miR-21 In Cells Research Articles

Immune Cells Release MicroRNA-155 Enriched Extracellular Vesicles That Promote HIV-1 Infection.

The hallmark of HIV-1 infection is the rapid dysregulation of immune functions. Recent investigations for biomarkers of such dysregulation in people living with HIV (PLWH) reveal a strong correlation between viral rebound and immune activation with an increased abundance of extracellular vesicles (EVs) enriched with microRNA-155. We propose that the activation of peripheral blood mononuclear cells (PBMCs) leads to an increased miR-155 expression and production of miR-155-rich extracellular vesicles (miR-155-rich EVs), which can exacerbate HIV-1 infection by promoting viral replication. PBMCs were incubated with either HIV-1 (NL4.3Balenv), a TLR-7/8 agonist, or TNF. EVs were harvested from the cell culture supernatant by differential centrifugation, and RT-qPCR quantified miR-155 in cells and derived EVs. The effect of miR-155-rich EVs on replication of HIV-1 in incubated PBMCs was then measured by viral RNA and DNA quantification. HIV-1, TLR7/8 agonist, and TNF each induced the release of miR-155-rich EVs by PBMCs. These miR-155-rich EVs increased viral replication in PBMCs infected in vitro. Infection with HIV-1 and inflammation promote the production of miR-155-rich EVs, enhancing viral replication. Such autocrine loops, therefore, could influence the course of HIV-1 infection by promoting viral replication.

The protective effects of miR-210 modified endothelial progenitor cells released exosomes in hypoxia/reoxygenation injured neurons

We have previously demonstrated that endothelial progenitor cells (EPCs) provide beneficial effects on ischemic stroke by reducing oxidative stress, which could be through EPCs-released exosomes (EPC-EXs). EXs are emerging as a bioagent for mediating cell-cell communications via their carried microRNAs (miR). miR-210 is shown to provide a neuroprotection effect against ischemic stroke. Here, we aimed to determine whether the combination of EPC-EXs and miR-210 would provide an enhanced protective effect on neurons. The hypoxia and reoxygenation (H/R) model were applied to neurons to mimic the ischemic injury of neurons. EPCs were transfected with miR-210 mimic to elevate the level of miR-210 in cells and EPC-EXs (miR210-EPC-EXs). For functional studies, EPC-EXs were co-incubated with H/R-injured neurons, then the cell viability and reactive oxygen species (ROS) production were determined. The results showed 1) H/R induced apoptosis and ROS overproduction in neurons; 2) miR-210 mimic increased the level of miR-210 in both EPCs and EPC-EXs; 3) EPCs cultured in serum-free medium released more exosomes in comparison with cells grown in complete growth media, suggesting serum starving induce the release of EXs; 4) After transfection, EPCs grown in complete media had almost 50 times higher miR-210 level than EPCs had in serum-free media, while the EPCs-EXs isolated from the complete media has lower miR-210 expression than from the serum-free media in a time-dependent manner, suggesting the transfer of miR-210 through EXs; 5) After co-incubation, EPC-EXs and miR210-EPC-EXs were uptaken by neurons, and the miR-210 level in neurons was elevated by miR210-EPC-EXs; 6) miR210-EPC-EXs were more effective in promoting cell viability and decreasing apoptosis and ROS production than EPC-EXs. The present study demonstrated that EPCs-carried miR-210 could be released and transferred to neurons in a time-dependent manner and that miR-210 loading can enhance the protective effects of EPC-EXs on H/R-induced neuron apoptosis, oxidative stress, and decreased viability.

LncRNA SNHG16 is Downregulated in Pneumonia and Downregulates miR-210 to Promote LPS-Induced Lung Cell Apoptosis.

Long non-coding RNA Small Nucleolar RNA Host Gene 16 (SNHG16) has been reported to participate in Lipopolysaccharide (LPS)-induced inflammatory pathway, which contributes to pneumonia. This study was therefore conducted to explore the role of SNHG16 in pneumonia. In this study, expression of SNHG16 and microRNA (miR)-210 in pneumonia plasma samples (n = 56) and control samples (n = 60) was detected by RT-qPCR. The potential crosstalk between SNHG16 and miR-210 was analyzed by performing overexpression experiments. MSP was performed to study the role of SNHG16 in methylation of miR-210 gene. Cell apoptosis was analyzed by cell apoptosis assay. Decreased expression levels of SNHG16 and increased expression levels of miR-210 were observed in pneumonia. SNHG16 showed an inverse correlation to miR-210. LPS treatment led to downregulated SNHG16 and upregulated miR-210 in Human Bronchial Epithelial Cells (HBEpCs). In HBEpCs, SNHG16 downregulated miR-210 and increased miR-210 DNA gene methylation. Moreover, SNHG16 suppressed the role of miR-210 in cell apoptosis under LPS treatment. In conclusion, SNHG16 is downregulated in pneumonia, and it downregulates miR-210 possibly through methylation to promote lung cell apoptosis induced by LPS.

CircCFL1/MiR-107 Axis Targeting HMGB1 Promotes the Malignant Progression of Diffuse Large B-Cell Lymphoma Tumors.

ObjectiveThe pathogenesis of diffuse large B-cell lymphoma (DLBCL) has not yet been fully elucidated. An increasing number of studies have shown that circular RNAs (circRNAs) play an important role in tumorigenesis and development. The aim of this study was to investigate the effect of CircCFL1 on the malignant progression of DLBCL.MethodsRT-qPCR was used to detect the expression levels of CircCFL1 and miR-107. A dual-luciferase reporter gene experiment was conducted to verify that CircCFL1 targeted miR-107 and the miR-107 target gene HMGB1. BrdU, transwell, and MTT tests were performed to detect cell invasion and proliferation. Western blot analysis was used to detect the phosphorylation of proteins. Xenograft models were established to confirm the effect of CircCFL1 on DLBCL tumor growth in vivo.ResultsThe expression of CircCFL1 in cells transfected with the CircCFL1 overexpression vector was higher than that in the control group. After overexpressing CircCFL1, the expression of miR-107 in cells decreased significantly, and the protein level of HMGB1 increased. The dual-luciferase reporter gene experiment showed that CircCFL1 directly bound to miR-107 and reduced the inhibition of the target gene HMGB1. After CircCFL1 was overexpressed, cell migration and proliferation were enhanced. The tumor volume and weight in the lentivirus CircCFL1 group were higher than those in the lentivirus NC group.ConclusionResults showed that the circRNA CircCFL1 could regulate the expression of HMGB1 through miR-107 to promote the proliferation and migration of DLBCL.

Stimulation of the Gαq/phospholipase Cβ1 signaling pathway returns differentiated cells to a stem-like state.

Phospholipase Cβ1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCβ1 has a cytosolic population that helps to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCβ1 or reducing its cytosolic population by activating Gαq to localize it to the plasma membrane returns differentiated PC12 and SK-N-SH cells to an undifferentiated state. In this state, PC12 cells have a spherical morphology, resume proliferation, and express the stem cell transcription factors nanog and Oct4. Similar changes are seen when C3PO is down-regulated. This return to a stem-like state is accompanied by shifts in multiple miR populations. Surprisingly, de-differentiation can be induced by extended stimulation of Gαq where cells return to a spherical morphology and levels of specific miRs return to their undifferentiated values. In complementary studies, we followed the real-time hydrolysis of a fluorescent-tagged miR in cells where PLCβ1 or C3PO were down-regulated in PC12 cells and find substantial differences in miR processing in the undifferentiated and differentiated states. Taken together, our studies suggest that PLCβ1, through its ability to regulate C3PO and endogenous miR populations, mediates the differentiation of two types of cultured neuronal cells.

MicroRNA-100 Enhances Autophagy and Suppresses Migration and Invasion of Renal Cell Carcinoma Cells via Disruption of NOX4-Dependent mTOR Pathway.

Renal cell carcinoma (RCC) is the most common kidney malignancy and has a poor prognosis owing to its resistance to chemotherapy. Recently, microRNAs (miRNAs or miRs) have been shown to have a role in cancer metastasis and potential as prognostic biomarkers in cancer. In the present study, we aim to explore the potential role of miR‐100 in RCC by targeting nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) through the mammalian target of rapamycin (mTOR) pathway. Initially, microarray‐based gene expression profiling of RCC was used to identify differentially expressed genes. Next, the expression of miR‐100 and NOX4 was examined in RCC tissues and cell lines. Then, the interaction between miR‐100 and NOX4 was identified using bioinformatics analysis and dual‐luciferase reporter assay. Gain‐of‐function or loss‐of‐function approaches were adopted to manipulate miR‐100 and NOX4 in order to explore the functional roles in RCC. The results revealed the presence of an upregulated NOX4 and a downregulated miR‐100 in both RCC tissues and cell lines. NOX4 was verified as a target of miR‐100 in cells. In addition, overexpression of miR‐100 or NOX4 silencing could increase autophagy while decreasing the expression of mTOR pathway‐related genes and migration and invasion. Conjointly, upregulated miR‐100 can potentially increase the autophagy and inhibit the invasion and migration of RCC cells by targeting NOX4 and inactivating the mTOR pathway, which contributes to an extensive understanding of RCC and may provide novel therapeutic options for this disease.

MiR-218 Targeting Bmi-1 Inhibits Proliferation of Acute Promyelocytic Leukemia Cells

To explore the molecular mechanism by which miR-218 targeting Bmi-1 inhibits the proliferation of acute promyelocytic leukemia (APL) cells. APL cell line HL-60 was transfected by miR-218 and RNA-negative control sequences, respectively. The expression of miR-218 in cells was detected by real-time fluorescence quantitative PCR. The effect of transfected miR-218 on the proliferation of APL cells was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The regulation effect of miR-218 on Bmi-1 expression was determined by Western blot. The correlation of miR-218 expressions with Bmi-1 was analyzed by Spearman test. The targeted relationship between miR-218 and Bmi-1 was verified by luciferase assay. MTT assay showed that the proliferation of HL-60 cells in vitro was inhibited by high expression miR-218 significantly. Flow cytometry showed that the G1 and G2 phase cells increased while the S phase cells decreased after transfected by miR-218. Western blot showed that the level of Bmi-1 protein in HL-60 cells decreased significantly after transfection of miR-218 (P＜0.05). Spearman correlation analysis showed that the mRNA level of miR-218 negatively correlated with the protein content of Bmi-1 (r=-0.326, P＜0.01). Luciferase assay indicated that Bmi-1 could targeted on miR-218 directly. miR-218 can inhibit the proliferation, metastasis and invasion of APL cells, which can be related with the down-regulated of Bmi-1.

Improved Therapeutic Effect of Puerarin-Encapsulated PEG-PLGA Nanoparticle on an In Vitro Cerebral Infarction Model

This study was to explore the therapeutic effect and mechanism of puerarin (PUE) combined with PEGylated nanoparticles on a rat cerebral infarction cell model. In this context, PEG-PLGA/PUE nanoparticles were prepared by the thin-film hydration method, and the toxicity of PEG-PLGA/PUE nanoparticles to brain capillary endothelial cell (BCEC) was detected by MTT. The BCEC/TF cell model was obtained by induction of BCEC cells with TNF-α. The BCEC/TF cell model was identified by immunofluorescence; the protein expression was detected by western blotting; the expression level of miR-424 in cells was measured by RT-qPCR; the targeting relationship between miR-424 and PDCD4 was confirmed by dual-luciferase reporter assay. We found that PEG-PLGA/PUE nanoparticles prepared by the thin-film hydration method had uniform particle size, regular shape, and good stability and were not toxic to cells. The vWF was widely expressed in the cytoplasm in BCECs. The BCEC/TF cell model was obtained after TNF-α treatment, and tissue factor (TF) was widely expressed on the cell membrane of BCEC/TF cells. Furthermore, it was observed that the PEG-PLGA/PUE nanoparticles showed better therapeutic effect on the BCEC/TF cell model than PUE. PEG-PLGA/PUE nanoparticles and PUE inhibited the expression of PDCD4 protein by increasing the expression of miR-424 in BCEC/TF cells. In summary, the therapeutic effect of PEG-PLGA/PUE nanoparticles on the in vitro cell model of cerebral infarction is better than that of PUE. Moreover, PEG-PLGA/PUE inhibits the expression of PDCD4 protein by lowering the expression level of miR-424 in cells, thereby reducing the hazard of cerebral infarction.

Extracellular vesicle-derived circ_SLC19A1 promotes prostate cancer cell growth and invasion through the miR-497/septin 2 pathway.

The occurrence and development of prostate cancer (PCa) is complex, and the related mechanism is not fully understood. Current studies have found that extracellular vesicles (EVs) and circular RNAs (circRNAs) have important functions in various tumours and other diseases. In this study, the detection of circRNAs in PCa showed that circ_SLC19A1 was increased in PCa cells and their secreted EVs. EVs with high expression of circ_SLC19A1 could be taken up by PCa cells, which promoted cell proliferation and invasion. The sequence of circ_SLC19A1 contained multiple binding sites for miR-497, and circ_SLC19A1 could bind directly to miR-497 in cells. The expression of miR-497 was downregulated in PCa cells, while the expression of its target gene septin 2 (SEPT2) was upregulated significantly. Transfection of circ_SLC19A1 small interfering RNA (siRNA) or miR-497 mimics could significantly inhibit the expression of SEPT2 and the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). After co-transfection of circ_SLC19A1 siRNA and miR-497 inhibitors or SEPT2 overexpression vector, the expression of SEPT2 and ERK1/2 phosphorylation levels showed no significant changes. Similar results were obtained with co-transfection of miR-497 mimics and the SEPT2 overexpression vector. Therefore, cancer cells can regulate the expression of SEPT2 through miR-497 by secreting EVs with high expression of circ_SLC19A1, thus affecting the activation of the downstream ERK1/2 pathway and ultimately regulating PCa cell growth and invasion. Therefore, EV-derived circ_SLC19A1 plays an important regulatory role in PCa and may be an important target for PCa prevention and treatment.

The microRNA‐155 mediates hepatitis B virus replication by reinforcing SOCS1 signalling–induced autophagy

As small conserved RNAs without a coding function, microRNAs are expressed in multicellular organisms and contribute to the modulation of multiple cellular reactions, such as viral replication, as well as autophagy. microRNAs can regulate host gene expression and inhibit or reinforce hepatitis B virus (HBV) replication. Hepatic cells express miR-155 noticeably. Consequently, our study explored miR-155 modulation of HBV replication and investigated the potential mechanism involved. miR-155 was inhibited on HBV infection. miR-155 transfection remarkably reinforced HBV replication, antigen expression, and progeny secretion in HepG2215 cells. Moreover, miR-155 impaired the inhibition of the cytokine signalling 1 (SOCS1)/Akt/mTOR axis and reinforced HepG2215 autophagy. Additionally, the autophagy inhibitor (3-MA) eliminated HBsAg secretion triggered by miR-155. Taken together, miR-155 reinforced HBV replication by reinforcing SOCS1-triggered autophagy. SIGNIFICANCE OF THE STUDY: The research studied the potential mechanism involved in HBV replication and miR-155 that miR-155 reinforces HBV replication by reinforcing the SOCS1/Akt/mTOR axis-stimulated autophagy, and therefore, it can provide medical practitioners with the inspiration that chronic HBV might be cured or improved by regulating the activation of miR-155 in cells. In the study, the experiments show that autophagy inhibitors (3-MA) counteracted miR-155 contribution to HBV replication, and it might be a practicable way to improve HBV through some therapies that can repress the autophagy in related cells.

Lung myofibroblast transition and fibrosis is regulated by circ0044226.

Idiopathic pulmonary fibrosis (IPF) is a life-threatening progressive disease characterized by aberrant fibroblast activation. This study aims to explore the role of the circ0044226 on fibroblast-to-myofibroblast transition (FMT). Bleomycin and TGF-β1 were respectively used to induce the IPF mice model and human lung fibroblasts to myofibroblast differentiation. The mRNA and protein levels were examined by qRT-PCR and western blot. Localization of α-SMA was evaluated by immunofluorescence staining. Cell viability and proliferation were evaluated by CCK8 and EDU test. Dual-luciferase reporter assay was used to analyze the interaction between miR-7 and circ0044226 or sp1. Fluorescence in situ hybridization (FISH) assay was used for the identification of sub-location of circ0044226 and miR-7 in cells. The IPF model mice received intratracheal injection of AAV-sh-NC and AAV-sh- circ0044226, and lung fibrosis was detected by HE staining, Masson staining and immunohistochemistry assay. The circ0044226 was upregulated while miR-7 was downregulated in IPF mice model and FMT-derived myofibroblasts. miR-7 was a target of circ0044226 and sp1 was a target of miR-7. circ0044226 was distributed mostly in the cytoplasm and functioned as a miR-7 sponge to positively regulate the expression of sp1. Intervention of circ0044226 could ameliorate FMT and suppress fibroblast viability and proliferation by functioning as an endogenous miR-7 sponge. Circ0044226 knockdown alleviates fibroblast proliferation and FMT by functioning as a competing endogenous RNA, which may represent a promising therapy for pulmonary fibrosis.

MiR-124 participates in the proliferation and differentiation of brain glioma stem cells through regulating Nogo/NgR expression.

The effect of miR-124 on the proliferation and differentiation of brain glioma stem cells and Nogo/NgR signaling pathway were investigated. miR-124 mimic, miR-124 inhibitor and miR-control expression vector were designed and produced to transfect U87 glioma stem cells. The results of transfection were tested via RT-qPCR and the expression of protein was detected by western blot analysis. Cell proliferation was detected by MTT proliferation and the proportion of CD133+ cells was detected by immunomagnetic beads to determine cell differentiation. The correlation between miR-124 and Nogo-A, and NgR protein expression was analyzed by Spearman correlation analysis. The relative expression of miR-124 in cells of miR-124 mimic group was significantly higher than that of miR-124 inhibitor and miR-control groups (P<0.05). The relative expression of Nogo-A and NgR protein in cells of the miR-124 mimic group was significantly lower than that of miR-124 inhibitor and miR-control groups (P<0.05). Absorbance values of the cells in the miR-124 mimic and miR-control groups were significantly lower than those in the miR-124 inhibitor group at each time point (P<0.05), while the values of the cells in the miR-124 mimic group were significantly lower than that in miR-control group (P<0.05). The level of CD133+ cells in miR-124 mimic group was significantly lower than that in miR-124 inhibitor and miR-control groups (P<0.05), while the level of CD133+ cells in miR-124 inhibitor group was higher than that in miR-control group (P<0.05). Correlation analysis revealed that there was a negative correlation between miR-124 and the expression of Nogo-A and NgR protein (P<0.05). miR-124 may participate in the differentiation of brain glioma stem cells through the Nogo/NgR pathway, which may bring a new direction for the clinical treatment of brain glioma.

Retracted: The long noncoding RNA cardiac hypertrophy‐related factor plays oncogenic roles in hepatocellular carcinoma by downregulating microRNA‐211

Hepatocellular carcinoma (HCC) is the most major type of primary hepatic cancer. This study aimed to explore the possible oncogenic effects of the long noncoding RNA cardiac hypertrophy-related factor (CHRF) on HCC, as well as the underlying possible mechanism. The expression levels of CHRF and microRNA-211 (miR-211) in HCC tissues and/or cell lines HepG2 and Huh-7 were measured using quantitative reverse transcription polymerase chain reaction. Cell transfection was conducted to change the expression levels of CHRF and miR-211 in cells. Cell viability and apoptosis were assessed using the cell counting kit-8 assay and annexin V-phycoerythrin staining, respectively. The pull-down assay and RNA immunoprecipitation were performed to analyze the association between CHRF and miR-211. The expression of the key factors involving in cell proliferation, cell apoptosis, and epithelial-mesenchymal transition (EMT) process, as well as the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways, were evaluated by Western blot analysis. CHRF was highly expressed in HCC tissues and positively associated with the TNM stage, differentiation, and size of tumors. Overexpression of CHRF promoted HepG2 cell viability, proliferation, and EMT process. CHRF knockdown had opposite effects. Moreover, CHRF negatively regulated the expression of miR-21, and miR-21 was a direct target of CHRF. Overexpression of miR-211 reversed the effects of CHRF on HepG2 and Huh-7 cell viability, proliferation, and EMT process. Furthermore, overexpression of CHRF activated the PI3K/AKT and Wnt/β-catenin pathways in HepG2 cells by downregulating miR-211. CHRF played oncogenic roles in HCC. The overexpression of CHRF promoted HepG2 and Huh-7 cell viability, proliferation, and EMT process by downregulating miR-211 and then activating the PI3K/AKT and Wnt/β-catenin pathways.

Effect of miR-132 on bupivacaine-induced neurotoxicity in human neuroblastoma cell line

BackgroundLocal anesthetics (LAs) may generate neurotoxicity in neurons. In the current study, we explored the mechanisms by which microRNA-132 (miR-132) regulated the neurotoxicity of human neuroblastoma cells (SH-SY5Y) induced by bupivacaine (BUP). MethodsCCK-8, flow cytometry, EdU detection, qRT-PCR and western blotting were used to explore the cell viability, apoptosis and gene expression, respectively. ResultsIn this study, we found that 600 μM BUP dramatically inhibited SH-SY5Y cells viability. In addition, BUP induced cell apoptosis and neurotoxicity via increasing active caspase-3 and cleaved PARP1 levels. More importantly, the level of miR-132 was significantly up-regulated in BUP-treated cells, which was significantly reversed by miR-132 inhibitor. In addition, dual-luciferase assay indicated IGF1R was the directly binding target of miR-132 in cells. Our study further indicated that the level of IGF1R was markedly decreased by BUP interference, while miR-132 inhibitor exerted the opposite effect. Furthermore, BUP induced apoptosis and neurotoxicity in SH-SY5Y cells were attenuated by IGF1, which further confirmed IGF1R was the downstream target of BUP in SH-SY5Y cells. ConclusionIn the present study, miR-132 played important roles in regulating BUP-induced neurotoxicity through IGF1R and may act as a promising molecular target for the treatment of human neurotoxicity induced by BUP.

Clinical significance of miR-210 expression in breast cancer tissues and its influence on malignant behavior of triple negative breast cancer cells

Objective To investigate the expression and clinical significance of microRNA-210 (miR-210) in breast cancer tissues, and to investigate its effect on the proliferation and metastasis of human triple negative breast cancer cell line MDA-MB-231 in vitro. Methods The breast cancer tissues and paracancerous tissues in 82 patients were collected in the Department of Pathology of Hunan Cancer Hospital from December 2013 to September 2015. Quantitative real-time polymerase chain reaction (qRT-PCR) technique was used to detect the expression level of miR-210 in tissues and cells. The relationship between the expression of miR-210 and clinical data and prognosis of patients were analyzed. The triple negative breast cancer cell line MDA-MB-231 transfected with full-length miR-210 plasmid was regarded as test group, and the cell transfected with blank vector was regarded as control group. CCK-8 assay was used to detect the proliferation ability of cells in both groups. Transwell invasion and migration assays were used to detect the metastasis and invasion ability of cells. Results The results of qRT-PCR showed that the expression level of miR-210 was 0.198±0.014 in breast cancer tissues, which was significantly higher than that in paracancerous tissues (0.084±0.009), and the difference was statistically significant (t=8.141, P<0.001). The expression level of miR-210 in triple negative breast cancer tissues was 0.254±0.026, which was significantly higher than that in non-triple negative breast cancer tissues (0.167±0.015), and the difference was statistically significant (t=3.175, P=0.003). There were significant differences in TNM staging and molecular typing between the patients with high and low expression of miR-210 (χ2=7.859, P=0.005; χ2=7.053, P=0.008). The 4-year survival rate of patients with high expression of miR-210 was significantly lower than that of patients with low expression of miR-210 (49.37% vs. 76.80%), and the difference was statistically significant (χ2=4.743, P=0.024). The results of qRT-PCR showed that the expression of miR-210 in cells in test group was 0.517±0.038, which was significantly higher than that in control group (0.284±0.022), and the difference was statistically significant (t=9.280, P<0.001). The results of CCK-8 assay showed that the proliferation abilities of the test group were significantly higher than those of the control group in 48, 72 and 96 h (3.771±0.452 vs. 3.206±0.314; 7.662±0.619 vs. 6.736±0.552; 15.477±1.425 vs. 11.592±1.243), and the differences were statistically significant (t=2.296, P=0.025; t=2.496, P=0.019; t=4.594, P=0.001). The results of Transwell invasion assay showed that the cell number of test group in inferior surface was 107.8±13.0, which was significantly higher than that of control group (74.4±10.9), and the difference was statistically significant (t=3.732, P=0.001). The results of Transwell migration assay showed that the cell number of test group in inferior surface was 136.5±18.5, which was significantly higher than that of control group (87.4±15.7), and the difference was statistically significant (t=4.256, P<0.001). Conclusion The expression of miR-210 in breast cancer tissues is high, and its expression is closely related to progression, malignancy and prognosis of patients. In vitro, miR-210 can promote the malignant behavior of triple negative breast cancer cell line MDA-MB-231. It is a potential molecular marker and targeted treatment site. Key words: Breast neoplasms; Cell proliferation; Neoplasm invasiveness; Cell movement; MicroRNA-210

Triptolide inhibits the proliferation and migration of medulloblastoma Daoy cells by upregulation of microRNA-138.

Medulloblastoma is a primitive neuroectodermal-derived brain tumor and the most common malignant brain tumor in children. Triptolide (TPL) is the major active component extracted from Tripterygium wilfordii Hook F. This study aimed to explore the effects of TPL on medulloblastoma cell proliferation, migration, and apoptosis, as well as the underlying possible molecular mechanism. Viability, proliferation, and apoptosis of Daoy cells were measured using cell counting kit-8 assay, 5-bromo-2'-deoxyuridine incorporation assay, and Guava Nexin assay, respectively. Cell migration was detected using two-chamber transwell assay and wound healing assay. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to determine the relative expression of microRNA-138 (miR-138) in Daoy cells. Cell transfection was used to change the expression of miR-138 in cells. Western blot analysis was used to analyze the expression of key factors involved in cell apoptosis, cell migration, the phosphatidylinositol 3-kinase (PI3K)/protein kinase 3 (AKT) pathway, and the Notch pathway in Daoy cells. We found that TPL significantly inhibited the viability, proliferation, and migration of Daoy cells but promoted Daoy cell apoptosis. The expression levels of matrix metalloproteinases (MMP)-2 and MMP-9 after TPL treatment were decreased. The expression of miR-138 in Daoy cells after TPL treatment was increased. Suppression of miR-138 obviously reversed the TPL-induced Daoy cell proliferation, migration inhibition, and cell apoptosis enhancement, as well as the inactivation of the PI3K/AKT and Notch pathways. Cyclin-dependent kinase 6 (CDK6) was a direct target gene of miR-138, which might be involved in the antitumor effects of TPL on Daoy cells. In conclusion, our study verified that TPL exerted anticancer effects on medulloblastoma cells possibly via upregulating miR-138 and inactivating the PI3K/AKT and Notch pathways.

Decreased expression of microRNA-214 contributes to imatinib mesylate resistance of chronic myeloid leukemia patients by upregulating ABCB1 gene expression.

The aim of the present study was to determine the expression of adenosine triphosphate binding cassette subfamily B member 1 (ABCB1) gene and its protein P-glycoprotein (PGP) in bone marrow mononuclear cells from chronic myeloid leukemia (CML) patients with imatinib mesylate (IM) resistance, or IM-resistant CML K562 cells. In addition, the molecular mechanism of action of microRNA (miR)-214 on ABCB1 in IM resistance was investigated. A total of 26 CML patients with IM resistance were included in the present study. In addition, 31 CML patients who did not have IM resistance were included as the control group. Bone marrow was collected from all subjects. The K562R cell line, which is a K562 cell line with IM resistance, was used for cellular studies. Reverse transcription-quantitative polymerase chain reaction was used to determine the expression of ABCB1 mRNA and miR-214 in cells. Western blotting was employed to determine the expression of PGP. Dual luciferase reporter assay was carried out to identify interactions between ABCB1 mRNA and miR-214. MTT assay was used to determine the survival rate of cells. ABCB1 mRNA and PGP expression was upregulated in bone marrow mononuclear cells from CML patients with IM resistance. K562R cells had higher ABCB1 and PGP expression than K562 cells, potentially due to their different sensitivity to IM. Expression miR-214 was decreased in bone marrow mononuclear cells from patients with IM resistance and K562R cells. Notably, miR-214 was able to bind with the 3′-untranslated region, seed region of ABCB1 mRNA to regulate its expression. In addition, elevated expression of miR-214 restored IM sensitivity to K562R cells potentially by affecting ABCB1 expression. The present study demonstrated that upregulated expression of ABCB1 mRNA and PGP in bone marrow mononuclear cells from CML patients with IM resistance may be associated with the downregulation of miR-214. In addition, miR-214 may participate in the IM resistance of CML patients by regulating ABCB1 expression.

LincRNA-ROR functions as a ceRNA to regulate Oct4, Sox2, and Nanog expression by sponging miR-145 and its effect on biologic characteristics of colonic cancer stem cells

Objective: To investigate the impact of lincRNA-ROR, a ceRNA by binding miR-145 on the expression of the downstream genes Oct4, Sox2 and Nanog, and related biological characteristics of colon cancer stem cells, and to elucidate the clinical significance of this molecular regulatory network. Methods: Fifty-two cases of colorectal cancer tissue and adjacent tissue were collected at Nanyang City Central Hospital and Nanyang Second Hospital, Henan Province, from 2014 to 2016. Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of lincRNA-ROR and miR-145 in colorectal cancer tissue and isolated colon cancer cells. The correlation between the expression of lincRNA-ROR, miR-145 and the clinicopathologic features of colon cancer was performed. CD44(-)CD133(-) and CD44(+) CD133(+) cells were isolated from SW1116 by using flow cytometry. The expression of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR and miR-145 in cells were detected by qPCR. The relationship between lincRNA-ROR, miR-145, Oct4, Sox2 and Nanog was analyzed by bioinformatics, dual luciferase reporter assay, qPCR and Western blot. The effects of silencing lincRNA-ROR on the proliferation and chemosensitivity of colon cancer stem cells were detected by MTT, colony formation. Results: LincRNA-ROR was frequently up-regulated and inversely correlated with miR-145 down-regulation in the colon cancer specimens(P<0.05). LincRNA-ROR was related to tumor size, lymph node involvement and distant metastasis(P<0.05), and miR-145 was found related to tumor size and tumor location(P<0.05). CD44(+) CD133(+) cells were successfully isolated from SW1116 by flow cytometry. The levels of CD44, CD133, Oct4, Sox2, Nanog, lincRNA-ROR in CD44(+) CD133(+) cells were significantly increased, while miR-145 was decreased compared with CD44(-)CD133(-)cells(P<0.05). The levels of CD44, CD133, lnc-ROR in CD44(+) CD133(+) cells were significantly reduced upon cell adherence, while miR-145 was significantly increased(P<0.05). Bioinformatics analysis revealed that lincRNA-ROR shared miRNA response elements with core transcription factors Oct4, Sox2 and Nanog. MiR-145 significantly inhibited the expression of lincRNA-ROR, Oct4, Sox2 and Nanog. Silencing lincRNA-ROR significantly inhibited colon cancer stem cells proliferation and increased the sensitivity to chemotherapy. Conclusions: Linc-ROR functions as a key ceRNA to prevent core TFs, e. g., Oct4, Sox2, Nanog, from miR-145-mediated suppression in colon cancer stem cells and regulates cell proliferation and chemosensitivity.The data may provide insights into the pathophysiological interactions of the components of genetic networks in the development of colon cancer and may lead to new therapies in the future.

Effects of miR-138 on invasion of brain glioma cells through targeting Sema4C

Objective To explore the effects and mechanism of microRNA-138 (miR-138) on brain glioma cells invasion and migration. Methods The expression of miR-138 was detected by real time quantitative polymerase chain reaction (qRT-PCR) in 60 cases of glioma tissues and para-carcinoma tissues, and the relationship between miR-138 expression and glioma grading was analyzed. Human glioma cells line U87 and U251 were transfected with miR-138 mimic and negative control (miR-NC). The expression of miR-138 was detected by qRT-PCR. The migration and invasion abilities were tested by Transwell assay, and the expression of semaphoring 4C (Sema4C) protein was tested by Western blotting. Results The expression level of miR-138 in glioma tissues (2.46±1.07) was significantly lower than that in normal brain tissues (4.83±1.16, t=-11.631, P<0.001), and miR-138 expression was negatively correlated with tumor grade (r=-0.563, P=0.001). The expression level of miR-138 in cells was significantly higher after being transfected with miR-138 mimic (U251: 3.96±0.16; U87: 4.43±0.96) than miR-NC (U251: 2.32±0.36; U87: 2.58±0.62, t=7.253, P<0.001; t=8.872, P<0.001). The ability of invasion and migration were lower after being transfected with miR-138 mimic (U251: 89±9; U87: 95±10) than miR-NC (U251: 206±15; U87: 240±20, t=36.629, P<0.001; t=35.521, P<0.001). The expression of Sema4C protein was lower after being transfected with miR-138 mimic (U251: 0.41±0.06; U87: 0.36±0.03) than miR-NC (U251: 1.01±0.08; U87: 1.03±0.13, t=-32.862, P<0.001; t=-27.512, P<0.001). Conclusion The up-regulated expression of miR-138 can suppress glioma cells migration and invasion, which might be related to the negative regulation expression of Sema4C protein. Key words: Glioma; Neoplasm invasiveness; Neoplasm metastasis; microRNA-138; Semaphoring-4C

MicroRNA-132 Negatively Regulates Palmitate-Induced NLRP3 Inflammasome Activation through FOXO3 Down-Regulation in THP-1 Cells.

Saturated fatty acids were proposed to activate the NLRP3 inflammasome, a molecular platform that mediates the processing of interleukin (IL)-1β and IL-18. However, the mechanisms underlying the miRNA-mediated regulation of palmitate (PA)-induced inflammasome activation are unclear. We examined the role of miR-132 in PA-induced NLRP3 inflammasome activation in THP-1 cells. To understand the regulatory role of miR-132 in inflammasome activation, we either overexpressed or suppressed miR-132 in THP-1 cells that expressed the NLRP3 inflammasome in response to stimulation by PA. We analyzed the mRNA and protein levels of NLRP3, caspase-1 p10, IL-18, and IL-1β; caspase-1 activity; and IL-1β secretion. The presence of PA activated the NLRP3 inflammasome and increased miR-132 expression. Overexpression of miR-132 reduced caspase-1 p10, IL-18, and IL-1β, while the suppression of miR-132 enhanced inflammasome activation. In addition, miR-132 regulated the mRNA and protein expression of FOXO3, which is a potential target of miR-132 in these cells. FOXO3 suppression by small interfering RNA decreased NLRP3 inflammasome activity stimulated by PA. Knockdown of FOXO3 attenuated NLRP3 inflammasome activation by the miR-132 inhibitor. Based on these findings, we conclude that miR-132 negatively regulates PA-induced NLRP3 inflammasome activation through FOXO3 down-regulation in THP-1 cells.