Long Noncoding RNA Regulator of Reprogramming Regulates Cell Growth, Metastasis, and Cisplatin Resistance in Gastric Cancer via miR-519d-3p/HMGA2 Axis.

Background: Gastric cancer (GC) is a common tumor found worldwide, and cisplatin is the first-line agent for the treatment of GC. However, the resistance to cisplatin is an obstacle. Here, we explored the biological mechanism of long noncoding RNA regulator of reprogramming (ROR) in the cisplatin resistance of GC. Materials and Methods: ROR, miR-519d-3p, and high mobility group protein A2 (HMGA2) expression in GC tissues and cells were measured by quantitative real-time polymerase chain reaction and Western blot. Cell viability, migration, invasion, and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, transwell assay, and flow cytometry, respectively. The relative protein expression was detected by Western blot. The interactions between miR-519d-3p and ROR, HMGA2 were predicted using miRcode and starBase v2.0 online database, and then verified by dual luciferase reporter assay and RNA immunoprecipitation assay. In addition, the xenograft tumor mouse model was constructed to verify the biological role of ROR in vivo. Results: The levels of ROR, HMGA2 were significantly upregulated, and miR-519d-3p was apparently downregulated in GC tissues and cells. The miRcode and starBase v2.0 online websites and dual luciferase reporter assay validated that miR-519d-3p directly interacted with ROR and HMGA2. Furthermore, ROR knockdown downregulated HMGA2 to restrain cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and cisplatin resistance in GC cells by targeting miR-519d-3p. In addition, the depletion of ROR repressed the xenograft tumor growth in vivo. Conclusion: In conclusion, we first found the ROR/miR-519d-3p/HMGA2 regulatory network to regulate cell proliferation, migration, invasion, EMT, and cisplatin resistance in GC, and this may shed light on the GC tumorigenesis.

miR-519d-3p alleviates high glucose-induced human retinal microvascular endothelial cells dysfunction and inhibits angiogenesis by targeting hypoxia inducible factor 1 subunit alpha / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To clarify the effect of miR-519d-3p on high glucose-induced human retinal microvascular endothelial cells(HRMEC)dysfunction and angiogenesis, and to elucidate the regulatory mechanism of miR-519d-3p on hypoxia inducible factor 1 subunit alpha(HIF-1α).METHODS: The normal glucose(NG)and high glucose(HG)cell models were established by inducing HRMEC with 5 and 30 mmol/L glucose, respectively. Control group: HG cell model was transfected with negative control mimics; mannitol group: the control group was added with 25 mmol/L mannitol; miR-519d-3p overexpression group: HG cell model was transfected with miR-519d-3p mimics; miR-519d-3p combined with HIF-1α overexpression group: HG cell model was co-transfected with miR-519d-3p mimics and HIF-1α overexpression vector. The expression of miR-519d-3p in each group was tested by real-time fluorescence quantitative PCR. The expression of HIF-1α protein in each group was tested by Western blotting. The binding sites between miR-519d-3p and HIF-1α were detected by luciferase reporter gene assay. The cell proliferation of each group was detected by CCK-8. The cell apoptosis of each group was tested by Hoechst 33342 staining. The protein expression of extracellular fluid inflammatory factors tumor necrosis factor-α(TNF-α), interleukin(IL)-1β and IL-6 in each group was tested by ELISA. The formation of new capillary lumen-like structures was detected by tubule formation assay.RESULTS: Compared with the NG, miR-519d-3p expression was significantly reduced in the HG cell model, while HIF-1α protein expression was significantly increased in the HG(all P<0.01). Compared with the control group, HIF-1α protein expression was significantly reduced in the miR-519d-3p overexpression group(P<0.01). The “CGUGAAA” sequence of miR-519d-3p could specifically bind to the “GCACUUU” sequence of HIF-1α 3'-untranslated region(3'-UTR). Compared with the control group, the miR-519d-3p overexpression group showed a significant increase in 24, 48 and 72h absorbance values, a significant decrease in cell apoptotic rate, a significant decrease in the concentrations of TNF-α, IL-1β and IL-6, and a significant decrease in the number of new capillary lumen-like structures(all P<0.01). Compared with the miR-519d-3p overexpression group, the miR-519d-3p combined with HIF-1α overexpression group showed a significant decrease in 24, 48 and 72h absorbance values, a significant increase in cell apoptotic rate, a significant increase in the concentrations of TNF-α, IL-1β and IL-6, and a significant increase in the number of new capillary lumen-like structures(all P<0.01). There was no difference between the control group and mannitol group in the comparison of the above indicators(all P>0.05).CONCLUSION: miR-519d-3p expression is down-regulated while HIF-1α protein expression is up-regulated in high glucose induced HRMEC model. HIF-1α is a target gene of miR-519d-3p. The miR-519d-3p targets HIF-1α to increase cell proliferation and reduce cell apoptosis and inflammation, thereby alleviating high glucose-induced HRMEC dysfunction and inhibiting angiogenesis.

MicroRNA-519d-3p antagonizes osteosarcoma resistance against cisplatin by targeting PD-L1.

Accumulating evidence indicates that a ligand of programmed cell death receptor-1 (PD-L1) participates in the progression and recurrence of multiple malignancies, including osteosarcoma. Nevertheless, the role of PD-L1 in chemoresistance development is not fully understood. In the current study, we aim to clarify the interaction of miR-519d-3p and PD-L1 in the development of cisplatin resistance. Immunohistochemistry, quantitative reverse-transcription polymerase reaction, and Western blot were used to evaluate PD-L1 expression. MTT and transwell migration assays were used to measure cell growth and motility, respectively. ENCORI, miRCode, and miRDB databases were recruited to predict candidate miRNAs targeting PD-L1. The binding sequences of miR-519d-3p and PD-L1 3' untranslated region were identified by dual-luciferase reporter and RNA immunoprecipitation assays. Flow cytometric analysis was conducted to measure the cycle distribution and cell apoptosis. Metastatic mouse models were generated with cisplatin-resistant sublines by intravenous injection. We found that PD-L1 expression was positively correlated to cisplatin resistance and metastasis, whereas miR-519d-3p expression was reduced in cisplatin-resistant specimens and was negatively correlated to cisplatin resistance and metastasis of osteosarcoma. We demonstrated that miR-519d-3p overexpression reversed cisplatin resistance, induced G1/S phase arrest and apoptosis. In addition, we proved that miR-519d-3p inhibited lung metastasis by establishing cisplatin-resistant MG63 metastatic xenograft models. The present findings suggest that miR-519d-3p/PD-L1 axis is a novel signaling pathway contributing to cisplatin resistance. Our study provides new clues for curing refractory osteosarcoma beyond immune checkpoint inhibitors.

Reduced miR-519d-3p levels in the synovium and synovial fluid facilitate the progression of post-traumatic osteoarthritis by targeting VEGF.

The present study aimed to investigate the expression and clinical significance of miR-519d-3p in patients with post-traumatic osteoarthritis (PTOA). The levels of miR-519d-3p in the synovium and synovial fluid (SF) of all subjects were detected by reverse transcription-quantitative polymerase chain reaction. The results of the present study demonstrated that the levels of miR-519d-3p in the synovium and SF of patients with PTOA were significantly lower, but that the VEGF content was significantly higher, compared with that of control group. Dual-luciferase reporter and Western blot assays demonstrated that VEGF was a target gene of miR-519d-3p. Furthermore, miR-519d-3p inhibitor-induced cell apoptosis, and cell cycle arrest could be partially reversed by silencing VEGF. Additionally, the level of miR-519d-3p in the synovium and SF of patients with PTOA was negatively correlated with the level of VEGF. ROC analysis demonstrated that miR-519d-3p levels in the synovium and SF could effectively differentiate patients with PTOA from healthy controls, with areas under the ROC curve of 0.928 and 0.896, respectively. In conclusion, reduction of miR-519d-3p in the synovium and SF resulted in the upregulation of VEGF in patients with PTOA, and miR-519d-3p may be a potential therapeutic target of PTOA.

LINC00839/miR-519d-3p/JMJD6 axis modulated cell viability, apoptosis, migration and invasiveness of lung cancer cells.

INTRODUCTION: Long noncoding RNAs are associated with progressions of lung cancer. LINC00839 has been dysregulated in osteosarcoma, breast cancer and lung cancer (LC). As an upregulated lncRNA, the roles of LINC00839 in lung cancer remain unclear. MATERIAL AND METHODS: RNA expressions of LINC00839, miR-519d-3p and JMJD6 were assessed using RT-qPCR and JMJD6 protein expression were analyzed through Western blot. Meanwhile, viabilities of A549 and H460 LC cells transfected by siNC, siLINC00839, oeNC, oeLINC00839, NC mimics, miR-519d-3p mimics and oeLINC00839 with siJMJD6 were examined with CCK-8 assay while apoptosis was examined using flow cytometry. Meanwhile, migration and invasiveness were analyzed using transwell assays. Bindings between LINC00839 and miR-519d-3p, miR-519d-3p and JMJD6 were measured by luciferase reporter assays. RESULTS: LINC00839 was upregulated in LC cells and its knockdown resulted in reduced cell viability, migratory ability and invasion with increased cell apoptosis. MiR-519d-3p was the target gene of LINC00839 and its expression was reduced by LINC00839 overexpression. JMJD6 was directly targeted and suppressed at the level of mRNA and protein expression by miR-519d-3p. Moreover, miR-519d-3p overexpression resulted in low LC cell viability, migration, invasiveness but a high apoptosis rate. Furthermore, mRNA and protein expressions of JMJD6 were upregulated by LINC00839 overexpression. LINC00839 competitively sponged miR-519d-3p, increasing JMJD6 expression, LC cell viability, invasion, migratory abilities and decreasing apoptosis rates in A549 and H460 lung cancer cells, which were hindered after JMJD6 knockdown. CONCLUSIONS: LINC00839/miR-519d-3p/JMJD6 axis mediated cell viability, apoptosis, and migration and invasiveness of H460 lung cancer cells.

miR-519d-3p suppresses tumorigenicity and metastasis by inhibiting Bcl-w and HIF-1α in NSCLC.

Bcl-w, a member of the Bcl-2 family, is highly expressed in various solid tumor, including lung cancer, suggesting that it is involved in cancer cell survival and carcinogenesis. Solid cancer-induced hypoxia has been reported to increase angiogenesis, growth factor, gene instability, invasion, and metastasis. Despite many studies on the treatment of non-small cell lung cancer (NSCLC) with a high incidence rate, the survival rate of patients has not improved because the cancer cells acquired resistance to treatment. This study investigated the correlation between Bcl-w expression and hypoxia in tumor malignancy of NSCLC. Meanwhile, microRNAs (miRNAs) are involved in a variety of key signaling mechanisms associated with hypoxia. Therefore, we discovered miR-519d-3p, which inhibits the expression of Bcl-w and hypoxia-inducing factor (HIF)-1α, and found that it reduces hypoxia-induced tumorigenesis. Spearman's correlation analysis showed that the expression levels of miR-519d-3p and Bcl-w/HIF-1α were negatively correlated, respectively. This showed that miR-519d-3p can be used as a diagnostic biomarker and target therapy for NSCLC.

H19 promotes aerobic glycolysis, proliferation, and immune escape of gastric cancer cells through the microRNA-519d-3p/lactate dehydrogenase A axis.

Long noncoding RNAs (lncRNAs) have been investigated in multiple human cancers including gastric cancer (GC). Our research aims to explore the role of H19 in aerobic glycolysis, proliferation, and immune escape of GC cells. The expression of H19 in GC samples was analyzed using Gene Expression Profiling Interactive Analysis, Gene Expression Omnibus data, and real-time quantitative PCR analysis. Relative quantification of glucose consumption and lactate production from cell supernatant were applied to assess the aerobic glycolysis of GC cells. Subcellular fractionation, luciferase reporter, and western blot assays certified the binding between genes. Cell Counting Kit-8 and colony formation assays were used to determine GC cell proliferation. Flow cytometry, ELISA, and real-time quantitative PCR assays were applied to analyze the immunosuppressive effect of H19. H19 was highly expressed in samples of patients with GC, and associated with tumor growth in vivo. H19 knockdown suppressed glucose consumption, lactate production, and proliferation of GC cells by regulating the microRNA (miR)-519d-3p/lactate dehydrogenase A (LDHA) axis. Both miR-519d-3p depletion and LDHA overexpression could reverse the H19 knockdown-induced decrease in aerobic glycolysis and proliferation. Moreover, conditioned medium from stable knockdown H19 GC cells modulated the activity of immune cells including γδT cells, Jurkat cells, and tumor-associated macrophages in a miR-519d-3p/LDHA/lactate axis-dependent manner. The H19/miR-519d-3p/LDHA axis mainly contributed to aerobic glycolysis, proliferation, and immune escape of GC cells.

LncRNA BLACAT1/miR-519d-3p/CREB1 Axis Mediates Proliferation, Apoptosis, Migration, Invasion, and Drug-Resistance in Colorectal Cancer Progression.

BACKGROUND: Colorectal cancer (CRC) is a common severe disease around the world. The merging papers reported that long noncoding RNAs (lncRNAs) took part in the diversified pathological processes of CRC. This study aimed to uncover the role and the potential mechanism of lncRNA bladder cancer-associated transcript 1 (BLACAT1) in CRC progression. METHODS: LncRNA BLACAT1, micro-519d-3p (miR-519d-3p), and cAMP-responsive element binding protein 1 (CREB1) levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in CRC tissues and cells. The bio-functional effects were examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), flow cytometry assay, and transwell assay. The susceptibility testing was determined by oxaliplatin (OXA) administration. The potential binding sites between miR-519d-3p and BLACAT1 or CREB1 were predicted by online software starBase and confirmed by dual-luciferase reporter analysis. The relative proteins expression in CRC cells was determined by Western blot analysis. Xenograft tumor model was used to evaluate biological function of BLACAT1 in vivo. RESULTS: The expression of BLACAT1 was promoted in CRC tissues and cells, and correlated to the TNM (tumor, node, metastasis) stage, distant metastasis, and overall survival rate. Silencing of BLACAT1 limited the proliferation, migration, and invasion, facilitated the apoptosis, and re-sensitized OXA-resistance in CRC cells. MiR-519d-3p was a target of BLACAT1. Furthermore, miR-519d-3p deletion reversed the positive effects of BLACAT1 deletion on CRC cells. Moreover, our data showed that miR-519d-3p directly targeted CREB1 and BLACAT1 sponged miR-519d-3p to regulate CREB1 expression. Besides, CREB1 disrupted the bio-functional results above from BLACAT1 suppression. Additionally, BLACAT1 knockdown promoted CRC cells sensitivity to OXA in vivo. CONCLUSION: BLACAT1 mediated the progression of CRC and OXA-resistance by miR-519d-3p/CREB1 axis.

miR-519d-3p Overexpression Inhibits P38 and PI3K/AKT Pathway via Targeting VEGFA to Attenuate the Malignant Biological Behavior of Non-Small Cell Lung Cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a heterogeneous tumor that accounts for approximately 85% of all lung cancer cases worldwide. microRNAs (miRNAs) are believed to play an important role in regulating a variety of biological processes, including immunity and cancer. We investigated the effect of miR-519d-3p on the mitigation of NSCLC in vitro and in vivo. METHODS: RT-PCR or immunohistochemical assays were used to assess the expression of miR-519d-3p. Colony formation, flow cytometry, and transwell assay were respectively used to detect proliferation, apoptosis, and invasion of A549 and NCI-H661 cell lines. Luciferase reporter assay was used to verify targeting the relationship between mir-519d-3p and VEGFA. Western blot was used to examine the expression of Ki67, caspase-3, E-cadherin, N-cadherin, VEGF, P38, and PI3K/AKT. Animal models were established by BABL/c mice to research the effect of mir-519d-3p overexpression in vivo. RESULTS: In vitro, miR-519d-3p overexpression inhibited A549 and NCI-H661 cells proliferation, invasion, and also promoted apoptosis. In addition, miR-519d-3p overexpression downregulated VEGFA expression and decreased the P38 and PI3K/AKT phosphorylation level. In vivo, miR-519d-3p overexpression significantly restrained tumor volume (2087±265 mm3 vs 599±135 mm3, *P< 0.05) and tumor weight (0.45±0.08 g vs 0.13±0.06 g, *P<0.05) compared with the control group. Overexpression of miR-519d-3p downregulated levels of Ki67 and N-cadherin significantly. CONCLUSION: The data indicated that miR-519d-3p could be a novel therapy or adjuvant against NSCLC.

Upregulation of miR-519d-3p Inhibits Viability, Proliferation, and G1/S Cell Cycle Transition of Oral Squamous Cell Carcinoma Cells Through Targeting CCND1.

Background: MicroRNA (miR)-519d-3p suppresses tumor development, however, its role in oral squamous cell carcinoma (OSCC) waited to be further determined. Materials and Methods: OSCC and adjacent tissues were collected (n = 45 for adjacent; n = 21 for Stage I-II OSCC; n = 24 for Stage III-IV OSCC). The cell viability, proliferation, and cell cycle of OSCC were, respectively, assessed by the Cell Counting Kit-8 (CCK-8), colony formation assay, and flow cytometry. Relative expressions of cell cycle-regulated proteins (Cyclin D1 [CCND1], CDK4, and CDK6) and miR-519d-3p were measured with western blot and quantitative real-time polymerase chain reaction as needed. Dual-luciferase reporter assay was performed to verify the prediction of TargetScan that miR-519d-3p and CCND1 shared potential binding sites. Correlation analysis between miR-519d-3p and CCND1 was performed with Pearson's correlation test. Results: In OSCC tissues, downregulating miR-519d-3p expression correlated with a higher tumor grade. Upregulating miR-519d-3p expression inhibited OSCC cell viability and proliferation, increased cells in G0/G1 phase and reduced those in S/G2 phase, and downregulated the expressions of cell cycle-related protein (CDK4, CDK6). CCND1 was the target gene of miR-519d-3p, and overexpressed CCND1 reversed the effects of upregulation of miR-519d-3p on suppressing the viability, proliferation, and cell cycle of OSCC cells. Conclusions: miR-519d-3p upregulation suppressed the cell viability, proliferation, and G1/S cell cycle transition of OSCC through targeting CCND1. The current findings provide a possible clinical option for OSCC treatment.

circ_0007385 served as competing endogenous RNA for miR-519d-3p to suppress malignant behaviors and cisplatin resistance of non-small cell lung cancer cells.

BACKGROUND: Circular RNAs (circRNAs) have been closely implicated in competing endogenous RNA (ceRNA) network among human cancers including non-small cell lung cancer (NSCLC). However, the role of most circRNAs in NSCLC remains to be determined. Here, we aimed to investigate the role of hsa_circ_0007385 (circ_0007385) in NSCLC cells. METHODS: Expression of hsa_circ_0007385 (circ_0007385), miRNA (miR)-519d-5p and high-mobility group box 1 (HMGB1) was measured by real-time quantitative PCR and western blotting. Functional experiments were evaluated by cell counting kit (CCK)-8, flow cytometry, fluorescein active caspase-3 staining kit, transwell assays, western blotting, and xenograft experiment. The relationship among circ_0007385,miR-519d-5p and HMGB1 was testified by dual-luciferase reporter assay. Kaplan-Meiersurvival curve identified overall survival in NSCLC patients. RESULTS: circ_0007385 expression was higher in NSCLC tissues and cell lines, and was associated with poor overall survival. Silencing circ_0007385 could suppress cell proliferation, migration and invasion in A549 and H1975 cells, as well as cisplatin (DDP) resistance. Moreover, circ_0007385 silence retarded tumor growth of A549 cells in vivo. Molecularly, there was a direct interaction between miR-519d-3p and either circ_0007385 or HMGB1; expression of miR-519d-3p was downregulated in NSCLC tumors in a circ_0007385-correlated manner, and circ_0007385 could indirectly regulate HMGB1 via miR-519d-3p. Functionally, both inhibiting miR-519d-3p and restoring HMGB1 could overturn the suppressive effect of circ_0007385 knockdown on cell proliferation, migration, invasion, and DDP resistance. CONCLUSIONS: Collectively, circ_0007385 deletion could function anti-tumor role in NSCLC by suppressing malignant behaviors and DDP resistance in vitro and in vivo via circ_0007385/miR-519d-3p/HMGB1 axis. These outcomes might enhance our understanding of the molecular mechanisms underlying the malignant progression of NSCLC. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: circ_0007385 was upregulated in NSCLC tissues and cells, and was associated with poor overall survival. Silenced circ_0007385 suppressed NSCLC cell proliferation, migration, invasion, and DDP resistance in vitro, and tumor growth in vivo. circ_0007385 was upregulated in NSCLC tissues and cells, and was associated with poor overall survival. WHAT THIS STUDY ADDS: miR-519d-3p could directly interact with circ_0007385 and HMGB1 in NSCLC cells. A promising circ_0007385/miR-519d-3p/HMGB1 regulatory pathway was determined in NSCLC cells.

Upregulation of LncRNA PVT1 Facilitates Pancreatic Ductal Adenocarcinoma Cell Progression and Glycolysis by Regulating MiR-519d-3p and HIF-1A.

The long, noncoding RNA (lncRNA) PVT1, as an important epigenetic regulator, has a critical role in carcinogenesis. However, its role in pancreatic ductal adenocarcinoma (PDAC) has not been fully investigated. Here, the up-regulated expression of lncRNA PVT1 is found in our PDAC tumor samples. Knockdown of it suppressed PDCA cells growth and glycolysis. An inverse association between miR-519d-3p and PVT1 was found. RIP, RNA pulldown and luciferase assay showed that PVT1 directly targets miR-519d-3p by binding with microRNA binding site. Bioinformatics analysis and study indicated that HIF-1A is a target of miR-519d-3p. Collectively, our findings suggested that PVT1 could act as an oncogenic lncRNA, and promote tumor progression by regulating HIF-1A via competing with miR-519d-3p.

Down-regulation of lncRNA BLACAT1 inhibits ovarian cancer progression by suppressing the Wnt/ß-catenin signaling pathway via regulating miR-519d-3p.

Ovarian cancer has the highest mortality in gynecologic malignancies. LncRNA BLACAT1 serves crucial functions in various cancers, but its role in ovarian cancer has not been investigated. In this article, our team explored the role and the potential regulatory mechanism of BLACAT1 in ovarian cancer. Quantitative RT-PCR showed that BLACAT1 was aberrantly up-regulated in ovarian cancer tissues compared with normal tissues. In vitro, BLACAT1 knockdown induced cell cycle arrest and inhibited the proliferation, migration and invasion of ovarian cancer cells using flow cytometry, MTT and EdU assays, wound healing assay and transwell assay, respectively. Luciferase assay verified the binding relationship between microRNA-519d-3p and lncRNA BLACAT1, and BLACAT1 negatively regulated the expression of miR-519d-3p. We also found that miR-519d-3p overexpression could inhibit ovarian cancer cells proliferation, migration and invasion. Further, Western blot demonstrated that the expression of RPS15A and nuclear ß-catenin expression was markedly reduced by BLACAT1 knockdown, and cytoplasmic ß-catenin level was not obviously affected. In vivo, BLACAT1 knockdown inhibited the tumor growth, and immunohistochemistry showed that ki67 expression was decreased by BLACAT1 suppression. Inhibition of BLACAT1 was sufficient to down-regulate the expression of RPS15A and nuclear ß-catenin but did not cause an obvious change in cytoplasmic ß-catenin expression. Taken together, BLACAT1 knockdown inhibited the progression of ovarian cancer by suppressing the Wnt/ß-catenin signaling pathway via regulating miR-519d-3p. Our work provided a proper understanding of the critical roles of BLACAT1 in ovarian cancer.

MicroRNA-519d-3p inhibits cell proliferation and cell cycle G1/S transition in glioma by targeting CCND1.

Glioma is the most common highly malignant primary brain tumor. MicroRNA-519d-3p exerts important effects in several tumors, but its functional role in glioma remained poorly understood. In this study, we found miR-519d-3p expression was significantly decreased in glioma tissues and cell lines. Moreover, the in vitro experiments showed that overexpression of miR-519d-3p suppressed cell proliferation and induced cell cycle G0/G1 phase arrest using MTT and flow cytometry assays in glioma cell lines, U87 and U251. Mechanistically, Cyclin D1 (CCND1) was predicted and confirmed as the direct target genes of miR-519d-3p using luciferase report assay. In addition, knockdown of CCND1 imitated the suppressive effects of miR-519d-3p on cell proliferation and cell cycle progression. Furthermore, restoration of CCND1 reversed the effects of miR-519d-3p overexpression in glioma cells. Taken together, these data demonstrate that suppression of CCND1 by miR-519d-3p might be a therapeutic target for glioma.Abbreviations miR-519d-3p: microRNA-519d-3p; CCND1: Cyclin D1; ATCC: American Type Culture Collection; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PI: propidium iodide; WT: wild type; MUT: mutant type; SD: standard deviation.

lncRNA HOTAIR promotes malignant biological behaviors of breast cancer SKBR3 cells through miR-519d-3p/CCND1 axis / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To explore the effect of lncRNA HOTAIR/miR-519d-3p/cyclin D1 (CCND1) axis on the proliferation and metastasis of breast cancer cells and its underlying mechanism. Methods: A total of 50 pairs of breast cancer tissues and corresponding para-cancer tissues resected from breast cancer patients in the Department of Breast Surgery, the Third Hospital of Nanchang from March 2017 to February 2019 were collected for this study. The expression level of HOTAIR in breast cancer tissues and paired paracancer tissues was detected by qPCR, in addition, the expressions of HOTAIR and miR-519d-3p in normal breast epithelial cells and breast cancer cell lines were also detected. Breast cancer SKBR3 cells were divided into NC group (without any treatment), si-HOTAIR group, mir-519d-3p mimics group, miR-519d-3p mimic+pcHOTAIR group, miR-519d-3p mimic+pcCCND1 group, and si-HOTAIR+ pcCCND1 group. The proliferation ability of SKBR3 cells was detected by CCK-8. Invasion and migration of SKBR3 cells were detected by Transwell. The expression levels of E-cadherin, N-cadherin, Vimentin and CCND1 in SKBR3 cells were detected by Western blotting. The targeting relationship between HOTAIR and miR-519d-3p, miR-519d-3p and CCND1 was detected by Dualluciferase reporter gene system. Results: HOTAIR was highly expressed in breast cancer tissues and cell lines, with the highest expression in SKBR3 cells. HOTAIR knockdown significantly inhibited the proliferation, invasion and migration of SKBR3 cells, as well as increased the expression level of E-cadherin and decreased the expression levels of N-cadherin and Vimentin. Dual-luciferase reporter gene assay showed that HOTAIR targetedly down-regulated the expression of miR-519d-3p, and miR-519d-3p targetedly downregulated the expression of CCND1. Further studies showed that knockout of HOTAIR inhibited the EMT, proliferation, invasion and migration of SKBR3 cells through enhancing the inhibitory effect of miR-519d-3p on CCND1 expression (all P<0.05). Conclusion: HOTAIR knockdown inhibits proliferation and metastasis of SKBR3 cells by regulating the axis of miR-519d-3p/CCND1.

lncRNA HOTAIR Protects Myocardial Infarction Rat by Sponging miR-519d-3p.

Myocardial infarction (MI) is a cardiovascular disease with high morbidity and mortality. In this study, we focused on exploring the roles and underlying regulatory mechanisms of Hox transcript antisense intergenic RNA (HOTAIR) and miR-519d-3p in myocardial infarction. To comprehensively understand the role of microRNA in MI rat, we construct MI rat model by permanent ligation of the left anterior descending (LAD) coronary artery. Cardiac troponin I and creatine kinase-MB concentration measured by ELISA and infract size of heart section analyzed by TTC staining were served as evaluation indicators to confirmed the established model. Based on the bioinformatics assay and qRT-PCR, we found that the expression of miR-519d-3p was upregulated remarkably. Dual-luciferase reporter assays were performed to investigate the interaction of lncRNA HOTAIR and miR-519d-3p. In order to investigate the potential mechanism of lncRNA HOTAIR and miR-519d-3p, flow cytometry was applied to measure apoptotic cardiomyocytes and western blot was used to detect expressions of apoptotic related protein Bax, Bcl-2, and caspase-3 in cardiomyocytes in vitro and myocardial infraction in vivo. Downregulating miR-519d-3p or overexpressing HOTAIR alleviated MI or hypoxia-induced cardiomyocytes apoptosis. Taken together, our results showed that the interaction of miR-519d-3p and HOTAIR can protect MI and hypoxia-induced cardiomyocytes apoptosis, providing the potential therapeutic target for MI treatment.

Long noncoding RNA PVT1 promotes laryngeal squamous cell carcinoma development by acting as a molecular sponge to regulate miR-519d-3p.

OBJECTIVE: This study was designed to investigate the effects and mechanism of long noncoding RNA (lncRNA) PVT1 on cell migration, proliferation, and apoptosis of laryngeal squamous cell carcinoma (LSCC). METHODS: We screened lncRNAs expression profiles in four pair LSCC and matched noncancerous tissues by microarray assay. The messenger RNA levels of PVT1 in tissues and cells were evaluated by quantitative real-time polymerase chain reaction analysis. StarBase website was used to predict the target miRNAs for PVT1. And the interaction between PVT1 and target miRNA-519d-3p in LSCC cells was analyzed using dual-luciferase reporter assay. MTT assay was used to investigate the cell viability. Cell counting assay was used to explore the cell proliferation. Annexin-V propidium iodide flow cytometry was used to examine the cell apoptosis, and transwell assay was used to investigate the effects of lncRNA PVT1 on cell migration. RESULTS: PVT1 was significantly overexpressed in human LSCC tissues and several LSCC cell lines. Upregulation of lncRNA PVT1 markedly facilitated proliferation suppressed apoptosis and promoted cell migration in LSCC cells. We further demonstrated that silencing PVT1 strikingly suppressed proliferation, promoted apoptosis, and reduced migration in LSCC cells. Further bioinformatic analysis and dual-luciferase reporter assay revealed that PVT1 could function as an oncogenic transcript partly through sponging miR-519d-3p. Besides, mechanistic investigations indicated that PVT1 could promote cell and migration through interacting with miR-519d-3p. CONCLUSION: LncRNA PVT1 is consistently overexpressed in human LSCC, and overexpression of lncRNA PVT1 contributes to the proliferation and migration of LSCC through inhibiting miR-519d-3p expression.

MicroRNA-519d-3p inhibits cell proliferation and migration by targeting TROAP in colorectal cancer.

Increasing evidence suggests that miR-519d-3p functions as tumor suppressor in several tumors, including breast cancer. However, its biological role in the development of colorectal cancer (CRC) still remains unclear. In this study, we found that miR-519d-3p expression level was remarkably down-regulated in CRC tissues samples and cell lines when compared to adjacent normal tissues and cell line by using qRT-PCR detection. Lower miR-519d-3p expression was significantly correlated with TNM stage, tumor size and lymph node metastasis. CRC patients with high level of miR-519d-3p had higher five-year survival rate than those with low expression of miR-519d-3p (p = 0.01178) using Kaplan-Meier analysis. Moreover, multivariate analysis suggested that miR-519d-3p expression might be an independent prognostic indicator for the survival of CRC patients. The in vitro functional analysis, including MTT, flow cytometry and transwell assays indicated that miR-519d-3p overexpression significantly suppressed cell proliferation, migration and invasion, induced cell cycle G0/G1 phase arrest and cell apoptosis of CRC cells. Furthermore, bioinformatics and luciferase reporter assays verified that trophinin associated protein (TROAP) was a direct target of miR-519d-3p in CRC cells. Using Oncomine database analysis, TROAP was confirmed to be upregulated in human CRC tissues. In addition, we found knockdown of TROAP presented similar inhibitory effects of miR-519d-3p overexpression in CRC cell function. In conclusion, miR-519d-3p might be a promising therapeutic strategy against human CRC by directly targeting TROAP.

miR-519d-3p Inhibits Cell Proliferation and Invasion of Gastric Cancer by Downregulating B-Cell Lymphoma 6.

miR-519d inhibits cell growth, migration, and invasion, but its role in gastric cancer (GC) cells is obscure. We showed that miR-519d-3p was lowly expressed in GC tissues and was associated with the clinical stage and lymph node metastasis of GC tissues. We found that miR-519d-3p repressed cell proliferation and invasion of MGC803 cells and delayed the G1/S phase transition, resulting in decreased cyclin B1 and MMP2 and increased E-cadherin levels. Furthermore, miR-519d-3p targeted and downregulated B-cell lymphoma 6 (BCL6) expression. BCL6 overexpression partially abrogated the suppressive function of miR-519d in MGC803 cells. In conclusion, our study demonstrated that miR-519d-3p functions as a tumor suppressor by targeting and downregulating the expression of BCL6 in GC cells.

MiR-519d-3p suppresses breast cancer cell growth and motility via targeting LIM domain kinase 1.

Breast cancer is the most common female cancer in women, and its estrogen receptor (ER)-negative subtype (ENBC) and triple-negative subtype (TNBC) have unfavorable prognosis in comparison with ER-positive subtype. MiRNAs are small noncoding RNAs that bind to the 3'-UTR region of targeting mRNAs to regulate gene expression. Mir-519d-3p was found to be associated with breast cancer for its potential role in proliferation and metastasis. To explore its potential role and mechanism of miR-519d-3p in breast carcinogenesis, we determined whether miR-519d-3p regulates breast cancer cell proliferation and motility by performing wound-healing assays and migration-invasion assays. We found that miR-519d-3p significantly inhibits proliferation and motility of ENBC and TNBC cells. Overexpression of miR-519d-3p arrested breast cancer cells in the G0/G1 phase and reduced the expression of CDK4, 6/Cyclin D1, and CDK2/Cyclin E1. It was reported that miR-519d-3p or miR-519d-3p expression was associated with cancer metastasis and clinical staging. Since LIM domain kinase 1 (LIMK1) was highly expressed in breast cancer and a major regulator of breast cancer growth and metastasis, we further demonstrated that LIMK1 is a potential target of miR-519d-3p by dual-luciferase report assay. Mir-519d-3p decreases LIMK1 expression at mRNA and protein levels, and the protein level and phosphorylation of cofilin 1 (CFL1), one of the key downstream targets of LIMK1. Our findings suggest that miR-519d-3p regulates the LIMK1/CFL1 pathway in breast cancer and this new venue could be targeted for future breast cancer therapy.