Negative Control miRNA Research Articles

MicroRNA-126-3p Attenuates Intracerebral Hemorrhage-Induced Blood-Brain Barrier Disruption by Regulating VCAM-1 Expression.

BackgroundmiR-126 is closely related to the occurrence of various complications after intracerebral hemorrhage (ICH), but the molecular mechanism is not fully elucidated. This study aimed to explore the mechanism of miR-126-3p in alleviating brain injury after ICH.MethodsSerum miR-126-3p levels were compared between patients with IHC and healthy controls. A rat model of ICH was generated by intracerebral injection of Type VII collagenase. The rats were intracerebral injected with miR-126-3p mimics or negative control miRNA. Rat brain microvascular endothelial cells (BMECs) were used as a cell model of blood-brain barrier (BBB), and validated by immunofluorescence staining of Factor VIII. The BBB permeability of BMECs after miR-126-3p antagomir transfection was determined by FITC-dextran 20 through a confluent BMECs layer (measured over 120 min). The binding site of miR-126-3p in the 3′UTR of VCAM-1 was predicated by TargetScan, and verified by dual luciferase reporter assay. The expression levels of miR-126-3p and vascular cell adhesion molecule-1 (VCAM-1) in rat brain tissues and BMECs were measured by real-time PCR or western blotting.ResultsSerum miR-126-3p level was markedly down-regulated in patients with ICH. The rats with ICH had decreased miR-126-3p levels in serum and hemorrhagic area, while those changes were reversed by the treatment with miR-126-3p mimic. VCAM-1 is a direct target of miR-126-3p, and VCAM-1 expression in hemorrhagic area was down-regulated by the administration of miR-126-3p mimic in rats. Inhibition of miR-126-3p by anti-miR126 treatment in BMECs resulted in barrier leakage.ConclusionmiR-126-3p attenuates intracerebral hemorrhage-induced blood-brain barrier disruption, which is associated with down-regulated expression of VCAM-1 in hemorrhagic area.

Mechanism of miR-7 affecting migration ability of intestinal epithelial cells

Objective To investigate the effect of miR-7 on the migration ability of intestinal epithelial cells and its related mechanisms. Methods LS174T cells were transfected with miR-7 mimic and miR-7 inhibitor respectively.The effect of miR-7 on cell migration ability was detected by transwell chamber.The changes of PI3K, p-Akt and Akt protein in each group were detected by Western Blot. Results Compared with the control group, miRNA mimic negative control group and miRNA inhibitor negative control group, the number of cells in the miR-7 mimic group through the tranwell chamber decreased, the cell migration rate decreased, and the expression of PI3K and p-Akt protein decreased at the same time, the difference was statistically significant(P<0.05). In the miRNA inhibitor group, the number of cells in the tranwell chamber increased, the cell migration rate increased, and the expression of PI3K and p-Akt protein was increased at the same time, the difference was statistically significant(P<0.05). Conclusion miR-7 can inhibit the migration of LS174T cells, which may be through the regulation of the PI3K/Akt signaling pathway. Key words: miR-7; Intestinal epithelial cell; Migration; Mechanism

Effect of downregulation of microRNA-373 expression on cell cycle and apoptosis of a cutaneous squamous cell carcinoma cell line A431

Objective To evaluate the effect of downregulation of microRNA (miR) -373 expression on cell cycle and apoptosis of a cutaneous squamous cell carcinoma (CSCC) cell line A431. Methods A431 cells at exponential growth phase were classified into 3 groups: miR-373 inhibitor group and negative control group transfected with miR-373 inhibitor and negative control miRNA respectively, and untreated group receiving no treatment. At 48 hours after the transfection, real-time PCR was performed to determine the expression of miR-373 in the above 3 groups, cell counting kit-8 (CCK-8) assay to evaluate the effect of downregulated expression of miR-373 on the proliferation of A431 cells, flow cytometry to investigate the distribution of cell cycle and changes in apoptosis of A431 cells in different treatment groups, and colorimetric analysis to detect the changes in caspase-3 activity in different treatment groups. Statistical analysis was carried out with SPSS 17.0 software by using two-sample t test for the comparison between two groups, one-way analysis of variance (ANOVA) for the comparison among 3 groups, and least significant difference (LSD) -t test for multiple comparisons. Results The expression of miR-373 was significantly lower in the miR-373 inhibitor group (0.120 ± 0.036) than in the untreated group (1.002 ± 0.022) and negative control group (1.037 ± 0.028, LSD-t=36.21, 34.83, respectively, both P < 0.001) . At 48, 72 and 96 hours, the miR-373 inhibitor group showed significantly decreased proliferative activity of A375 cells compared with the untreated group and negative control group (F=10.805, 13.720 and 30.907 respectively, P=0.038, 0.010 and 0.001 respectively) . The proportion of A375 cells in G0/G1 phase was significantly higher in the miR-373 inhibitor group (64.69% ± 1.18%) than in the untreated group (52.74% ± 0.66%, t=15.51, P < 0.001) and negative control group (53.80% ± 0.80%, t=13.24, P < 0.001) . The proportion of total apoptotic cells and activity of caspase-3 in the miR-373 inhibitor group were 22.69% ± 1.24% and 1.238 ± 0.057 respectively, which were significantly higher than those in the untreated group (9.62% ± 1.14%, 0.413 ± 0.028 respectively, both P < 0.001) and negative control group (9.66% ± 0.97%, 0.437 ± 0.036 respectively, both P < 0.001) . Conclusion MiR-373 may play an important role in the regulation of cell cycle and induction of apoptosis of the CSCC cell line A431. Key words: Neoplasms, squamous cell; MicroRNAs; Cell cycle; Apoptosis; Caspase 3; microRNA-373

Abstract 1878: MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions

Abstract The epithelial-to-mesenchymal transition (EMT), and its reverse process, the mesenchymal-to-epithelial transition (MET), are associated with metastatic tumor progression in breast cancer (BC). E-selectin, a cell adhesion molecule expressed by cytokine-activated vascular endothelium, is also involved in metastasis via binding to counter-receptors (E-selectin ligands) on the surface of BC cells. Consequently, efforts have been made to investigate a link between the EMT/MET and the adhesive interactions between E-selectin and their ligands. Our lab previously demonstrated that BC cells with an epithelial phenotype exhibit more adhesive interactions between E-selectin/ligands under blood flow conditions than cells with a mesenchymal phenotype. Therefore, molecular factors in the blood that activate the EMT or the MET in circulating tumor cells (CTCs) have been of particular interest. These factors may regulate the potential for CTCs to traffic to bone marrow endothelium, a frequent site of BC metastasis that constitutively expresses E-selectin. To advance knowledge in this area, we hypothesized that miRNA-200c, associated with poor prognosis in metastatic BC patients, induces the MET in MDA-MB-231 BC cells and promotes adhesion between E-selectin/ligands. Microscopy images show that MDA-MB-231 cells transfected with miRNA-200c displayed a cell morphology similar to that of epithelial cells, whereas cells transfected with a negative control miRNA (NC miRNA) retained their mesenchymal phenotype. Furthermore, cells transfected with miRNA-200c exhibited greater mRNA expression of E-cadherin and lower expression of Vimentin compared to cells transfected with a NC miRNA by qRT-PCR. The expression of Zeb1/2, both targets for miRNA-200c, were down-regulated in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Both the microscopy images and mRNA expression analysis indicate induction of MET in miRNA-200c transfected cells. Shear flow adhesion assays and shear flow detachment assays were performed at bone marrow shear stresses to examine adhesive interactions between E-selectin/ligands. Using these assays, MDA-MB-231 cells transfected with miRNA-200c demonstrated slower rolling velocities, greater firm adhesion, lower detachment, and equivalent tethering activity when compared to cells transfected with a NC miRNA. Moreover, analysis of the mRNA expression of α1,3- and α1,4- fucosyltransferases (FT) revealed increased expression of FT3 and FT6 in cells transfected with miRNA-200c compared to cells transfected with a NC miRNA. Altogether, these results suggest that miRNA-200c increases fluid-shear resistant adhesion and that factors present in the blood may impact the metastatic potential of cancer cells in circulation by regulating cellular phenotypes. Citation Format: Christian A. Showalter, Monica M. Burdick. MicroRNA-200c (miRNA-200c) increases adhesive interactions between E-selectin ligands expressed by breast cancer cells and E-selectin under physiological blood flow conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1878.

β-catenin regulates effects of miR-24 on the viability and autophagy of glioma cells.

Mutations of the β-catenin gene are common in various cancer types. MicroRNA (miR)-24 suppresses gene expression during the cell cycle. However, the effects of miR-24 on the cell viability and autophagy of glioma cells, and how these biological processes are regulated by β-catenin are largely unclear. The current study aimed to investigate the role of β-catenin in regulating the effects of miR-24 on the cell viability and autophagy of glioma cells. The expression levels of microtubule-associated proteins 1A/1B light chain 3B (LC3B) and Beclin1 were detected by immunohistochemistry and western blotting. Glioma C6 cells were transfected with miR-24 mimics, miR-24 inhibitors and negative control miRNAs. C6 cells transfected with miR-24 mimics or negative control miRNAs were treated with the β-catenin inhibitor, XAV-939. An MTT assay was utilized to evaluate the viability of C6 cells. The expression of miR-24 and mRNA expression of autophagy related 4a cysteine peptidase (ATG4A) were detected by quantitative polymerase chain reaction analysis. The protein expression of LC3B and Beclin1 decreased significantly in glioma tissue and glioma C6 cells compared with normal brain tissue. Compared with the negative control group, C6 cells transfected with miR-24 mimics exhibited significantly higher cell viability at 24 and 48 h, and those transfected with miR-24 inhibitors exhibited significantly lower cell viability at 48 h. XAV-939 decreased the stimulatory effects of miR-24 mimics on the viability of C6 cells. The expression of miR-24 significantly decreased and ATG4A mRNA significantly increased in C6 cells transfected with XAV-939 compared with those transfected with the negative control miRNA. XAV-939 attenuated the miR-24-induced decrease of the protein expression of LC3B and Beclin1, and decreased the stimulatory effects of miR-24 mimics on cell viability. In addition, XAV-939 attenuated the miR-24-induced decrease of autophagy marker expression by attenuating miR-24 expression and increasing ATG4A mRNA expression in glioma C6 cells. To the best of our knowledge, the present study is the first to demonstrate whether β-catenin regulates the intracellular effects of miR-24 on the viability and autophagy of glioma cells. The results also provide some mechanistic basis to the pharmaceutical targeting of WNT signaling in high grade glial tumors.

Effcets of mesenchymal stromal cell-derived exosomes deliver microRNA-145-5p on pancreatic ductal adenocarcinoma progression

Objective To investigate whether human umbilical cord mesenchymal stem cells-derived exosomes could inhibit pancreatic ductal adenocarcinoma progression. Methods The human umbilical cord derived-mesenchymal stem cells (huc-MSCs) loaded with microRNA (miRNA, miR)-145-5p mimics and negative control miRNA were added to pancreatic ductal adenocarcinoma (PDAC) cells. The proliferative activity and invasion abilities of BxPC3 and PANC-1 cells were measured by cell counting kit-8 (CCK-8) and Transwell assays. The cell cycle and cell apoptosis were detected by flow cytometry analysis. The expression levels of Smad3 were detected by Western blotting. Results HucMSC exosomes were isolated and purified from the hucMSC-conditioned medium by ultracentrifugation. HucMSC exosomes had a characteristic 'saucer-like’ morphology with diameters that ranged from 52.5 nm to 185.5 nm, with an average of 119 nm and the concentration of hucMSC exosomes was 400 Particles/ml. Further results showed positive expression of exosomal markers, such as CD9 and CD81. The results of cell proliferation showed that the absorbance value of BxPC3 and PANC-1 cells in 145-exo group was significantly lower than that of the NC-exo group after 4 days [(2.681±0.049) vs. (3.683±0.003), t=20.460, P<0.05; (2.646±0.116) vs. (3.450±0.112), t=4.992, P<0.05]. The results of Transwell assays showed that the number of metastatic ceils in the 145-exo group was significantly decreased compared to the NC-exo group [(68.200±3.262) cells vs. (104.800±2.177) cells, t=9.333, P<0.05; (83.000±3.493) cells vs. (146.200±4.432) cells, t=11.200, P<0.05]. Flow cytometric analysis revealed that 145-exo group caused an increase in the percentage of the cells in the G0/G1 stage of the cell cycle compared to the NC-exo group [(43.030±1.060)% vs. (31.730±0.843)%, t=8.346, P<0.05)]. Flow cytometric analysis revealed that 145-exo group increase in apoptotic cells, relative to NC-exo group [(10.970±0.405)% vs. (6.863±0.099)%, t=9.830, P<0.05; (2.400±0.166)% vs. (1.513±0.045)%, t=5.167, P<0.05]. The expression of Smad3 protein in 145-exo group was reduced by 38.0% and 37.4%, respectively (t=6.362, P<0.05; t=11.430, P<0.05). Conclusion HucMSCs-exo can effectively deliver miR-145-5p to inhibit pancreatic ductal adenocarcinoma progression, which may be related to downregulation of Smad3 expression. Key words: Human umbilical cord mesenchymal stromal cell; Exosomes; MicroRNA-145-5p; PANCreatic ductal adenocarcinoma; Treatment

Exosomal transfer of miR-501 confers doxorubicin resistance and tumorigenesis via targeting of BLID in gastric cancer.

Exosomal transfer of oncogenic miRNAs can enhance recipient cell growth, metastasis and chemoresistance. Currently we found that microRNA-501-5p (miR-501) was overexpressed in doxorubicin-resistant gastric cancer (GC) SGC7901/ADR cell-secreted exosomes (ADR Exo) than that in SGC7901 cell-secreted exosomes (7901 Exo). ADR Exo was internalized by SGC7901, and a Cy3-miR-501 mimic was transferred from SGC7901/ADR to SGC7901 via exosomes. ADR Exo conferred doxorubicin resistance, proliferation, migration and invasion abilities to negative control miRNA inhibitor-expressing GC cells, whereas it inhibited apoptosis. MiR-501 knockdown or BH3-like motif-containing protein, cell death inducer (BLID) overexpression could reverse the effects of ADR Exo on recipient cells. SGC7901 cells cocultured with SGC7901/ADR prior to treatment with GW4869 or transfection of a miR-501 inhibitor were sensitive to doxorubicin and exhibited attenuated proliferation, migration and invasion and increased apoptosis. The intratumoral injection of ADR Exo into negative control miRNA inhibitor-expressing SGC7901 cells induced rapid subcutaneous tumor growth and resistance to doxorubicin compared to that of miR-501 knockdown or BLID-overexpressing cells. This effect is possibly achieved by exosomal miR-501-induced downregulation of BLID, subsequent inactivation of caspase-9/-3 and phosphorylation of Akt. Exosomal miR-501 might be a therapeutic target for GC.

MiR-186-5p promotes cell growth, migration and invasion of lung adenocarcinoma by targeting PTEN

ObjectiveTo explore the expression of miR-186-5p in lung adenocarcinoma (LUAD) and its possible function associated with cancer cell proliferation, migration and invasion. MethodsMiR-186-5p expression levels in LUAD samples, human LUAD cell lines H1299 and NCI-H1975, and normal human lung epithelial cell line BEAS-IB were assessed by quantitative real-time PCR (qRT-PCR). H1299 and NCI-H1975 cells were transfected with miR-186-5p mimic or miRNA negative control. CCK-8 assay was performed to evaluate the cell proliferation. Transwell assay and transwell-matrigel™ invasion assay were applied to assess the migration and invasion abilities of H1299 and NCI-H1975 cells. ResultsmiR-186-5p expression was significantly up-regulated in LUAD tumor tissues and LUAD cell lines as compared with tumor-adjacent tissues and normal human lung epithelial cells, respectively. MiR-186-5p overexpression remarkably promoted the proliferation, migration and invasion of LUAD cells. Furthermore, phosphatase and tensin homolog (PTEN) was a direct target of miR-186-5p verified by luciferase reporter assay. Overexpression of PTEN significantly suppressed LUAD cells to proliferate, migrate and invade. MiR-186-5p overexpression-induced LUAD cell phenotype could be partially rescued by co-overexpression of miR-186-5p and PTEN. ConclusionThis study demonstrated that miR-186-5p is up-regulated in LUAD, and functionally associated with cell proliferation, migration and invasion. MiR-186-5p promotes the proliferation, migration and invasion of LUAD cells by targeting PTEN. MiR-186-5p may be utilized as a novel molecular marker and therapeutic target of LUAD.

BMP‑2 and miR‑29c in osteosarcoma tissues on proliferation and invasion of osteosarcoma cells

Expression of bone morphogenetic protein (BMP)-2 and microRNA (miR)-29c in osteosarcoma tissues and effects on proliferation and invasion of osteosarcoma cells were investigated. A retrospective analysis of 75 patients with osteosarcoma who underwent surgery in Tianjin Baodi Hospital from May 2013 to June 2017 was conducted. A total of 75 osteosarcoma tissues and 51 normal paraneoplastic tissues were collected. RT-PCR was used to compare the expression difference of BMP-2 and miR-29c. miR-29c mimics (experimental group A) and BMP-2 siRNA plasmid (experimental group B) were transfected into human osteosarcoma cells MG-63, respectively. The transfected cells were divided into miRNA negative control (miR-NC) group (cells transfected with miR-negative control), siRNA negative control group (cells transfected with non-silent siRNA) and blank group (cells without any transfection). MTT assay was used to detect cell proliferation in each group at different time periods. Transwell insert was used to detect invasion of cells in vitro. The relative expression of BMP-2 in osteosarcoma tissue was significantly higher than that in paraneoplastic tissue (P<0.05). Τhe relative expression of miR-29c in osteosarcoma tissue was significantly lower than that in paraneoplastic tissue (P<0.05). The cell survival rates in experimental groups A and B were significantly lower than those in the blank, miR-NC negative control and siRNA negative control groups on day 5 (P<0.05). The number of cell transmembranes in experimental groups A and B was significantly lower than those in the blank, miR-NC negative control and siRNA negative control groups (P<0.05). BMP-2 is over-expressed in osteosarcoma tissues, and miR-29c is under-expressed in osteosarcoma tissues. Interfering with the expression of BMP-2 and overexpression of miR-29c can inhibit the proliferation and invasion of osteosarcoma cells, indicating that BMP-2 and miR-29c may be involved in the regulation of proliferation and metastasis of osteosarcoma cells and could be used as new molecular target markers for the diagnosis and treatment of osteosarcoma.

Age‐ and Salt‐dependent Changes in miRNA Profile in FBN rat and C57BL/6J mice: Role of sodium hydrogen exchanger regulatory factor‐1 (NHERF1)

Previous studies from our laboratory demonstrated decreased NHERF1 expression in aging F344. Loss of NHERF1 expression prevented dopamine‐dependent inhibition of NKA in renal PTCs from aged F344. Our laboratory demonstrated increased expression of miRNAs regulating markers of inflammation in NHERF‐1 deficient mice and rat models. We hypothesized that loss of NHERF1 expression prevents salt‐sensitive hypertension due to changes in miRNA expression regulating Na‐transporters in renal tubules. To address this hypothesis, we analyzed the miRNA expression profile in 4mo and 24mo old FBN rats and 18mo old C57BL/6J wild‐type and NHERF1 knock‐out mice fed a normal salt or 8% salt diet (HSD). Total RNA from kidney cortex was isolated using Trizol (miRVana, Ambion) and miRNA profile was analyzed using kits and software from Nanostring. The top 100 miRNAs with cut‐off set at counts three times the SD of the negative control miRNA counts were considered for analysis. Counts from normal diet fed animals (n=8, 4M4F) were averaged together and considered as 1 to calculate changes in HSD animals. In 4‐mo old FBN rats 23 miRNAs significantly increased in HSD (miR196c, miR466b, miR144, and miR142‐5p), while miR547, miR375, and 6 others significantly decreased. In 24‐mo old FBN rats 30 miRNAs increased significantly in HSD (miR‐192, miRNA141, and miR21), while 10 miRNAs decreased significantly (miR328a and miR‐let‐7a and f). Similar results were observed in WT mice. However, in NHERF‐1 KO mice the miRNAs increased strongly regulate markers of inflammation. The data was confirmed by measuring the expression of down‐stream targets using western blotting and ELISA. These data may suggest that NHERF1 plays a major role in regulation of renal inflammation and salt‐sensitive hypertension.Support or Funding Information7R21AG047474‐03 NIH; R25AG047843‐NIH; 16GRNT31030019‐AHAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Upregulation of miR-216a exerts neuroprotective effects against ischemic injury through negatively regulating JAK2/STAT3-involved apoptosis and inflammatory pathways.

Ischemic stroke remains a significant cause of death and disability in industrialized nations. Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) of the JAK2/STAT3 pathway play important roles in the downstream signal pathway regulation of ischemic stroke-related inflammatory neuronal damage. Recently, microRNAs (miRNAs) have emerged as major regulators in cerebral ischemic injury; therefore, the authors aimed to investigate the underlying molecular mechanism between miRNAs and ischemic stroke, which may provide potential therapeutic targets for ischemic stroke. The JAK2- and JAK3-related miRNA (miR-135, miR-216a, and miR-433) expression levels were detected by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis in both oxygen-glucose deprivation (OGD)-treated primary cultured neuronal cells and mouse brain with middle cerebral artery occlusion (MCAO)-induced ischemic stroke. The miR-135, miR-216a, and miR-433 were determined by bioinformatics analysis that may target JAK2, and miR-216a was further confirmed by 3' untranslated region (3'UTR) dual-luciferase assay. The study further detected cell apoptosis, the level of lactate dehydrogenase, and inflammatory mediators (inducible nitric oxide synthase [iNOS], matrix metalloproteinase-9 [MMP-9], tumor necrosis factor-α [TNF-α], and interleukin-1β [IL-1β]) after cells were transfected with miR-NC (miRNA negative control) or miR-216a mimics and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) damage with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V-FITC/PI, Western blots, and enzyme-linked immunosorbent assay detection. Furthermore, neurological deficit detection and neurological behavior grading were performed to determine the infarction area and neurological deficits. JAK2 showed its highest level while miR-216a showed its lowest level at day 1 after ischemic reperfusion. However, miR-135 and miR-433 had no obvious change during the process. The luciferase assay data further confirmed that miR-216a can directly target the 3'UTR of JAK2, and overexpression of miR-216a repressed JAK2 protein levels in OGD/R-treated neuronal cells as well as in the MCAO model ischemic region. In addition, overexpression of miR-216a mitigated cell apoptosis both in vitro and in vivo, which was consistent with the effect of knockdown of JAK2. Furthermore, the study found that miR-216a obviously inhibited the inflammatory mediators after OGD/R, including inflammatory enzymes (iNOS and MMP-9) and cytokines (TNF-α and IL-1β). Upregulating miR-216a levels reduced ischemic infarction and improved neurological deficit. These findings suggest that upregulation of miR-216a, which targets JAK2, could induce neuroprotection against ischemic injury in vitro and in vivo, which provides a potential therapeutic target for ischemic stroke.

MiR-302 cluster inhibits angiogenesis and growth of K562 leukemia cells by targeting VEGFA.

BackgroundmiR-302 cluster has been reported as a tumor suppressor in many human cancers; yet, its function in chronic myeloid leukemia (CML) tumorigenesis remains largely unclear. The study was aimed to explore the functional roles of miR-302 cluster in CML progression.Materials and methodsQuantitative reverse transcriptase PCR and Western blot were performed to evaluate miR-302 cluster and vascular endothelial growth factor A (VEGFA) expression levels. Cell Counting Kit-8 assay, colony formation assay and human umbilical vein endothelial cell line capillary tube formation were used to determine the influence of miR-302 cluster on the growth and angiogenesis of K562 cells, respectively. Luciferase reporter assay was employed to confirm the direct target interaction between miR-302 cluster and VEGFA.ResultsThis study demonstrated that miR-302 cluster was frequently downregulated in CML samples and cell lines and high level of miR-302 cluster was significantly associated with good prognosis of CML patients. Compared with miRNA negative control, miR-302 cluster mimics obviously suppressed cell growth, colony formation and angiogenesis. Further studies revealed that VEGFA was a direct target gene of miR-302 cluster. Moreover, overexpression of VEGFA dramatically abated the inhibition of miR-302 cluster on cell growth and angiogenesis.ConclusionThe present study, for the first time, identified miR-302 cluster as a tumor suppressor, and overexpression of miR-302 cluster inhibited growth and angiogenesis in K562 cells. miR-302 cluster may be a potential therapeutic target in CML to develop the adjuvant antiangiogenic therapy based on VEGFA.

Mechanism of miR-221 contributes to gefitinib resistance in PC-9 cells

Objective: To investigate the effect of microRNA-221 (miR-221) overexpression on gefitinib resistance in PC-9 cells and study its underlying mechanisms. Methods: PC-9 cells were transfected with LV-NC and LV-miR-221 to establish cell stabilizing strains (PC-9/NC and PC-9/miR-221), then they were used to detect the relative expression of miR-221 and apoptotic protease activating factor-1 (APAF-1) mRNA by real-time fluorescence quantitative PCR (qRT-PCR). The effects of gefitinib (0-4 μmol/L) on the growth and proliferation of cell stabilizing strains were detected by CCK-8 Assay. After gefitinib treatment, cell apoptosis was detected by Flow Cytometry Assays. The expression of epidermal growth factor receptor (EGFR), phosphorylated epidermal growth factor receptor (p-EGFR), APAF-1 and cleaved cysteinyl aspartate specific proteinase-3 (Cleaved-caspase-3) were detected by Western blot. Dual-Luciferase Reporter Assay was used to evaluate the relationship between APAF-1 and miR-221. Results: The relative expression of miR-221 in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[(1.00±0.082) vs (40.24±0.017)](P<0.01). The half maximal inhibitory concentration (IC(50)) in PC-9/NC cells was significantly lower than that in PC-9/miR-221 cells[(IC(50)=0.1 μmol/L) vs (IC(50)>4 μmol/L)](P<0.05). Apoptosis rate of PC-9/NC cell was significantly higher than PC-9/miR-221[(33.42±4.28)% vs (10.27±1.12)%](P<0.05); APAF-1 mRNA expression was significantly higher in PC-9/NC cells than PC-9/miR-221[(1.000+ 0.069) vs (0.701±0.072)](P<0.05), and the expression of APAF-1 protein in PC-9/NC cells was significantly higher than that of PC-9/miR-221 cells. The dual luciferase reporter gene results showed that miR-221a inhibited luciferase activity significantly stronger than transfected miRNA negative control group after co-transfection of luciferase plasmids pmir-REPORT-APAF-1-wt and miR-221a mimics (P<0.01). p-EGFR was down-regulated in both PC-9/NC and PC-9/miR-221 cells after treatment with gefitinib. APAF-1 and Cleaved-caspase-3 proteins were significantly down-regulated in PC-9/miR-221 cells compared with PC-9/NC cells, while APAF-1 and Cleaved-caspase-3 proteins were up-regulated in PC-9/NC cells treated with gefitinib compared with PC-9/miR-221 cells (P<0.05). Conclusion: miR-221 induces resistance to gefitinib in PC-9 cells by downregulating APAF-1 expression.

Abstract 16934: Targeted Cardiac Delivery of miR-29b Mimic via Ultrasound-Mediated Microbubble Cavitation Attenuates Myocardial Fibrosis

Introduction: Cardiac fibrosis contributes to adverse ventricular remodeling in ischemic or hypertensive heart disease, increases heart failure risk, and is associated with loss of microRNA-29b (miR-29b). Plasmid or i.v. injection of miR-29b mimic has proven the concept that restoring miR-29b blunts cardiac fibrosis, but these are inefficient, non-targeted delivery strategies with low clinical translational potential. We hypothesized that ultrasound (US) mediated cavitation of microbubbles (MBs) loaded with miR-29b mimic would inhibit cardiac fibrosis. We first tested effects of US-targeted cavitation of miR29b-MBs (UTMC) on cardiac fibroblasts in vitro , then performed in vivo studies in a mouse model of angiotensin II (ANGII)-induced hypertension/fibrosis. Methods: Cationic lipid MBs were loaded with miR-29b mimic or negative control (NC) miRNA, placed with cultured neonatal cardiac fibroblasts, then treated with 10 sec pulsed US ( f =1.3 MHz, MI 1.6, 4 frames/burst, 4 bursts/second). Cells were harvested at 24 hrs to measure downstream fibrotic mediators. Mice received continuous ANGII infusion via osmotic pump and UTMC with miRNA-loaded MBs at days 0, 3, 7. Serial echo was performed; the heart was harvested on day 10. Results: In cardiac fibroblasts, UTMC treatment with MBs loaded with miR-29b mimic vs NC miRNA caused a 543±51% increase in miR-29b ( p &lt;0.01) and knockdown in transcripts for collagen 1A1 (26±6%, p &lt;0.01), collagen 1A2 (53±8%, p &lt;0.01), collagen III (31±5%, p &lt;0.01), and fibrillin (43±10%, p &lt;0.05) ( n =4/group). ANGII mice treated with UTMC + miR-29b mimic vs. NC miRNA ( n =8-9/group) had downregulated cardiac fibrillin (37±8%, p &lt;0.05); trends of lower collagen IA1, 1A2, and III by RT-PCR; decreased cardiac α-SMA protein by immunoblot ( p &lt;0.05); and preservation of ejection fraction and fractional shortening ( p &lt;0.01). Conclusion: UTMC-mediated delivery of miR-29b mimic blunts pathologic expression of fibrosis markers and is associated with preservation of left ventricular function in ANGII-induced murine cardiac fibrosis. This is the first demonstration of UTMC-delivery of a miRNA mimic for attenuating cardiac fibrosis, and has promising implications for targeted RNA-based approaches for treating cardiac disease.

Low expression of miR-199 in hepatocellular carcinoma contributes to tumor cell hyper-proliferation by negatively suppressing XBP1.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide, and microRNAs (miRs) are considered to serve important functions in the pathogenesis of HCC by regulating the expression of specific target genes. The present study was conducted to investigate the role of miR-199 and its putative target X-box binding protein 1 (XBP1) in HCC, as well as of the downstream gene cyclin D. The expression levels of miR-199, XBP1 and cyclin D were detected in clinical HCC specimens. The effect of miR-199 on the regulation of HCC cell proliferation and its underlying mechanism were examined in Hep3B2.1–7 cells, through expression assays and measurement of cell proliferation (via Cell Counting Kit-8, and 5-ethynyl-2′-deoxyuridine and DAPI double-staining assays) coupled with gain- and lose- of function experiments. The expression of XBP1 and cyclin D was significantly increased in HCC tissues when compared with adjacent non-HCC tissues, while the expression of miR-199 was decreased. Exogenous miR-199 significantly suppressed the expression of XBP1 and cyclin D in Hep3B2.1–7 cells. However, the expression of XBP1 and cyclin D significantly increased on treatment with miR-199 inhibitor. Consistently, Hep3B2.1–7 cells co-transfected with a wild type reporter plasmid [XBP1-3′untranslated region (UTR)-WT] and exogenous miR-199 exhibited lower relative luciferase enzyme activity than cells co-transfected with negative control miRNA and XBP1-3′UTR-WT, while cells co-transfected with mutated plasmid (XBP1-3′UTR-MU) and miR-199 exhibited no change. It was further observed that knockdown of XBP1 by small interfering RNA significantly decreased the expression of cyclin D in Hep3B2.1–7 cells. Additionally, exogenous miR-199 decreased the proliferation of Hep3B2.1–7 cells, which was contrary to the effect of miR-199 inhibitor. In conclusion, it was demonstrated that miR-199 negatively regulated the expression of XBP1 by directly binding to its 3′UTR and that XBP1 impacted cyclin D expression, which was associated with the cell cycle regulation in Hep3B2.1–7 cells. These findings suggested that a miR-199/XBP1/cyclin D axis may serve an important role in the pathogenesis of HCC.

MiR-155 Aggravates Liver Ischemia/reperfusion Injury by Suppressing SOCS1 in Mice

Liver ischemia/reperfusion injury (IRI) occurs during partial liver resection and liver transplantation. Activation of Toll-like receptors (TLRs) is a key event triggered by a range of proinflammatory cytokines during liver I/R. Although it has been reported that miR-155 takes part in both innate and adaptive immune responses, the potential role of miR-155 in liver IRI remains unknown. In this study, we found that expression of miR-155 was upregulated during liver I/R by many inflammatory cytokines, and forced expression of miR-155 aggravated hepatocyte injury following liver I/R both in vivo and in vitro. Mice transfected with Ago-miR-155—a chemically modified miR-155—showed enhanced liver severity compared to those transfected with negative control miRNA by inhibiting the expression of SOCS1, the target of miR-155. Thus by the inhibition of SOCS1, the overexpression of miR-155 promoted activation of NF-κB, and elevating the production of proinflammatory cytokines, such TNF-α and IL-6. In conclusion, miR-155 aggravates liver I/R injury in vivo and hepatocyte hypoxia/reoxygenation injury by suppressing the expression of SOCS1.

MiRNA-708/CUL4B axis contributes into cell proliferation and apoptosis of osteosarcoma.

The functions of miRNA-708 for various diseases have been confirmed. However, its roles in osteosarcoma are unclear. In this study, we aimed to explore the role of miRNA-708 in osteosarcoma. Detection of the expression of miRNA-708 and CUL4B was used by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cells were transfected with miRNA-708 mimics (mimics group) and miRNA negative control (NC group). Detection of cell growth curve at 24 h, 48 h, 72 h, and 96 h was made by cell counting kit-8 (CCK-8). Examination of the apoptosis rate was made by flow cytometry. The identification of the regulatory function was made by the luciferase reporter assay. The expression level of CUL4B was detected by Western blot. MiRNA-708 expression was reduced in the tumor cell lines. Compared with NC group, miRNA-708 expression was up-regulated by transfecting with mimics. Lower proliferation efficiency and higher cell apoptosis were showed in miRNA-708 mimics group relative to NC group. MiRNA-708 could regulate the expression of CUL4B by binding to its 3'UTR area. Furthermore, lower miRNA-708 and higher CUL4B were expressed in tumor tissues. MiRNA-708 expression was lower in tissues with IIB-III stage than that in IA-IIA stage. MiRNA-708/CUL4B axis contributes into cell proliferation and apoptosis of osteosarcoma.

Reduced expression of microRNA-199a-3p is associated with vascular endothelial cell injury induced by type 2 diabetes mellitus.

The aim of the present study was to investigate the function and mechanism of action of microRNA (miRNA or miR)-199a-3p in vascular endothelial cell injury induced by type 2 diabetes mellitus (T2DM). A total of 36 patients with T2DM (26 males and 10 females; mean age, 52.5±7.0 years) and 20 healthy subjects (10 males and 10 females; mean age, 55.6±4.5 years) were included in the present study. Peripheral blood samples were obtained from all participants and total RNA was extracted Reverse transcription-quantitative polymerase chain reaction was performed to determine the expression of miR-199a-3p. Following the transfection of human umbilical vein endothelial cells (HUVECs) with a negative control (NC) miRNA or miR-199a-3p mimics, cell proliferation was assessed using a Cell Counting kit-8 assay. Cell migration was investigated using Transwell assays and flow cytometry was performed to detect the apoptosis of HUVECs. HUVECs were infected with Ad-GFP-LC3B and laser-scanning confocal microscopy was performed to observe autophagosomes in HUVECs. Western blotting was used to measure the expression of proteins associated with autophagy and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/nuclear factor (NF)-κB signaling pathway. MiR-199a-3p was downregulated in peripheral blood from patients with T2DM compared with healthy subjects. Transfection with miR-199a-3p mimics promoted the proliferation and migration of HUVECs. However, miR-199a-3p overexpression inhibited the apoptosis of HUVECs. MiR-199a-3p facilitated HUVEC autophagy by affecting autophagy-associated signaling pathways. Furthermore, miR-199a-3p regulated the biological functions of HUVECs via the PI3K/AKT/NF-κB signaling pathway. The results of the present study suggest that miR-199a-3p expression was reduced in patients with T2DM compared with healthy subjects and may be associated with vascular endothelial cell injury. In addition, miR-199a-3p promoted the proliferation, migration and autophagy of HUVECs, potentially by regulating the PI3K/AKT/NF-κB signaling pathway. Therefore, miR-199a-3p may function as protector of vascular endothelia.

MicroRNA-466 (miR-466) functions as a tumor suppressor and prognostic factor in colorectal cancer (CRC).

MicroRNAs (miRNAs) have an important role in the regulation of tumor development and metastasis. In this study, we investigated the clinical and prognostic value as well as biological function of miR-466 in colorectal cancer (CRC). Tumor and adjacent healthy tissues were obtained from 100 patients diagnosed with CRC. miR-466 expression was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). mRNA and protein levels of cyclin D1, apoptosis regulator BAX (BAX), and matrix metalloproteinase-2 (MMP-2) were analyzed by qRT-PCR and Western blot, respectively, in SW-620 CRC cells transfected with miR-466 mimics or negative control miRNA. Effects of miR-466 on SW-620 cell proliferation, cell cycle and apoptosis, and invasion were investigated using CCK-8 assay, flow cytometry and Transwell assay, respectively. miR-466 expression was significantly downregulated in tumor tissues compared to matched adjacent non-tumor tissues. Low expression of miR-466 was significantly correlated with the tumor size, Tumor Node Metastasis stage, lymph node metastasis, and distant metastasis. The overall survival of CRC patients with low miR-466 expression was significantly shorter compared to high-miR-466 expression group (log-rank test: p = 0.0103). Multivariate analysis revealed that low miR-466 expression was associated with poor prognosis in CRC patients. The ectopic expression of miR-466 suppressed cell proliferation and migration/invasion, as well as induced G0/G1 arrest and apoptosis in SW-620 cells. Moreover, the ectopic expression of miR-466 decreased the expression of cyclin D1 and MMP-2, but increased BAX expression in SW-620 cells. In conclusion, our findings demonstrated that miR-466 functions as a suppressor miRNA in CRC and may be used as a prognostic factor in these patients.

MicroRNA-139-5P inhibits human prostate cancer cell proliferation by targeting Notch1.

Despite an improvement in the efficacy of chemotherapeutic agents, the outcome of patients with prostate cancer remains poor. MicroRNA (miRNA/miR)-139 expression is often downregulated in multiple types of tumor, including in prostate cancer. The aim of the present study was to investigate the inhibitory effect of miR-139 on the PC-3, C4-2B and LNCaP prostate cancer cell lines. Analysis of the cell cycle of PC-3, C4-2B and LNCaP cells transfected with miR-139 revealed a significantly increased percentage of cells in the G1 phase and a decreased percentage in the S and G2 phases compared with those transfected with a negative control miRNA. The growth inhibitory rate of miR-139-transfected cells 24, 48 and 72 h after transfection were 32.83±2.61, 52.58±3.2 and 62.36±4.55% in PC-3 cells; 30.28±2.25, 51.74±3.27 and 60.80±3.58% in C4-2B cells; and 33.20±2.67, 51.83±3.59 and 61.79±4.85% in LNCaP cells, respectively. The present study revealed that miR-139 inhibited the proliferation of prostate cancer cells by interfering with the cell cycle. Further study into the mechanism by which this happened suggested that miR-139 reduced cyclin D1 expression and inhibited cell proliferation through targeting Notch1.