Clinical Significance of MicroRNA-299-3p in Coronary Artery Disease Based on Bioinformatics Analysis.

The purpose of the research was to evaluate the diagnostic performance of microRNA-299-3p (miR-299-3p) in patients with coronary artery disease (CAD). The relative abundance of miR-299-3p in patients with CAD was verified by quantitative real time polymerase chain reaction (qRT-PCR) assay. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for analysis, and target genes were predicted and enriched by DAVID software. The protein-protein interaction (PPI) network was drawn by STRING database. Receiver operating characteristic (ROC) was adopted to appraise the diagnostic value of miR-299-3p in CAD. Bioinformatics analysis showed that the GO function of miR-299-3p target genes of miR-299-3p mainly focuses on specific granular membrane, regulation of apoptotic signaling pathway, growth factor binding and so on. KEGG analysis showed that the most abundant pathways involve fluid shear stress and atherosclerosis, as well as Notch signaling pathways. PPI network showed the seven predictive genes encoding the proteins play pivotal roles in maintaining the stability and interaction of the network, especially matrix metallopeptidase 2 (MMP2) and intercellular cell adhesion molecule-1 (ICAM1). Compared with the control group, serum miR-299-3p in the CAD group was distinctly up-regulated via qRT-PCR (p < 0.001). ROC analysis showed that miR-299-3p was an important index for detecting CAD patients and major adverse cardiovascular events (MACE) patients with an AUC of 0.931 and 0.758, respectively. MiR-299-3p is involved in the development of CAD, and might become a potential biomarker for monitoring CAD.

Restoration of miR-299-3p promotes efferocytosis and ameliorates atherosclerosis via repressing CD47 in mice.

Atherosclerotic plaque formation is largely attributed to the impaired efferocytosis, which is known to be associated with the pathologic upregulation of cluster of differentiation 47 (CD47), a key antiphagocytic molecule. By gene expression omnibus (GEO) datasets analysis, we identified that four miRNAs are aberrantly downregulated in atherosclerosis, coronary artery disease, and obesity. Of them, hsa-miR-299-3p (miR-299-3p) was predicted to target the 3'UTR of human CD47 mRNA by bioinformatics analysis. Further, we demonstrated that miR-299-3p negatively regulates CD47 expression by binding to the target sequence "CCCACAU" in the 3'UTR of CD47 mRNA through luciferase reporter assay and site-directed mutagenesis. Additionally, we found that miR-299-3p was downregulated by ~32% in foam cells in response to oxidized low-density lipoprotein (ox-LDL) stimulation, thus upregulating CD47 and contributing to the impaired efferocytosis. Whereas, restoration of miR-299-3p reversed the ox-LDL-induced upregulation of CD47, thereby facilitating efferocytosis. In high-fat diet (HFD) fed ApoE-/- mice, we discovered that miR-299-3p was downregulated thus leading to upregulation of CD47 in abdominal aorta. Conversely, miR-299-3p restoration potently suppressed HFD-induced upregulation of CD47 and promoted phagocytosis of foam cells by macrophages in atherosclerotic plaques, thereby reducing necrotic core, increasing plaque stability, and mitigating atherosclerosis. Conclusively, we identify miR-299-3p as a negative regulator of CD47, and reveal a molecular mechanism whereby the ox-LDL-induced downregulation of miR-299-3p leads to the upregulation of CD47 in foam cells thus contributing to the impaired efferocytosis in atherosclerosis, and propose miR-299-3p can potentially serve as an inhibitor of CD47 to promote efferocytosis and ameliorate atherosclerosis.

The upregulation and transcriptional regulatory mechanisms of Extra spindle pole bodies like 1 in bladder cancer: An immunohistochemistry and high-throughput screening Evaluation.

Background: This study aimed to explore the expression level and transcriptional regulation mechanism of Extra Spindle Pole Bodies Like 1 (ESPL1) in bladder cancer (BC). Methods: A multicentre database of samples (n = 1391) was assayed for ESPL1 mRNA expression in BC and validated at the protein level by immunohistochemical (IHC) staining of in-house samples (n = 202). Single-cell sequencing (scRNA-seq) analysis and enrichment analysis explored ESPL1 distribution and their accompanying molecular mechanisms. ATAC-seq, ChIP-seq and Hi-C data from multiple platforms were used to investigate ESPL1 upstream transcription factors (TFs) and potential epigenetic regulatory mechanisms. Immune-related analysis, drug sensitivity and molecular docking of ESPL1 were also calculated. Furthermore, upstream microRNAs and the binding sites of ESPL1 were predicted. The expression level and early screening efficacy of miR-299-5p in blood (n = 6625) and tissues (n = 537) were examined. Results: ESPL1 was significantly overexpressed at the mRNA level (p < 0.05, SMD = 0.75; 95 % CI = 0.09, 1.40), and IHC staining of in-house samples verified this finding (p < 0.0001). ESPL1 was predominantly distributed in BC epithelial cells. Coexpressed genes of ESPL1 were enriched in cell cycle-related signalling pathways, and ESPL1 might be involved in the communication between epithelial and residual cells in the Hippo, ErbB, PI3K-Akt and Ras signalling pathways. Three TFs (H2AZ, IRF5 and HIF1A) were detected upstream of ESPL1 and presence of promoter-super enhancer and promoter-typical enhancer loops. ESPL1 expression was correlated with various immune cell infiltration levels. ESPL1 expression might promote BC growth and affect the sensitivity and therapeutic efficacy of paclitaxel and gemcitabine in BC patients. As an upstream regulator of ESPL1, miR-299-5p expression was downregulated in both the blood and tissues, possessing great potential for early screening. Conclusions: ESPL1 expression was upregulated in BC and was mainly distributed in epithelial cells. Elevated ESPL1 expression was associated with TFs at the upstream transcription start site (TSS) and distant chromatin loops of regulatory elements. ESPL1 might be an immune-related predictive and diagnostic marker for BC, and the overexpression of ESPL1 played a cancer-promoting role and affected BC patients' sensitivity to drug therapy. miR-299-5p was downregulated in BC blood and tissues and was also expected to be a novel marker for early screening.

Restoration of miR-299-3p promotes macrophage phagocytosis and suppresses malignant phenotypes in breast cancer carcinogenesis via dual-targeting CD47 and ABCE1.

Immunoevasion has been a severe obstacle for the clinical treatment of breast cancer (BC). CD47, known as an anti-phagocytic molecule, plays a key role in governing the evasion of tumor cells from immune surveillance by interacting with signal-regulated protein α (SIRPα) on macrophages. Here, we report for the first time that miR-299-3p is a direct regulator of CD47 with tumor suppressive effects both in vitro and in vivo. miRNA expression profiles and overall survival of BC cohorts from the Cancer Genome Atlas, METABRIC, or GSE19783 datasets showed that miR-299-3p is downregulated in BC tissues and that BC patients with low levels of miR-299-3p have poorer prognoses. Using dual-luciferase reporter, qRT-PCR, Western blot, and phagocytosis assays, we proved that restoration of miR-299-3p can suppress CD47 expression by directly targeting the predicted seed sequence "CCCACAU" in its 3'-UTR, leading to phagocytosis of BC cells by macrophages, whereas miR-299-3p inhibition or deletion reversed this effect. Additionally, Gene Ontology (GO) analysis and a variety of confirmatory experiments revealed that miR-299-3p was inversely correlated with cell proliferation, migration, and the cell cycle process. Mechanistically, miR-299-3p can also directly target ABCE1, an essential ribosome recycling factor, alleviating these malignant phenotypes of BC cells. In vivo BC xenografts based on nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice further proved that restoration of miR-299-3p resulted in a significant suppression of tumorigenesis and a promotion of macrophage activation and infiltration. Overall, our study suggested that miR-299-3p is a potent inhibitor of CD47 and ABCE1 to exhibit bifunctional BC-suppressing effects through immune activation conjugated with malignant behavior inhibition in breast carcinogenesis and thus can potentially serve as a novel therapeutic target for BC.

MicroRNA-299-3p inhibits cell proliferation, motility, invasion and angiogenesis via VEGFA in upper tract urothelial carcinoma.

BACKGROUND: Upper tract urothelial carcinoma (UTUC) is a rare tumor with extraordinarily different features between Eastern and Western countries. Vascular endothelial growth factor-A (VEGFA) was originally identified as a secreted signaling protein and regulator of vascular development and cancer progression. In this study, we aimed to elucidate the molecular mechanisms underlying the regulation of VEGFA by microRNA in UTUC. METHODS: VEGFA expression was evaluated by immunohistochemistry in 140 human UTUC tissue samples. Next, we assessed the regulatory relationship between VEGFA and miR-299-3p by real-time PCR, western blotting, ELISA and dual-luciferase reporter assays using two UTUC cell lines. The role of miR-299-3p/VEGFA in cell proliferation, motility, invasion, and tube formation was analyzed in vitro. RESULTS: High VEGFA expression was significantly associated with tumor stage, grade, distant metastasis and cancer-related death and correlated with poor progression-free and cancer-specific survival. VEGFA knockdown repressed proliferation, migration, invasion and angiogenesis in UTUC cell lines. miR-299-3p significantly reduced VEGFA protein expression and miR-299-3p overexpression inhibited VEGFA mRNA and protein expression by directly targeting its 3'-UTR. Functional studies indicated that VEGFA overexpression reversed the miR-299-3p-mediated suppression of tumor cell proliferation, migration, invasion and angiogenesis. In addition, miR-299-3p/VEGFA suppressed cellular functions in UTUC by modulating the expression of P18 and cyclin E2. CONCLUSIONS: Our findings suggest that miR-299-3p possibly suppresses UTUC cell proliferation, motility, invasion and angiogenesis via VEGFA. VEGFA may act as a prognostic predictor, and both VEGFA and miR-299-3p could be potential therapeutic targets for UTUC.

DOK7, a target of miR-299-5p, suppresses the progression of bladder cancer.

OBJECTIVE: Bladder cancer (BLCA) is the 6th most common malignancy in males. microRNA (miRNAs) can function as tumor suppressors or oncogenic factors, which are of significance in the progression of BLCA. This study explored the mechanisms by which miR-299-5p modulates DOK7 (Docking Protein 7) expression and the functional role of DOK7 in the progression of BLCA. METHODS: The expression of the DOK7 in BLCA patient samples was examined by RT-qPCR (Real-time quantitative polymerase chain reaction), Western blotting and Immunohistochemical (IHC) staining. The malignant phenotype of BLCA cells upon DOK7 overexpression or silencing was assessed by functional assays including cell count kit-9 (CCK8), colony formation and 5-ethynyl-2'-deoxyuridine (Edu) staining assays, as well as Transwell migration and invasion assays. The miRNA regulators of DOK7 were identified through bioinformatics prediction, and the biological role of miR-299-5p/DOK7 axis was validated by functional assays. The impact of miR-299-5p/DOK7 axis on Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway was further examined by Western blotting. RESULTS: DOX7 was significantly downregulated in BLCA tumor tissues compared with normal tissues. Ectopic DOK7 expression suppressed the proliferation, migration and invasion of BLCA cells. DOK7 overexpression also attenuated the tumorigenesis of BLCA cells in nude mice. miR-299-5p was a negative regulator of DOK7 expression in BLCA cells. miR-299-5p/DOK7 axis impaired the malignancy of BLCA cells through regulating the JAK signaling pathway. CONCLUSION: Our data indicate that miR-299-5p/DOK7 axis suppresses BLCA progression possibly by regulating the JAK signaling pathway.

Circular RNA CDR1as Mediated by Human Antigen R (HuR) Promotes Gastric Cancer Growth via miR-299-3p/TGIF1 Axis.

Background: Gastric cancer (GC) remains a common malignancy worldwide with a limited understanding of the disease mechanisms. A novel circular RNA CDR1as has been recently reported to be a crucial regulator of human cancer. However, its biological role and mechanism in the GC growth are still far from clear. Methods: Small interfering RNAs (siRNAs), lentivirus or plasmid vectors were applied for gene manipulation. The CDR1as effects on the GC growth were evaluated in CCK8 and colony formation assays, a flow cytometry analysis and mouse xenograft tumor models. A bioinformatics analysis combined with RNA immunoprecipitation (RIP), RNA pull-down assays, dual-luciferase reporter gene assays, Western blot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and functional rescue experiments were used to identify the CDR1as target miRNA, the downstream target gene and its interaction with human antigen R (HuR). Results: The CDR1as overexpression promoted the GC growth in vitro and in vivo and reduced the apoptotic rate of GC cells. Its knockdown inhibited the GC cell proliferation and viability and increased the cell apoptotic rate. Proliferation-related proteins PCNA and Cyclin D1 and apoptosis-related proteins Bax, Bcl-2, Caspase-3 and Caspase-9 were regulated. Mechanically, the cytoplasmic CDR1as acted as a miR-299-3p sponge to relieve its suppressive effects on the GC cell growth. Oncogenic TGIF1 was a miR-299-3p downstream target gene that mediated the promotive effects of CDR1as and regulated the PCNA and Bax levels. HuR interacted with CDR1as via the RRM2 domain and positively regulated the CDR1as level and its oncogenic role as well as downstream target TGIF1. Conclusions: CDR1as promotes the GC growth through the HuR/CDR1as/miR-299-3p/TGIF1 axis and could be used as a new therapeutic target for GC.

Long noncoding RNA negative regulator of antiviral response contributes to pancreatic ductal adenocarcinoma progression via targeting miR-299-3p.

BACKGROUND: Pancreatic ductal cancer (PDAC) has high malignancy and poor prognosis. Long noncoding RNAs (lncRNAs) are associated with high levels of malignancy, including PDAC. However, the biological and clinical significance of negative regulator of antiviral response (NRAV) in PDAC is unclear. AIM: To study the regulatory role of lncRNA NRAV in PDAC. METHODS: GEPIA analyzed lncRNA NRAV and miRNA (miR-299-3p) expression levels in PDAC tissues and measured them in PDAC cells by quantitative measurements in real time. The specific role of NRAV and miR-299-3p in cell proliferation and transfer potential was evaluated by cell formation analysis, Cell Counting Kit-8 and Transwell analysis. The relationship between NRAV and miR-299-3p was studied by predictive bioinformatics, RNA immunoassay, and fluorescence enzyme analysis. In vivo experiments included transplantation of simulated tumor cells under naked mice. RESULTS: The expression level of lncRNA NRAV was higher in both tumor tissues and cell lines of PDAC and was negatively associated with the clinical survival of PDAC patients. Functionally, overexpression of NRAV promoted cell proliferation and metastasis of PDAC cells, while knockdown of NRAV reversed these effects. Finally, NRAV was performed as a molecular sponge of miR-299-3p. Moreover, overexpression of miR-299-3p could reverse the promoting effects of NRAV on cell proliferation and metastasis of PDAC cells. CONCLUSION: NRAV facilitates progression of PDAC as a molecular sponge of miR-299-3p and may be a potential molecular marker for diagnosis and treatment of PDAC.

LncRNA PTAR activates the progression of bladder cancer by modulating miR-299-3p/CD164 axis.

Bladder cancer (BC) occurs in the urinary system which has high incidence and mortality. During past decades, lots of long noncoding RNAs (lncRNAs) have been identified to function in cancer progression, including BC. In our research, we targeted at investigating the functions and mechanisms of lncRNA pro-transition associated RNA (PTAR) in BC. Functional assays were implemented to access the changes of BC cell phenotype. Mechanistic assays were applied for confirming the interaction between RNAs. Based on the collected data, PTAR expression was high in BC cells and silenced PTAR repressed BC cell proliferative, migratory and invasive abilities but improved cell apoptotic ability. In vivo study also verified PTAR depletion inhibited BC tumor growth. Furthermore, miR-299-3p was confirmed to bind with PTAR and its overexpression suppressed malignant behaviors of BC cells. Cluster of differentiation 164 (CD164) was proved to be miR-299-3p target. Rescue experiments implied overexpressed CD164 offset the inhibitory function of PTAR depletion on BC cell phenotype. Additionally, CD164 was uncovered to combine with C-X-C motif chemokine receptor 4 (CXCR4) to switch on PI3K/AKT pathway. To conclude, PTAR facilitates BC development via regulating miR-299-3p/CD164 axis and activating PI3K/AKT pathway.

FOXN3 Expression Regulated by miR-299-5p Inhibiting the Proliferation, Migration and Invasion of Oral Squamous Cell Carcinoma Cells.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the commonest malignancies of the oral cavity. FOXN3 is a tumor suppressor that represses the progression of many tumors. Nonetheless, its role in OSCC has not been elucidated. This work is performed to probe the role and dysregulation mechanism of FOXN3 in OSCC. METHODS: FOXN3 mRNA and miR-299-5p expressions were quantified by quantitative real-time polymerase chain reaction (qRT-PCR); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to detect OSCC cell growth; Transwell experiment was conducted to detect cell migration and invasion; dual-luciferase reporter experiment and bioinformatics were adopted to analyze the relationship between miR-299-5p and FOXN3; Western blot was implemented to detect FOXN3 protein expression. RESULTS: FOXN3 expression was remarkably down-modulated, and miR-299-5p expression was markedly up-modulated in OSCC tissues and cell lines compared with paracancerous tissues and normal oral epithelial cell line. FOXN3 overexpression impeded OSCC cell growth, migration and invasion. FOXN3 was proven to be a downstream target of miR-299-5p, and miR-299-5p mimics enhanced OSCC cell growth, migration and invasion. Moreover, FOXN3 overexpression partially reversed the promoting effects of miR-299-5p mimics on OSCC cell growth, migration and invasion. CONCLUSION: FOXN3 expression is remarkably down-modulated in OSCC tissues and cell lines, and miR-299-5p targets FOXN3 to facilitate OSCC cell growth, migration and invasion. These results imply that miR-299-5p/FOXN3 axis may be a potential target for OSCC treatment.

Silencing of circ_OSBPL10 affects the functional behaviors of oral squamous cell carcinoma cells by the miR-299-3p/CDK6 axis.

BACKGROUND: Circular RNAs (circRNAs) are increasingly implicated in the development of oral squamous cell carcinoma (OSCC). Here, we explored the precise role of circRNA oxysterol binding protein like 10 (circ_OSBPL10, circ_0008549) in OSCC pathogenesis. METHODS: Ribonuclease (RNase) R assay was performed to assess the stability of circ_OSBPL10. The levels of circ_OSBPL10, microRNA (miR)-299-3p and cyclin-dependent kinase 6 (CDK6) mRNA were gauged by qRT-PCR. CDK6 protein level was measured by western blot. Cell proliferation was detected by MTT and colony formation assays. Cell cycle distribution and apoptosis were measured by flow cytometry. Cell migration and invasion were evaluated using transwell assay. Dual-luciferase reporter assay was used to identify the relationship between miR-299-3p and circ_OSBPL10 or CDK6. Animal studies were performed to evaluate the role of circ_OSBPL10 in tumor growth in vivo. RESULTS: Circ_OSBPL10 was up-regulated in OSCC tissues and cells. Silencing of circ_OSBPL10 hindered cell proliferation, cell cycle progression, colony formation, migration, invasion, and promoted apoptosis in vitro and diminished tumor growth in vivo. Mechanistically, circ_OSBPL10 directly targeted miR-299-3p, and circ_OSBPL10 silencing affected cell functional properties in vitro by up-regulating miR-299-3p. CDK6 was a direct and functional target of miR-299-3p. The circ_OSBPL10/miR-299-3p axis regulated cell functional properties in vitro via CDK6. Moreover, circ_OSBPL10 acted as a competing endogenous RNA (ceRNA) to regulate CDK6 expression through miR-299-3p. CONCLUSION: Our present findings first demonstrate that circ_OSBPL10 can regulate the functional behaviors of OSCC cells at least partially by miR-299-3p/CDK6 axis, highlighting circ_OSBPL10 as a promising therapeutic target for OSCC.

LncRNA SNHG5 promotes the glycolysis and proliferation of breast cancer cell through regulating BACH1 via targeting miR-299.

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors in women. Accumulating studies have been reported that long non-coding RNA (lncRNA) SNHG5 is highly expressed in BC. However, the specific molecular mechanism of SNHG5 in BC is unclear. METHODS: Gene and protein expressions in BC cell were detected by qRT-PCR and western blotting. The proliferation and cell cycle were measured using colony formation assay and flow cytometry analysis, separately. The glucose consumption and lactate production were determined by using the glucose assay kit and lactate assay kit. A dual-luciferase reporter assay was performed to measure the interaction between miR-299 and SNHG5 or BACH1. RESULTS: SNHG5 and BACH1 expressions were increased in BC cell while miR-299 level was decreased. SNHG5 increased BACH1 expression by directly targeting miR-299. SNHG5 silencing or miR-299 overexpression suppressed the proliferation of BC cell, arrested the cell cycle in the G1 cell phase, and decreased the glucose consumption and lactate production of BC cell. However, inhibition of miR-299 or overexpression of BACH1 could reverse the inhibitory effects of sh-SNHG5 on cell proliferation and glycolysis in BC. CONCLUSION: SNHG5 promoted the BC cell growth and glycolysis through up-regulating BACH1 expression via targeting miR-299. These findings may improve the diagnostic and therapeutic approaches to BC.

Berberine affects activity of HSV-1 virus infected HEp-2 cells and its molecular mechanism / 中国药理学通报

Aim To discuss the effect of berberine ( BE) on the activity of HSV-1 virus infected HEp-2 cells and its related molecular mechanisms.Methods Hie infected cell model was constructed and divided into control group, infection group, low concentration group ( 5 (xmol • L 1 -BE) , medium concentration group ( 10 (xmol • L '-BE) and high concentration group ( 15 (xmol • L '-BE) ) , and then incubated for 24 hours.qRT-PCR was used to determine HSV-1 infection-related genes ( gD, ICP-4, ICP-8, ICP-27 ) and mRNA expression levels of LncBNA NRAV, miR- 299-3p, RAB5C.CCK-8 method and flow cytometry were applied to detect cell viability and apoptotic rate.The expression levels of PI3K/AKT signaling pathway and JNK/p38 MAPK signaling pathway related protein were analysed by WB.Results It was found that BE j j reduced the mRNA expression of gD, ICP-4, ICP-8, anrl ICP-27, improved cell viability, and inhibited eell apoptosis.BE promoted the expression of miR-299-3p by inhibiting LncRNA NRAV and RAB5C.BE inhibited the protein expression levels of PBK/AKT signaling pathway and JNK/p38 MAPK signaling pathway proteins PI3K, AKT, JNK, and P38.Conclusions The mechanism that BE enhances the activity of HEp-2 cells after HSV-1 infection and suppresses its apoptosis may be related to LncRNA NRAV and RAB5C targeting competitive binding to miH-299-3p, inhibiting the activation of PBK/AKT signaling pathway and JNK/ p38 MAPK signaling pathway.

miR-299-3p suppresses cell progression and induces apoptosis by downregulating PAX3 in gastric cancer.

Gastric cancer (GC) is ranked the fourth leading cause of cancer-related death, with an over 75% mortality rate worldwide. In recent years, miR-299-3p has been identified as a biomarker in multiple cancers, such as acute promyelocytic leukemia, thyroid cancer, and lung cancer. However, the regulatory mechanism of miR-299-3p in GC cell progression is still largely unclear. Cell viability and apoptosis tests were performed by CCK8 and flow cytometry assay, respectively. Transwell assay was recruited to examine cell invasion ability. The interaction between miR-299-3p and PAX3 was determined by the luciferase reporter system. PAX3 protein level was evaluated by western blot assay. The expression of miR-299-3p was downregulated in GC tissues and cell lines (MKN-45, AGS, and MGC-803) compared with the normal tissues and cells. Besides, overexpression of miR-299-3p significantly suppressed proliferation and invasion and promoted apoptosis in GC. Next, we clarified that PAX3 expression was regulated by miR-299-3p using a luciferase reporter system, qRT-PCR, and western blot assay. Additionally, downregulation of PAX3 repressed GC cell progression. The rescue experiments indicated that restoration of PAX3 inversed miR-299-3p-mediated inhibition on cell proliferation and invasion. miR-299-3p suppresses cell proliferation and invasion as well as induces apoptosis by regulating PAX3 expression in GC, representing desirable biomarkers for GC diagnosis and therapy.

Long non-coding RNA ZEB1-AS1 promotes proliferation and metastasis of hepatocellular carcinoma cells by targeting miR-299-3p/E2F1 axis.

There is emerging evidence that dysregulation of long non-coding RNAs (lncRNAs) is associated with hepatocellular carcinoma (HCC). Zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) functions as an oncogenic regulator in various malignancies. Nonetheless, the potential role of ZEB1-AS1 in HCC remains poorly elucidated. Herein, qRT-PCR was employed for examining ZEB1-AS1, miR-299-3p and E2F transcription factor 1 (E2F1) mRNA expressions in HCC cells and tissues. MTT assay was performed to evaluate cell proliferation. Transwell assay was utilized for evaluating cancer cell migration and invasion. Western blot was employed for measuring E2F1 protein expression. What's more, dual-luciferase reporter assay was utilized for verifying the targeting relationships between ZEB1-AS1 and miR-299-3p, as well as E2F1 and miR-299-3p. It was demonstrated that, in HCC tissues and cells, ZEB1-AS1 expression was markedly increased, and meanwhile, its high expression level is related to the unfavourable clinicopathologic indicators. ZEB1-AS1 overexpression facilitated HCC cell proliferation, migration and invasion, while its knockdown led to the opposite effects. In terms of mechanism, we discovered that ZEB1-AS1 could decoy miR-299-3p and upregulate E2F1 expression. This work reveals the functions and mechanism of ZEB1-AS1 in HCC tumourigenesis and progression, which provides novel biomarkers and therapeutic targets for HCC.

LncRNA MIR205HG regulates melanomagenesis via the miR-299-3p/VEGFA axis.

In this study, we investigated the role of lncRNA MIR205HG in melanomagenesis. Quantitative real-time PCR (qRT-PCR) analysis showed that MIR205HG levels were significantly upregulated in melanoma cell lines compared to normal human melanocytes. Similarly, MIR205HG levels were significantly higher melanoma tissues than adjacent normal skin tissues (n=30). CCK-8 and flow cytometry assays showed that MIR205HG knockdown significantly decreased the viability of melanoma cells. Dual luciferase reporter and RNA pull-down assays confirmed that MIR205HG directly binds to microRNA (miR)-299-3p. Targetscan analysis and dual luciferase reporter assays showed that miR-299-3p directly binds to the 3'UTR of VEGFA mRNA. Wound healing and transwell invasion assays showed that MIR205HG knockdown decreased in vitro migration and invasiveness of melanoma cells, and these effects were reversed by treatment with miR-299-3p inhibitor. MIR205HG-silenced melanoma cells showed increased miR-299-3p expression and lower levels of both VEGFA mRNA and protein. Tumor volumes were significantly smaller in nude mice xenografted with MIR205HG knockdown melanoma cells than the controls. These results demonstrate that MIR205HG supports melanoma growth via the miR-299-3p/VEGFA axis. This makes MIR205HG a potential therapeutic target for the treatment of melanoma.

Circ_0110805 Knockdown Enhances Cisplatin Sensitivity and Inhibits Gastric Cancer Progression by miR-299-3p/ENDOPDI Axis.

BACKGROUND: Gastric cancer is a prevalent primary stomach tumor. Cisplatin is frequently used to treat gastric cancer. However, the resistance of cisplatin in gastric cancer often occurs, which brings a heavy burden to gastric cancer treatment. METHODS: In this study, we revealed a novel underlying mechanism about cisplatin-resistant effect in gastric cancer. A Cell Counting Kit-8 (CCK-8) cell viability assay and a xenograft model were performed to evaluate the function of circRNA in the cisplatin resistance of gastric cancer. RESULTS: Compared with control groups, we observed that circ_0110805 was highly expressed, the mRNA and protein expression levels of ENDOPDI were dramatically upregulated, and the expression of miR-299-3p was significantly downregulated in gastric cancer cells, cisplatin-resistant gastric cancer tissues or cells. Functionally, circ_0110805 knockdown improved cisplatin sensitivity, induced cell apoptosis, whereas repressed cell viability, migration and invasion in AGS/DDP and HGC-27/DDP cells, which was reversed by miR-299-3p inhibitor. Additionally, ENDOPDI overexpression hindered the effects of miR-299-3p on cisplatin sensitivity and gastric cancer progression. Circ_0110805 knockdown enhanced cisplatin sensitivity in vivo. Mechanistically, circ_0110805 acted as a sponge of miR-299-3p and its targeted ENDOPDI. CONCLUSION: We showed that circ_0110805 knockdown increased the sensitivity of gastric cancer to cisplatin, which also repressed gastric cancer progression by sponging miR-299-3p to downregulate ENDOPDI expression. It might provide a new insight for future studying cisplatin-resistant gastric cancer.

Knockdown of lncRNA HCP5 Suppresses the Progression of Colorectal Cancer by miR-299-3p/PFN1/AKT Axis.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system. The lncRNA HCP5 has been reported to affect the progression of tumor in several types of cancer. Here, in this research, we focus on the role and function of lncRNA HCP5 in human colorectal cancer. MATERIALS AND METHODS: Tissue samples from colorectal cancer patients were used for detecting the expression of HCP5 by qRT-PCR. Proliferation, migration, invasion and apoptotic cells were assessed by CCK-8, colony formation, transwell assays and flow cytometry in SW480 and HCT-116 cells. The interactions between miR-299-3p and HCP5 or PFN1 were analyzed and confirmed by online database and luciferase reporter assays. The changes in PFN1 and AKT proteins were measured by Western blot. In vivo experiment was used to confirm the role of HCP5 in CRC. RESULTS: The expression of HCP5 had a higher level in colorectal cancer samples and cells by qRT-PCR, comparing with the normal colorectal tissues and human normal colon epithelial cell. It was revealed that knockdown of HCP5 inhibited viabilities, migration and invasion, while inducing apoptosis in SW480 and HCT-116 cells. Then, HCP5 negatively regulated the expressions of miR-299-3p, which negatively regulated the expressions of PFN1 by targeting PFN1. Furthermore, miR-299-3p inhibitor could alleviate the inhibiting effect by si-HCP5 on cell process of SW480 and HCT-116 cells. In addition, the lncHCP5/miR-299-3p/PFN1 axis could affect the progression of CRC through activating the AKT signaling. Last, we confirmed that knockdown of HCP5 inhibited the progression of CRC with an in vivo experiment. CONCLUSION: The experiments and analyses support our hypothesis that knockdown of lncRNA HCP5 suppresses the progression of colorectal cancer by miR-299-3p/PFN1/AKT axis.

MIR205HG acts as a ceRNA to expedite cell proliferation and progression in lung squamous cell carcinoma via targeting miR-299-3p/MAP3K2 axis.

INTRODUCTION: Long noncoding RNAs (lncRNAs) have been associated with many types of cancers, but their molecular mechanisms in lung squamous cell carcinoma (LUSC) have not been fully studied. Therefore, the current study investigated the regulation role of microRNA-205 host gene (MIR205HG) in LUSC and recognized the target genes managed by this lncRNA. METHODS: MIR205HG expression was assessed by the quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The effects of silenced MIR205HG on cell biological behaviors were detected by colony formation assay, transwell assay, flow cytometry analysis and western blot analysis. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized to proof the binding relationship between miR-299-3p and MIR205HG/mitogen-activated protein kinase kinase kinase 2 (MAP 3 K2). RESULTS: The expression levels of MIR205HG in LUSC tissues and cell lines were obviously up-regulated. Down-regulation of MIR205HG expression remarkably reduced cell proliferation, migration and epithelial-to-mesenchymal transition (EMT) progression, whereas promoted cell apoptosis. MIR205HG could bind with miR-299-3p and down-regulation of MIR205HG elevated miR-299-3p expression. MAP 3 K2 acted as the target gene of miR-299-3p and was up-regulated by MIR205HG overexpression. Overexpressing MAP 3 K2 could counteract the effects of down-regulating MIR205HG on LUSC progression to some degree. CONCLUSION: MIR205HG acts as a competing endogenous RNA (ceRNA) to expedite cell proliferation and progression via targeting miR-299-3p in LUSC.