High Expression of miR-218-5p in the Peripheral Blood Stream and Tumor Tissues of Pediatric Patients with Sarcomas.

Sarcomas are malignant tumors that may metastasize and the course of the disease is highly aggressive in children and young adults. Because of the rare incidence of sarcomas and the heterogeneity of tumors, there is a need for non-invasive diagnostic and prognostic biomarkers in sarcomas. The aim of the study was to investigate the level of miR-218-5p in peripheral blood and tumor tissue samples of Ewing's sarcoma, osteosarcoma, spindle cell sarcoma patients, and healthy controls, and assessed whether the corresponding molecule was a diagnostic and prognostic biomarker. The study was performed patients (n = 22) diagnosed and treated with Ewing's sarcoma and osteosarcoma and in a control group of 22 healthy children who were matched for age, gender, and ethnicity with the patient group. The expression level of miR-218-5p in RNA samples from peripheral blood and tissue samples were analyzed using the RT-PCR and the expression level of miR-218-5p was evaluated by comparison with the levels in patients and healthy controls. The expression level of miR-218-5p was found to be statistically higher (3.33-fold, p = 0.006) in pediatric patients with sarcomas and when the target genes of miR-218-5p were investigated using the bioinformatics tools, the miR-218-5p was found as an important miRNA in cancer. In this study, the miR-218-5p was shown for the first time to have been highly expressed in the peripheral blood and tumor tissue of sarcoma patients. The results suggest that miR-218-5p can be used as a diagnostic and prognostic biomarker in sarcomas and will be evaluated as an important therapeutic target.

LncRNA HOXA-AS3 promotes cell proliferation and invasion via targeting miR-218-5p/FOXP1 axis in osteosarcoma.

Osteosarcoma is an aggressive form of bone cancer and affects the health in children and adolescents. Although conventional treatment improves the osteosarcoma survival, some patients have metastasis and drug resistance, leading to a worse prognosis. Therefore, it is necessary to explore the molecular mechanism of osteosarcoma occurrence and progression, which could discover the novel treatment for osteosarcoma. Long noncoding RNAs (lncRNAs) have been reported to regulate osteosarcoma occurrence and malignant progression. LncRNA HOXA-AS3 facilitates the tumorigenesis and progression in a variety of human cancers. However, the underlying mechanism of lncRNA HOXA-AS3-induced oncogenesis is poorly determined in osteosarcoma. To address this point, we utilized several cellular biological strategies and molecular approaches to explore the biological functions and mechanisms of lncRNA HOXA-AS3 in osteosarcoma cells. We found that lncRNA HOXA-AS3 facilitates cell proliferation and invasion via targeting miR-218-5p/FOXP1 axis in osteosarcoma. In conclusion, lncRNA HOXA-AS3 could be a promising target for osteosarcoma treatment.

miR-218-5p promotes hepatic lipogenesis through targeting Elovl5 in non-alcoholic fatty liver disease.

Investigating and identifying pathogenic molecules of non-alcoholic fatty liver disease (NAFLD) has become imperative, which would serve as effective targets in the future. We established high-fat diet (HFD)-induced NAFLD model in mice and palmitic acid (PA)-induced model in mouse AML12 cells. The level of miR-218-5p was examined by qRT-PCR, and Elovl5 was identified as the potential target gene of miR-218-5p. The binding relationship between miR-218-5p and Elovl5 was validated by double luciferase reporter gene assay, and inhibition/overexpression of miR-218-5p in vitro. The functional mechanisms of miR-218-5p/Elovl5 in regulating lipogenesis in NAFLD were investigated in vivo and in vitro through gain- and loss-of-function studies. MiR-218-5p was significantly increased, and Elovl5 was decreased in model group. According to the double luciferase reporter and gene interference experiments in AML12 cells, Elovl5 was a target gene of miR-218-5p and its expression was regulated by miR-218-5p. The SREBP1-mediated lipogenesis signaling pathway regulated by Elovl5 was upregulated in model group. Moreover, silencing of miR-218-5p significantly upregulated Elovl5 expression, and suppressed SREBP1 signaling pathway in PA-induced AML-12 cells. Correspondingly, the cell injury, elevated TC, TG contents and lipid droplet accumulation were ameliorated. Furthermore, the effect of miR-218-5p on lipogenesis in vitro and in vivo was obstructed by si-Elovl5, implicating that miR-218-5p promotes lipogenesis by targeting ELOVL5 in NAFLD. miR-218-5p could promote fatty acid synthesis by targeting Elovl5, thereby accelerating the development of NAFLD, which is one of the key pathogenic mechanisms of NAFLD and provides a new molecular target for the management of NAFLD.

miR-218-5p, miR-124-3p and miR-23b-3p act synergistically to modulate the expression of NACC1, proliferation, and apoptosis in C-33A and CaSki cells.

Background: In cervical cancer (CC), miR-218-5p, -124-3p, and -23b-3p act as tumor suppressors. These miRNAs have specific and common target genes that modulate apoptosis, proliferation, invasion, and migration; biological processes involved in cancer. Methods: miR-218-5p, -124-3p, and -23b-3p mimics were transfected into C-33A and CaSki cells, and RT-qPCR was used to quantify the level of each miRNA and NACC1. Proliferation was assessed by BrdU and apoptosis by Annexin V/PI. In the TCGA and The Human Protein Atlas databases, the level of NACC1 mRNA and protein (putative target of the three miRNAs) was analyzed in CC and normal tissue. The relationship of NACC1 with the overall survival in CC was analyzed in GEPIA2. NACC1 mRNA and protein levels were higher in CC tissues compared with cervical tissue without injury. Results: An increased expression of NACC1 was associated with lower overall survival in CC patients. The levels of miR-218-5p, -124-3p, and -23b-3p were lower, and NACC1 was higher in C-33A and CaSki cells compared to HaCaT cells. The increase of miR-218-5p, -124-3p, and -23b-3p induced a significant decrease in NACC1 mRNA. The transfection of the three miRNAs together caused more drastic changes in the level of NACC1, in the proliferation, and in the apoptosis with respect to the individual transfections of each miRNA. Conclusion: The results indicate that miR-218-5p, -124-3p, and -23b-3p act synergistically to decrease NACC1 expression and proliferation while promoting apoptosis in C-33A and CaSki cells. The levels of NACC1, miR-218-5p, -124-3p, and -23b-3p may be a potential prognostic indicator in CC.

Circular RNA circRPS19 promotes chicken granulosa cell proliferation and steroid hormone synthesis by interrupting the miR-218-5p/INHBB axis.

Ovarian follicle development is an important physiological activity for females and makes great significance in maintaining female health and reproduction performance. The development of ovarian follicle is mainly affected by the granulosa cells (GCs), whose growth is regulated by a variety of factors. Here, we identified a novel circular RNA (circRNA) derived from the Ribosomal protein S19 (RPS19) gene, named circRPS19, which is differentially expressed during chicken ovarian follicle development. Further explorations identified that circRPS19 promotes GCs proliferation and steroid hormone synthesis. Furthermore, circRPS19 was found to target and regulate miR-218-5p through a competitive manner with endogenous RNA (ceRNA). Functionals investigation revealed that miR-218-5p attenuates GCs proliferation and steroidogenesis, which is opposite to that of circRPS19. In addition, we also confirmed that circRPS19 upregulates the expression of Inhibin beta B subunit (INHBB) by binding with miR-218-5p to facilitate GCs proliferation and steroidogenesis. Overall, this study revealed that circRPS19 regulates GCs development by releasing the repression of miR-218-5p on INHBB, which suggests a novel mechanism in respect to circRNA and miRNA regulation in ovarian follicle development.

MicroRNA-218-5p-Ddx41 axis restrains microglia-mediated neuroinflammation through downregulating type I interferon response in a mouse model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Microglia-mediated neuroinflammation has been largely considered one of main factors to the PD pathology. MicroRNA-218-5p (miR-218-5p) is a microRNA that plays a role in neurodevelopment and function, while its potential function in PD and neuroinflammation remains unclear. METHODS: We explore the involvement of miR-218-5p in the PD in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model. The miR-218-5p agomir used for overexpression was delivered into the substantia nigra (SN) by bilateral stereotaxic infusions. The loss of dopaminergic (DA) neurons and microglial inflammation in the SN was determined using Western blotting and immunofluorescence. Motor function was assessed using the rotarod test. RNA sequencing (RNA-seq) was performed to explore the pathways regulated by miR-218-5p. The target genes of miR-218-5p were predicted using TargetScan and confirmed using dual luciferase reporter assays. The effects of miR-218-5p on microglial inflammation and related pathways were verified in murine microglia-like BV2 cells. To stimulate BV2 cells, SH-SY5Y cells were treated with 1-methyl-4-phenylpyridinium (MPP+) and the conditioned media (CM) were collected. RESULTS: MiR-218-5p expression was reduced in both the SN of MPTP-induced mice and MPP+-treated BV2 cells. MiR-218-5p overexpression significantly alleviated MPTP-induced microglial inflammation, loss of DA neurons, and motor dysfunction. RNA sequence and gene set enrichment analysis showed that type I interferon (IFN-I) pathways were upregulated in MPTP-induced mice, while this upregulation was reversed by miR-218-5p overexpression. A luciferase reporter assay verified that Ddx41 was a target gene of miR-218-5p. In vitro, miR-218-5p overexpression or Ddx41 knockdown inhibited the IFN-I response and expression of inflammatory cytokines in BV2 cells stimulated with MPP+-CM. CONCLUSIONS: MiR-218-5p suppresses microglia-mediated neuroinflammation and preserves DA neurons via Ddx41/IFN-I. Hence, miR-218-5p-Ddx41 is a promising therapeutic target for PD.

Influences of CircBICD2 on the proliferation,apoptosis and epithelial-mesenchymal transition of oral squa-mous cell carcinoma cells by regulating miR-218-5p/RhoA axis / 实用口腔医学杂志

Objective:To investigate the influences of circular RNA(circRNA)BICD2 on the proliferation,apoptosis and epitheli-al-mesenchymal transition(EMT)of oral squamous cell carcinoma(OSCC)cells by regulating the miR-218-5p/Ras homolog gene family member A(RhoA)axis.Methods:qRT-PCR and Western blot were applied to detect CircBICD2,miR-218-5p gene expres-sion and RhoA protein expression in OSCC tissue,paracancerous tissue,human oral epithelial cell line HOEC and OSCC cell line HSC-4,CAL-27 and SCC-15,respectively.SCC-15 cells were divided into 7 groups of Ct(control,A),si-NC(B),si-CircBICD2(C),miR-NC(D),miR-218-5p(E),si-CircBICD2+anti-NC(F)and si-CircBICD2+anti-miR-218-5p(G).qRT-PCR was ap-plied to detect the expression of CircBICD2 and miR-218-5p in the cells;CCK-8 assay and plate cloning assay were applied to detect the cell proliferation;flow cytometry was applied to detect cell apoptosis;Western blot was applied to detect the protein expressions of RhoA,E-cadherin,Vimentin and N-cadherin;dual luciferase was applied to verify the relationship between CircBICD2 and miR-218-5p,miR-218-5p and RhoA,respectively.Results:In OSCC tissues and cells,CircBICD2 and RhoA proteins were highly ex-pressed,miR-218-5p was lowly expressed.In SCC-15 cells,the expression of CircBICD2 and RhoA protein was the highest,and the expression of miR-218-5p was the lowest(P＜0.05),SCC-15 cells in the 7 groups showed that,compared with group B,the expres-sion of CircBICD2 and RhoA in group C was decreased,and the expression of miR-218-5p was increased(P＜0.05);compared with group D,in group E RhoA protein expression level was decreased,and the miR-218-5p expression level was increased(P＜0.05);compared with group C and group F,the expression of miR-218-5p decreased in group G,and the expression of RhoA protein in-creased(P＜0.05).Down-regulation of CircBICD2 or over-expression of miR-218-5p inhibited SCC-15 cell proliferation and EMT,and induced cell apoptosis;down-regulation of miR-218-5p attenuated the inhibition of SCC-15 cell proliferation and EMT,the cell apoptosis was promoted by silencing CircBICD2.Cir-cBICD2 targeted the miR-218-5p/RhoA axis.Conclusion:Silencing CircBICD2 may inhibit RhoA expression by up-regulating miR-218-5p,inhibits SCC-15 cell proliferation and EMT,and induces cell apoptosis.

miR-218-5p regulates glycolysis in human non-small cell lung cancer A549 cells by targeting PDE7A / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究miR-218-5p靶向磷酸二酯酶7A（PDE7A）调节人非小细胞肺癌（NSCLC）A549细胞糖酵解过程的机制。方法：常规培养A549细胞，用Lipo3000将miR-218-5p mimic、mimic-NC、PDE7A过表达质粒（PDE7A-oe）和PDE7A对照质粒（PDE7A-NC）转染A549细胞，记为miR-218-5p mimic组、mimic-NC组、PDE7A-oe组和PDE7A-NC组。qPCR法验证转染效率，WB法检测糖酵解关键酶蛋白的表达，葡萄糖测定法和乳酸生成测定法检测各转染组A549细胞中2脱氧葡萄糖和乳酸含量，双萤光素酶报告基因实验验证miR-218-5p与PDE7A靶向结合关系，用TCGA数据库数据分析PDE7A mRNA在肺癌组织中的表达水平。结果：在A549细胞中成功地过表达了miR-218-5p（P<0.01）。过表达miR-218-5p均能显著抑制A549细胞中PDE7A、HK2、PKM2蛋白的表达（均P<0.01）、葡萄糖摄取量和乳酸生成量（均P<0.01）。过表达PDE7A均可显著促进A549细胞中PDE7A、HK2、PKM2蛋白的表达（均P<0.01），以及葡萄糖摄取量和乳酸生成量（均P<0.01）。A549细胞中miR-218-5p可与PDE7A mRNA的3´-UTR直接结合。数据库数据分析结果显示，PDE7A mRNA在肺鳞状细胞癌组织中呈高表达（P<0.01）。结论： miR-218-5p靶向PDE7A调控A549细胞中HK2和PKM2的表达水平，进而抑制糖酵解过程，miR-218-5p/PDE7A可能是NSCLC临床诊断和治疗的潜在靶点。

[miR-218-5p Targeting TPX2 Regulates p53 Pathway and Inhibits Malignant Progression of Lung Adenocarcinoma].

BACKGROUND: Lung adenocarcinoma (LUAD) is a major subtype of lung cancer, and its treatment and diagnosis remain a hot research topic. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is highly expressed in a variety of cancer cells and may be associated with the progression of LUAD. This study aimed to investigate the effect of TPX2 on the malignant progression of LUAD cells and the regulatory mechanisms. METHODS: The expression of gene TPX2 in LUAD tissues from The Cancer Genome Atlas (TCGA) database was analyzed by bioinformatics analysis techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TPX2 and miR-218-5p in human lung normal cell lines and human LUAD cell lines. Western blot was used to detect TPX2 protein expression in cell lines and its effect on the expression of key proteins in the p53 signaling pathway. The relationship between TPX2 and miR-218-5p was predicted using bioinformatics and verified by dual luciferase reporter gene assay. Cell counting kit-8 (CCK-8) assay, cell clone formation, cell scratching, Transwell assay, and flow cytometry were used to detect the effects of miR-218-5p and TPX2 on LUAD cell function. RESULTS: TPX2 was significantly overexpressed in LUAD cells, and knockdown of TPX2 inhibited LUAD cell proliferation, migration, and invasion, promoted apoptosis and induced G2/M phase block, and promoted the expression of key proteins in the p53 signaling pathway. miR-218-5p, an upstream regulator of TPX2, could inhibit its expression. Overexpression of miR-218-5p eliminated the malignant development caused by high expression of TPX2, inhibited the malignant processes of LUAD cells such as proliferation and migration as well as promoted the p53 signaling pathway. CONCLUSIONS: miR-218-5p targets and inhibits TPX2 expression and exerts an inhibitory effect on the malignant progression of LUAD cells via p53.

SNAI2/FTH1P3/miR-218-5p Positive Feedback Loop Promotes Colorectal Cancer Metastasis.

Colorectal cancer (CRC) is a type of intestinal cancer that causes more than 600,000 deaths every year. Overcoming the problems of metastasis requires detailed studies to reveal the potential molecular mechanisms. This study aims to reveal the molecular mechanism of CRC metastasis involving non-coding RNA regulation. The expression profile of FTH1P3 was analyzed based on the data of TCGA-COAD patient cohorts. Q-PCR analysis was performed to validate the expression of FTH1P3 in colorectal cancer cells. JASPR was used to screen transcription factors of FTH1P3. q-ChIP analysis was used to validate the target between FTH1P3 and transcription factor. Scratch assay and transwell assay were used to evaluate the migration and invasion ability of colorectal cancer cells. FTH1P3 is highly expressed in CRC patient cohort. FTH1P3 induced migration and invasion of SW480 cell through regulating epithelial-mesenchymal transition (EMT). In addition, FTH1P3 is a direct target of SNAI2. SNAI2 promotes the expression of FTH1P3. Both FTH1P3 and SNAI2 were directly targeted and repressed by miR-218-5p. Interestingly, ectopic FTH1P3 caused a decreased miR-218-5p level and an elevated nucleic SNAI2 protein expression level. Of note, only ectopic SNAI2 protein resulted in a repressed miR-218-5p and an increased FTH1P3, whereas SNAI2 3'UTR failed to affect the expression of miR-218-5p and FTH1P3. SNAI2 transcriptionally activates FTH1P3 expression. Both SNAI2 and FTH1P3 are targets of miR-218-5p. SNAI2/FTH1P3/miR-218-5p form a positive feedback loop in the regulation of CRC metastasis.

MiR-218-5p promotes trophoblast infiltration and inhibits endoplasmic reticulum/oxidative stress by reducing UBE3A-mediated degradation of SATB1.

This research evaluated the effects of miR-218-5p on trophoblast infiltration and endoplasmic reticulum/oxidative stress during preeclampsia (PE). The expression of miR-218-5p and special AT-rich sequence binding protein 1 (SATB1) in placental tissues from 25 patients with PE and 25 normal pregnant subjects was determined using qRT-PCR and western blotting. Cell invasion and cell migration were detected by performing Transwell assays and scratch assays, respectively. MMP-2/9, TIMP1/2, HIF-1α, p-eIF2α, and ATF4 expression in cells was assessed through western blotting. Intracellular reactive oxygen species were detected using 2,7-dichlorodihydrofluorescein diacetate, and intracellular malondialdehyde and superoxide dismutase activities were determined with kits. Dual-luciferase and RNA pull-down assays were performed to verify the interaction between miR-218-5p and UBE3A. Co-immunoprecipitation and western blotting were used to detect the ubiquitination levels of SATB1. A rat model of PE was established, and an miR-218-5p agomir was injected into rat placental tissues. The pathological characteristics of placental tissues were detected via HE staining, and MMP-2/9, TIMP1/2, p-eIF2α, and ATF4 expression in rat placental tissues was determined through western blotting. MiR-218-5p and SATB1 were expressed at low levels, while UBE3A was highly expressed in the placental tissues of patients with PE. The transfection of an miR-218-5p mimic, UBE3A shRNA, or an SATB1 overexpression vector into HTR-8/SVneo cells promoted trophoblast infiltration and inhibited endoplasmic reticulum/oxidative stress. It was determined that UBE3A is a target of miR-218-5p; UBE3A induces ubiquitin-mediated degradation of SATB1. In PE model rats, miR-218-5p alleviated pathological features, promoted trophoblast infiltration, and inhibited endoplasmic reticulum/oxidative stress. MiR-218-5p targeted and negatively regulated UBE3A expression to inhibit ubiquitin-mediated SATB1 degradation, promote trophoblast infiltration, and inhibit endoplasmic reticulum/oxidative stress.

Liver-derived extracellular vesicles from patients with hepatitis B virus-related acute-on-chronic liver failure impair hepatic regeneration by inhibiting on FGFR2 signaling via miR-218-5p.

BACKGROUND: Impaired liver regeneration in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) patients is closely related to prognosis; however, the mechanisms are not yet defined. Liver-derived extracellular vesicles (EVs) may be involved in the dysregulation of liver regeneration. Clarifying the underlying mechanisms will improve the treatments for HBV-ACLF. METHODS: EVs were isolated by ultracentrifugation from liver tissues of HBV-ACLF patients (ACLF_EVs) after liver transplantation, and their function was investigated in acute liver injury (ALI) mice and AML12 cells. Differentially expressed miRNAs (DE-miRNAs) were screened by deep miRNA sequencing. The lipid nanoparticle (LNP) system was applied as a carrier for the targeted delivery of miRNA inhibitors to improve its effect on liver regeneration. RESULTS: ACLF_EVs inhibited hepatocyte proliferation and liver regeneration, with a critical role of miR-218-5p. Mechanistically, ACLF_EVs fused directly with target hepatocytes and transferred miR-218-5p into hepatocytes, acting by suppressing FGFR2 mRNA and inhibiting the activation of ERK1/2 signaling pathway. Reducing the level of miR-218-5p expression in the liver of ACLF mice partially restored liver regeneration ability. CONCLUSION: The current data reveal the mechanism underlying impaired liver regeneration in HBV-ACLF that promotes the discovery of new therapeutic approaches.

MicroRNA-218-5p regulates inflammation response via targeting TLR4 in atherosclerosis.

BACKGROUND: To investigate the expression of miR-218-5p in atherosclerosis patients and its effect on ox-LDL induced THP-1-derived macrophage inflammatory response. METHODS: RT-qPCR detected the expression of serum miR-218-5p, and the diagnostic value of miR-218-5p was analyzed by ROC curve. Pearson correlation coefficient was used to evaluate the correlation between miR-218-5p and CIMT and CRP. THP-1 cells were treated with ox-LDL to construct foam cell model. The expression of miR-218-5p was regulated by in vitro transfection technique, and the effects of miR-218-5p on cell viability, apoptosis and inflammation were investigated. Luciferase reporter genes were used to analyze target genes of miR-218-5p in cell models. RESULTS: The expression of miR-218-5p in the atherosclerosis cohort was significantly reduced, and miR-218-5p showed a good ability to distinguish patients from healthy people. Correlation analysis showed that the level of miR-218-5p was negatively correlated with the levels of CIMT and CRP. Cytological studies showed that the expression of miR-218-5p in macrophages decreased after ox-LDL induction. ox-LDL treatment on macrophages resulted in decreased cell viability, increased cell apoptosis and production of inflammatory cytokines, which contributed to the exacerbation of plaque formation. However, the above situation was reversed after upregulation of miR-218-5p. Bioinformatics analysis showed that TLR4 may be the target gene of miR-218-5p, and this hypothesis was proved by luciferase reporter gene assay. CONCLUSIONS: The expression of miR-218-5p is reduced in atherosclerosis, and it may regulate the inflammatory response of atherosclerotic foam cells by targeting TLR4, suggesting that miR-218-5p may be a promising target for clinical atherosclerosis therapy.

CircRNA-104718 promotes glioma malignancy through regulation of miR-218-5p/HMGB1 signalling pathway.

Increasing number of studies have proven that circular RNAs (circRNAs) play a major role in the biological processes of many different cancers, including glioma, especially as competitive molecular sponges of microRNAs (miRNAs). However, the clear molecular mechanism of the circRNA network in glioma is still not well understood. The expression level of circRNA-104718 and microRNA (miR)-218-5p in glioma tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The target protein's expression level was assessed by western blotting. Bioinformatics systems were used to predict the possible microRNAs and target genes of circRNA-104718, after which dual-luciferase reporter assays were used to confirm the predicted interactions. The proliferation, invasion, migration and apoptosis of glioma cells were detected by CCK, EdU, transwell, wound-healing and flow cytometry assays. CircRNA-104718 was upregulated in human glioma tissues, and a higher level of circRNA-104718 indicated poorer outcomes in glioma patients. In contrast, in glioma tissues, miR-218-5p was downregulated. Knockdown of circRNA-104718 suppressed migration and invasion while boosting the apoptosis rate of glioma cells. In addition, the upregulation of miR-218-5p in glioma cells caused the same suppression. Mechanistically, circRNA-104718 inhibited the protein expression level of high mobility group box-1 (HMGB1) by acting as a molecular sponge for miR-218-5p. CircRNA-104718 is a suppressive factor in glioma cells and might represent a new target for the treatment of glioma patients. CircRNA-104718 modulates glioma cell proliferation through the miR-218-5p/HMGB1 signalling axis. CircRNA-104718 provides a possible mechanism for understanding the pathogenesis of glioma.

Targeting TRIM9 by miR-218-5p Restricts Cell Proliferation and Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer.

OBJECTIVE: Tripartite motif-containing protein 9 (TRIM9) has been studied in several human tumors. MicroRNA-218-5p (miR-218-5p) was predicted to target TRIM9. We aimed to investigate the roles of miR-218-5p/TRIM9 axis in non-small cell lung cancer (NSCLC). METHODS: TRIM9 and miR-218-5p expression levels in NSCLC tissues and cell lines (95D and H1299) were determined by reverse transcription quantitative PCR. UALCAN and Kaplan-Meier (KM) plotter were employed to analyze the expression level of TRIM9 in lung cancer. The interaction between TRIM9 and miR-218-5p was explored by luciferase reporter assay and Spearman correlation test. Immunohistochemistry assay was used to confirm the protein expression of TRIM9 in NSCLC tissues. Regulatory effects of TRIM9 or miR-218-5p on cell proliferation, migration and invasion, and epithelial-mesenchymal transition (EMT) process of NSCLC cells were assessed by CCK-8 assay, transwell assay and western blot analysis. RESULTS: MiR-218-5p was predicted to specifically target TRIM9 and confirmed to negatively regulate TRIM9 expression in NSCLC cells. Online bioinformatics analysis showed TRIM9 overexpression in lung cancer and predicted poor prognosis. The data from collected clinical specimens showed that miR-218-5p was downregulated, while TRIM9 was upregulated in NSCLC tissues, whose expression levels were negatively correlated. The in vitro experiments demonstrated that TRIM9 knockdown imitated the suppressive effects miR-218-5p overexpression on cell proliferation, migration, invasion and EMT process. In addition, overexpression of TRIM9 reversed the effects of miR-218-5p in NSCLC cells. CONCLUSIONS: Our results suggest that TRIM9 functions as an oncogene in NSCLC in vitro and is regulated by miR-218-5p.

MiR-218-5p-dependent SOCS3 downregulation increases osteoblast differentiation inpostmenopausal osteoporosis.

BACKGROUND: Postmenopausal osteoporosis (POP) is a prevalent skeletal disease among elderly women. Previous study indicated that suppressor of cytokine signaling 3 (SOCS3) participates in the regulation of bone marrow stromal cell (BMSC) osteogenesis. Here, we further investigated the exact function and mechanism of SOCS3 in POP progression. METHODS: BMSCs were isolated from Sprague-Dawley rats and treated with Dexamethasone (Dex). Alizarin Red staining and ALP activity assays were applied to assess the osteogenic differentiation of rat BMSCs under the indicated conditions. Osteogenic genes (ALP, OPN, OCN, COL1) mRNA levels were determined using quantitative RT-PCR. Luciferase reporter assay verified the interaction between SOCS3 and miR-218-5p. Rat models of POP were established in ovariectomized (OVX) rats to detect the in vivo effects of SOCS3 and miR-218-5p. RESULTS: We found that silencing SOCS3 antagonized the suppressive effects of Dex on the osteogenic differentiation of BMSCs. SOCS3 was found to be targeted by miR-218-5p in BMSCs. The SOCS3 levels were negatively modulated by miR-218-5p in femurs of POP rats. MiR-218-5p upregulation promoted the BMSC osteogenic differentiation, while SOCS3 overexpression reversed the effects of miR-218-5p. Moreover, SOCS3 was highly expressed and miR-218-5p was downregulated in the OVX rat models, and silencing SOCS3 or overexpressing miR-218-5p alleviated POP in OVX rats by promoting osteogenesis. CONCLUSION: SOCS3 downregulation mediated by miR-218-5p increases osteoblast differentiation to alleviate POP.

MiR-218-5p/EGFR Signaling in Arsenic-Induced Carcinogenesis.

BACKGROUND: Arsenic is a well-known carcinogen inducing lung, skin, bladder, and liver cancer. Abnormal epidermal growth factor receptor (EGFR) expression is common in lung cancer; it is involved in cancer initiation, development, metastasis, and treatment resistance. However, the underlying mechanism for arsenic-inducing EGFR upregulation remains unclear. METHODS: RT-PCR and immunoblotting assays were used to detect the levels of miR-218-5p and EGFR expression. The Luciferase assay was used to test the transcriptional activity of EGFR mediated by miR-218-5p. Cell proliferation, colony formation, wound healing, migration assays, tube formation assays, and tumor growth assays were used to study the function of miR-218-5p/EGFR signaling. RESULTS: EGFR and miR-218-5p were dramatically upregulated and downregulated in arsenic-induced transformed (As-T) cells, respectively. MiR-218-5p acted as a tumor suppressor to inhibit cell proliferation, migration, colony formation, tube formation, tumor growth, and angiogenesis. Furthermore, miR-218-5p directly targeted EGFR by binding to its 3'-untranslated region (UTR). Finally, miR-218-5p exerted its antitumor effect by inhibiting its direct target, EGFR. CONCLUSION: Our study highlights the vital role of the miR-218-5p/EGFR signaling pathway in arsenic-induced carcinogenesis and angiogenesis, which may be helpful for the treatment of lung cancer induced by chronic arsenic exposure in the future.

Nardosinone suppresses monoiodoacetate-induced osteoarthritis in rats: The key role of the miR-218-5p/NUMB axis.

Nardosinone is a bioactive compound with a sesquiterpenoid structure isolated from Nardostachys jatamansi. The compound has shown treatment effects against skeletal disorders. In the current study, the effects of nardosinone on osteoarthritis (OA) were first assessed and the mechanism underlying the effects was explored by detecting changes in the miR-218-5p/NUMB axis. The miR, as a potential target mediating the effects of nardosinone on OA, was first determined with microarray and RT-qPCR detections. Then, OA symptoms were induced in rats using monoiodoacetate (MIA) and treated with nardosinone. The anti-OA effects of nardosinone were assessed via the detection of the histological structure and inflammation. The role of miR-218-5p was delineated by modulating its levels in OA-affected rats. Based on the results of microarray and RT-qPCR detections, miR-218-5p was selected as the therapeutic target for nardosinone. The induction of OA resulted in tissue destruction and the production of cytokines in rat joint tissues, which was associated with the up-regulation of miR-218-5p and the downregulation of NUMB. For OA-affected rats treated with nardosinone, the joint structure was improved and the inflammatory response was suppressed, along with the restored expression levels of miR-218-5p and NUMB. The re-induced level of miR-218-5p compromised the anti-OA effects of nardosinone, indicating that the inhibition of the miR played an indispensable role in the anti-OA function of nardosinone. Collectively, the findings of our study demonstrated that nardosinone exerts treatment effects against OA by modulating the miR-218-5p/NUMB axis. Future studies will provide more detailed information on the interaction between nardosinone and miR in the attenuation of OA.

miR-218-5p Targeting TPX2 Regulates p53 Pathway and Inhibits Malignant Progression of Lung Adenocarcinoma / 中国肺癌杂志

BACKGROUND@#Lung adenocarcinoma (LUAD) is a major subtype of lung cancer, and its treatment and diagnosis remain a hot research topic. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is highly expressed in a variety of cancer cells and may be associated with the progression of LUAD. This study aimed to investigate the effect of TPX2 on the malignant progression of LUAD cells and the regulatory mechanisms.@*METHODS@#The expression of gene TPX2 in LUAD tissues from The Cancer Genome Atlas (TCGA) database was analyzed by bioinformatics analysis techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of TPX2 and miR-218-5p in human lung normal cell lines and human LUAD cell lines. Western blot was used to detect TPX2 protein expression in cell lines and its effect on the expression of key proteins in the p53 signaling pathway. The relationship between TPX2 and miR-218-5p was predicted using bioinformatics and verified by dual luciferase reporter gene assay. Cell counting kit-8 (CCK-8) assay, cell clone formation, cell scratching, Transwell assay, and flow cytometry were used to detect the effects of miR-218-5p and TPX2 on LUAD cell function.@*RESULTS@#TPX2 was significantly overexpressed in LUAD cells, and knockdown of TPX2 inhibited LUAD cell proliferation, migration, and invasion, promoted apoptosis and induced G2/M phase block, and promoted the expression of key proteins in the p53 signaling pathway. miR-218-5p, an upstream regulator of TPX2, could inhibit its expression. Overexpression of miR-218-5p eliminated the malignant development caused by high expression of TPX2, inhibited the malignant processes of LUAD cells such as proliferation and migration as well as promoted the p53 signaling pathway.@*CONCLUSIONS@#miR-218-5p targets and inhibits TPX2 expression and exerts an inhibitory effect on the malignant progression of LUAD cells via p53.

miR-218-5p Inhibits the Development of Colon Cancer by Regulating LAYN / 昆明医科大学学报

Objective To investigate the expression level of miR-218-5p and LAYN in colon cancer and the effects of their regulatory relationship on the progress of colon cancer.Methods RT-qPCR was used to detect the expression of miR-218-5p in colon cancer tissues,adjacent tissues,and cell lines.Western blot was used to detect the expression level of LAYN protein in colon cancer tissues,adjacent tissues,and cell lines.The in situ hybridization(ISH)was used to detect the expression of miR-218-5p in colon cancer and adjacent tissues.Immunohistochemical(IHC)experiment was used to detect the distribution and expression of LAYN protein in colon cancer tissues and adjacent tissues.The dual luciferase experiment was used to detect the binding relationship between miR-218-5p and 3'-rn-translation region(UTR)of LAYN.CCK-8 was used to detect the cell proliferation.Transwell was used to detect the invasion ability of cells.The flow cytometry was used to detect the apoptosis level of cells.Western blot was used to detect the expression level of cleaved Caspase3 in cells.Results The expression of miR-218-5p decreased in colon cancer tissues and colon cancer cell lines,with the lowest expression in HT-29 cells(P<0.0001),and the expression of LAYN increased and the highest expression in HT-29 cells(P<0.05).The double luciferase reporter gene experiment(P<0.01)combined with Western blot experiment(P<0.01)showed that miR-218-5p could bind to the 3'UTR region of LAYN and inhibit its protein expression.In cell experiments,overexpression of miR-218-5p inhibited cell proliferation(P<0.05),invasion(P<0.001),and increased apoptosis(P<0.01),and the expression of cleaved Caspase3 protein(P<0.01).Overexpression of LAYN promoted cell proliferation(P<0.05),invasion(P<0.001),and decreased apoptosis(P<0.0001)and the expression of cleaved Caspase3 protein(P<0.01),while overexpression of miR-218-5p and LAYN partially reversed the above changes.Conclusion The expression level of miR-218-5p is decreased in colon cancer tissues.Cell experiments show that miR-218-5p inhibits cell invasion and proliferation and promotes cell apoptosis through targeted regulation of LAYN.