling pathways of CRC.

MicroRNA-296-5p (miR-296-5p) functions as a tumor suppressor in prostate cancer by directly targeting Pin1.

Upregulation of Pin1 was shown to advance the functioning of several oncogenic pathways. It was recently shown that Pin1 is potentially an excellent prognostic marker and can also serve as a novel therapeutic target for prostate cancer. However, the molecular mechanism of Pin1 overexpression in prostate cancer is still unclear. In the present study, we showed that the mRNA expression levels of Pin1 were not correlated with Pin1 protein levels in prostate cell lines which indicated that Pin1 may be regulated at the post-transcriptional level. A key player in post-transcriptional regulation is represented by microRNAs (miRNAs) that negatively regulate expressions of protein-coding genes at the post-transcriptional level. A bioinformatics analysis revealed that miR-296-5p has a conserved binding site in the Pin1 3'-untranslated region (UTR). A luciferase reporter assay demonstrated that the seed region of miR-296-5p directly interacts with the 3'-UTR of Pin1 mRNA. Moreover, miR-296-5p expression was found to be inversely correlated with Pin1 expression in prostate cancer cell lines and prostate cancer tissues. Furthermore, restoration of miR-296-5p or the knockdown of Pin1 had the same effect on the inhibition of the ability of cell proliferation and anchorage-independent growth of prostate cancer cell lines. Our results support miR-296-5p playing a tumor-suppressive role by targeting Pin1 and implicate potential effects of miR-296-5p on the prognosis and clinical application to prostate cancer therapy.

Diagnostic and Prognostic Value of Serum miR-296-5p and miR-28-3p in Human Gastric Cancer.

Background: Previous studies reported the use of microRNAs (miRNAs) as diagnostic and/or prognostic biomarkers for various cancers, including gastric cancer (GC). This study evaluated the diagnostic and prognostic significance of serum miR-296-5p and miR-28-3p in GC. Materials and Methods: Serum samples of 90 patients with GC and 90 healthy individuals, and 20 pairs of tissue specimens from patients with GC were collected. The expression of miR-296-5p and miR-28-3p in both the serum and tissue samples were detected using quantitative real-time polymerase chain reaction analysis. The diagnostic and prognostic values of miR-296-5p and miR-28-3p were evaluated by using receiver operating characteristic curve and Kaplan-Meier analyses, respectively. Results: Compared with the healthy controls, the expression of miR-296-5p in the serum and tissues of patients with GC was significantly upregulated, whereas that of miR-28-3p was significantly downregulated. High miR-296-5p and low miR-28-3p levels in the serum significantly correlated with larger tumor size (>5 cm), lymph node metastasis, and TNM stage III+IV. The area under the curve values of miR-296-5p and miR-28-3p were 0.919 and 0.911, respectively, with high sensitivity and specificity. Kaplan-Meier survival curves showed that patients with GC with high level of miR-296-5p or low level of miR-1236-3p in the serum had the poorest overall survival. COX analysis showed that lymphatic metastasis, high miR-296-5p expression, and low miR-28-3p expression are independent parameters indicating poor prognosis in GC. Conclusion: Our findings indicate that serum miR-296-5p and miR-28-3p levels are potential biomarkers in the diagnosis and prognosis of GC.

Unveiling the role of miR-137-3p/miR-296-5p/SERPINA3 signaling in colorectal cancer progression: integrative analysis of gene expression profiles and in vitro studies.

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy worldwide, with increasing incidence and mortality rates. Although treatment options have improved, CRC remains a leading cause of death due to metastasis. Early intervention can significantly improve patient outcomes, making it crucial to understand the molecular mechanisms underlying CRC metastasis. In this study, we performed bioinformatics analysis to identify potential genes associated with CRC metastasis. METHODS: We downloaded and integrated gene expression datasets (GSE89393, GSE100243, and GSE144259) from GEO database. Differential expression analysis was conducted, followed by Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The hub gene SERPINA3 was selected for further in vitro functional studies. Additionally, the role of miR-137-3p/miR-296-5p/ Serpin family A member 3 (SERPINA3) in CRC cell function was investigated using in vitro assays. RESULTS: Analysis of the gene expression datasets revealed differentially expressed genes (DEGs) associated with CRC metastasis. GO analysis showed enrichment in biological processes such as blood coagulation regulation and wound healing. Cellular component analysis highlighted extracellular matrix components and secretory granules. Molecular function analysis identified activities such as serine-type endopeptidase inhibition and lipoprotein receptor binding. KEGG analysis revealed involvement in pathways related to complement and coagulation cascades, cholesterol metabolism, and immune responses. The common DEGs among the datasets were further investigated. We identified SERPINA3 as a hub gene associated with CRC metastasis. SERPINA3 exerted enhanced effects on migration, proliferation and epithelial-mesenchymal transition (EMT) and inhibitory effects on caspase-3/-9 activities in HT29 and SW620 cells. MiR-137-3p overexpression increased activities of caspase-3/-9, decreased migration and proliferation, and also repressed EMT in HT29 cells, which were obviously attenuated by SERPINA3 enforced overexpression. Consistently, SERPINA3 enforced overexpression also largely reversed miR-296-5p mimics-induced increased in activities of caspase-3/-9, decrease in migration, proliferation and EMT in HT29 cells. CONCLUSION: Through bioinformatics analysis, we identified potential genes associated with CRC metastasis. The functional studies focusing on SERPINA3/miR-137-3p/miR-296-5p further consolidated its role in regulating CRC progression. Our findings provide insights into novel mechanisms underlying CRC metastasis and might contribute to the development of effective treatment strategies. However, the role of SERPINA3/miR-137-3p/miR-296-5p signaling in CRC still requires further investigation.

Whole-transcriptome sequencing with ceRNA regulation network construction and verification in glioblastoma.

OBJECTIVES: To explore the key genes involved in the occurrence and development of glioblastoma (GBM) by analyzing whole-transcriptome sequencing and biologic data from GBM and normal cerebral cortex tissues and to search for important noncoding RNA (ncRNA) molecular markers based on the competitive endogenous RNA (ceRNA) network. METHODS: Ten GBM and normal cerebral cortex tissues were collected for full transcriptome sequencing, screened for differentially expressed (DE) mRNAs, miRNAs, lncRNAs, and circRNAs, and subjected to bioinformatic analysis. We constructed a Protein-Protein Interaction (PPI) network and a circRNA/lncRNA-miRNA-mRNA regulatory network and identified them using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases were used to validate and conduct a survival analysis of the target genes. RESULTS: A total of 5341 DEmRNAs, 259 DEmiRNAs, 3122 DElncRNAs, and 2135 DEcircRNAs were identified. Enrichment analysis showed that target genes regulated by DEmiRNA, DElncRNA, and DEcircRNA were closely related to chemical synaptic transmission and ion transmembrane transport. A PPI network analysis screened 10 hub genes that directly participate in tumor cell mitosis regulation. In addition, the ceRNA composite network showed that hsa-miR-296-5p and hsa-miR-874-5p were the central nodes of the network, and the reliability of relevant key molecules was successfully verified through RT-qPCR identification and the TCGA database. The CGGA database survival analysis produced 8 DEmRNAs closely related to GBM patient survival prognosis. CONCLUSIONS: This study revealed the important regulatory functions and molecular mechanisms of ncRNA molecules and identified hsa-miR-296-5p and hsa-miR-874-5p as key molecules in the ceRNA network. They may play an important role in GBM pathogenesis, treatment, and prognosis.

Circ_0000514 promotes breast cancer progression by regulating the miR-296-5p/CXCL10 axis.

The biological function of circular RNA 0000514 (circ_0000514) in breast cancer (bc) is still unknown. In this study, we downloaded the microarray dataset GSE101123 from Gene Expression Omnibus database and then analysed the differentially expressed circular RNAs in bc tissues compared with adjacent tissues, and we demonstrated that circ_0000514 was up-regulated in bc tissues. Circ_0000514, miR-296-5p and CXC chemokine ligand10 (CXCL10) expressions in bc tissues and cell lines were probed by quantitativereal-time polymerase chain reaction and Western blot. Cell counting kit-8, 5-bromo-2'-deoxyuridine and transwell assays were adopted to determine the cell viability, proliferation, migration and invasion. The targeting relationship between miR-296-5p and circ_0000514 or CXCL10 3'-UTR was predicted by bioinformatics and validated by dual-luciferase reporter assay. We demonstrated that circ_0000514 and CXCL10 expressions were raised in bc tissues and cell lines while miR-296-5p expression was declined. Circ_0000514 knockdown could inhibit the proliferation, migration and invasion of bc cells and miR-296-5p overexpression also suppressed the malignant phenotypes of bc cells. Mechanistically, miR-296-5p was identified as the downstream target of circ_0000514 and could be inhibited by circ_0000514. Moreover, CXCL10 was the target of miR-296-5p, whose expression could be indirectly and positively regulated by circ_0000514. In conclusion, circ_0000514 is involved in bc progression via regulating miR-296-5p/CXCL10 axis. Graphical Abstract.

Hsa_circRNA_102541 regulates the development of atherosclerosis by targeting miR-296-5p/PLK1 pathway.

BACKGROUND: Cardiovascular disorders pose great threat to public health. As a common type of cardiovascular disease, atherosclerosis is characterized by high morbidity and mortality/recurrence rate. However, the pathogenesis of atherosclerosis is complex and not fully understood. The aim of this study was to investigate the influences of hsa_circRNA_102541 (circ_102541) on proliferation and apoptosis of HUVEC cells and to identify the underlying mechanisms. METHODS: RT-PCR was used to determine the expression levels of circ_102541, miR-296-5p, and PLK1 in atherosclerosis and healthy blood samples. Following the transfection with sh-circ_102541, LV-circ_102541, miR-296-5p mimics, miR-296-5p inhibitors, and si-PLK1, cell proliferation was evaluated using CCK8 assay; cell apoptosis was determined by flow cytometry; dual luciferase assay was performed to examine the interaction between abovementioned molecules. The levels of associated markers including PCNA and caspase-3 were assessed by western blotting and RT-qPCR. RESULTS: The expression of circRNA_102541 and PLK1 were significantly elevated in atherosclerosis specimens, where the level of miR-296-5p was reduced. Furthermore, circRNA_102541 could bind miR-296-5p and subsequently target PLK1. Following treatment with sh-circRNA_102541 or miR-296-5p mimics, proliferative ability and levels of PCNA were remarkably reduced in HUVEC cells, while apoptosis was significantly enhanced. Co-transfection with miR-296-5p mimics abrogated the effects induced by the overexpressed circ_102541. Additionally, treatment with si-PLK1 attenuated the biological behavior changes caused by miR-296-5p inhibitors in HUVEC cells. Moreover, transfection with LV-PLK1 reversed the effects triggered by miR-296-5p mimics. CONCLUSION: Taken together, circRNA_102541 was upregulated in atherosclerosis, and knockdown of circRNA_102541 suppressed cell proliferation while promoted apoptosis of HUVEC cells via miR-296-5p/PLK1. This novel pathway may serve essential roles on the development of atherosclerosis, and circRNA_102541 could be a promising therapeutic candidate for the treatment of atherosclerosis.

Aloperine inhibits colorectal cancer cell proliferation and metastasis progress via regulating miR-296-5p/STAT3 axis.

Anti-tumorous effect of Aloperine (ALO) has been previously found. This study examined the role and the underlying mechanism of ALO in colorectal cancer (CRC). CRC cells were processed by different concentrations of ALO, and subsequently the cell proliferation was detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and miR-296-5p expression was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Moreover, the target gene of miR-296-5p was predicted by TargetScan and confirmed by dual-luciferase reporter assay. The expressions of signal transducer and activator of transcription 3 (STAT3), apoptosis-related proteins and epithelial-mesenchymal transition (EMT)-related markers were measured by Western blot. Clone formation assay, flow cytometry, wound-healing and Transwell assays were respectively employed to detect cell proliferation, apoptosis, migration and invasion. ALO inhibited CRC cell proliferation in a dose-dependent manner. MiR-296-5p was low-expressed in CRC tissues and cells, and ALO promoted miR-296-5p expression. STAT3 was targeted by miR-296-5p. Up-regulation of miR-296-5p and ALO treatment both suppressed STAT3 expression, inhibited CRC cell proliferation, migration, invasion as well as the expressions of Bcl-2 and N-cadherin, but promoted apoptosis and expressions of Bax and E-cadherin, which were all reversed by overexpressed STAT3. ALO inhibited CRC cell proliferation, metastasis and EMT but promoted apoptosis via regulating miR-296-5p/STAT3 axis.

CircRPPH1 serves as a sponge for miR-296-5p to enhance progression of breast cancer by regulating FOXP4 expression.

Circular RNAs (circRNAs) have been demonstrated to play critical roles in the initiation and development of breast cancer (BC). This study aimed to uncover the regulatory roles of a novel circRNA, circRPPH1 (hsa_circ_0000514) in BC progression. CircRPPH1, miR-296-5p and FOXP4 levels were determined by qRT-PCR. CircRPPH1 stability was detected in response to ribonuclease (RNase) R digestion and actinomycin D treatment. Cell growth, migration and invasion were evaluated using various functional experiments. Protein levels of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 9 (MMP-9), hexokinase 2 (HK2) and forkhead box protein 4 (FOXP4) were measured by Western blotting. Metabolic alterations of BC cells were evaluated using commercial kits. The interaction between miR-296-5p and circRPPH1/FOXP4 was assessed using dual-luciferase assay, RNA pull-down, and RNA immunoprecipitation (RIP) assay. The in vivo tumorigenesis was assessed in nude mice. According to the results, up-regulation of circRPPH1 was closely correlated with the poor prognosis of BC patients. Functional experiments showed that knockdown of circRPPH1 repressed BC cell growth, migration, invasion, glycolysis, and in vivo tumor growth. In addition, circRPPH1 could sponge miR-296-5p to enhance FOXP4 expression in BC cells. miR-296-5p inhibition or FOXP4 overexpression restored the malignant properties of circRPPH1-silenced BC cells. Thus, circRPPH1 promoted BC malignant progression through regulating miR-296-5p/FOXP4 axis, indicating a possible novel therapeutic strategy involving circRNA for BC patients.

Circ_0067835 Knockdown Enhances the Radiosensitivity of Colorectal Cancer by miR-296-5p/IGF1R Axis.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant cancers globally. Circular RNAs (circRNAs) have been implicated in the development of CRC. In this paper, we set to explore the precise action of circ_0067835 in CRC progression and radioresistance. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to evaluate the expression of circ_0067835, microRNA-296-5p (miR-296-5p) and insulin-like growth factor 1 receptor (IGF1R). Western blot was used to measure the level of IGF1R protein. Cell proliferation, cell cycle distribution and apoptosis were determined by Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry and caspase-3 activity assays, respectively. The direct relationship between miR-296-5p and circ_0067835 or IGF1R was verified by dual-luciferase reporter assays. Additionally, in vivo assays were applied to confirm the role of circ_0067835 in vivo. RESULTS: Exosomal circ_0067835 was upregulated in the serum of CRC patients after radiotherapy. Exosome-mediated circ_0067835 knockdown repressed cell proliferation, cell cycle progression, and enhanced cell apoptosis and radiosensitivity in vitro. Circ_0067835 sponged miR-296-5p to regulate IGF1R expression in CRC cells. Moreover, the knockdown of circ_0067835 regulated CRC cell behaviors by up-regulating miR-296-5p and down-regulating IGF1R in vitro. Furthermore, circ_0067835 knockdown diminished tumor growth and promoted cell radiosensitivity in vivo. CONCLUSION: Circ_0067835 knockdown suppressed CRC progression and enhanced CRC cell radiosensitivity partially by the miR-296-5p/IGF1R axis. The findings established a rationale that targeting circ_0067835 might be a promising point for improving CRC treatment.

MiR-296-5p ameliorates deep venous thrombosis by inactivating S100A4.

Deep venous thrombosis is one of the most common venous thromboembolic diseases and has a low cure rate and a high postoperative recurrence rate. Furthermore, emerging evidence indicates that microRNAs are involved in deep venous thrombosis. miR-296-5p is an important microRNA that plays a critical role in various cellular functions, and S100A4 is closely related to vascular function. miR-296-5p is downregulated in deep venous thrombosis patients, and its predicted target S100A4 is upregulated in deep venous thrombosis patients. Therefore, it was hypothesized that miR-296-5p may play a vital role in the development of deep venous thrombosis by targeting S100A4. An Ox-LDL-stimulated HUVEC and deep venous thrombosis mouse model was employed to detect the biological functions of miR-296-5p and S100A4. Dual luciferase reporter assays and pull-down assays were used to authenticate the interaction between miR-296-5p and S100A4. ELISA and Western blotting were employed to detect the protein levels of thrombosis-related factors and the endothelial-to-mesenchymal transition (EndMT)-related factors. The miR-296-5p levels were reduced, while the S100A4 levels were enhanced in deep venous thrombosis patients, and the miR-296-5p levels were negatively correlated with the S100A4 levels in deep venous thrombosis patients. miR-296-5p suppressed S100A4 expression by targeting the 3' UTR of S100A4. MiR-296-5p knockdown accelerated ox-LDL-induced HUVEC apoptosis, oxidative stress, thrombosis-related factor expression, and EndMT, while S100A4 knockdown antagonized these effects in ox-LDL-induced HUVECs. S100A4 knockdown reversed the effect induced by miR-296-5p knockdown. Moreover, the in vivo studies revealed that miR-296-5p knockdown in deep venous thrombosis mice exacerbated deep venous thrombosis formation, whereas S100A4 knockdown had the opposite effect. These results indicate that elevated miR-296-5p inhibits deep venous thrombosis formation by inhibiting S100A4 expression. Both miR-296-5p and S100A4 may be potential diagnostic markers and therapeutic targets for deep venous thrombosis.

Exosomal circ_IFT80 Enhances Tumorigenesis and Suppresses Radiosensitivity in Colorectal Cancer by Regulating miR-296-5p/MSI1 Axis.

BACKGROUND: Exosomal circular RNAs (circRNAs) can act as biomarkers and play crucial roles in colorectal cancer (CRC) and radiosensitivity. The aim of this study was to explore the functions and regulatory mechanism of exosomal circRNA intraflagellar transport 80 (circ_IFT80) in tumorigenesis and radiosensitivity of CRC. METHODS: Exosomes were detected using transmission electron microscopy (TEM). Protein levels were determined by Western blot assay. The expression of circ_IFT80, microRNA-296-5p (miR-296-5p) and musashi1 (MSI1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell cycle distribution, cell apoptosis, and cell proliferation were detected by flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. Colony formation assay was used to determine the radiosensitivity of cells. The interaction between miR-296-5p and circ_IFT80 or MSI1 was verified by dual-luciferase reporter assay. A xenograft tumor model was established to explore the role of exosomal circ_IFT80 in vivo. RESULTS: Circ_IFT80 was upregulated in exosomes derived from CRC patient serum and CRC cells. Exosomal circ_IFT80 or circ_IFT80 overexpression facilitated tumorigenesis by increasing cell proliferation and reducing apoptosis, and inhibited radiosensitivity via promoting colony formation and inhibiting apoptosis. Additionally, circ_IFT80 acted as a sponge of miR-296-5p, and miR-296-5p reversed the effects of circ_IFT80 on tumorigenesis and radiosensitivity. Moreover, MSI1 was a direct target of miR-296-5p. Furthermore, miR-296-5p overexpression inhibited tumorigenesis and promoted radiosensitivity by downregulating MSI1. Exosomal circ_IFT80 also accelerated tumor growth in vivo. CONCLUSION: Exosomal circ_IFT80 promoted tumorigenesis and reduced radiosensitivity by regulating miR-296-5p/MSI1 axis, which might provide a novel avenue for treatment of CRC.