microRNA-210 Research Articles

MiR34a: a master regulator in the pathogenesis of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is the most common chronic lung disease in infants with lifelong pulmonary and neurodevelopmental consequences. The pathogenesis of BPD is contributed by genetic and environmental factors; among the latter, a critical contributor is exposure of the developing lung to hyperoxia. We have recently reported (Nat Comm 8:1173) that hyperoxia exposure in our in vitro and in vivo modeling systems of hyperoxia-induced lung injury (HALI) and BPD leads to an upregulation of the microRNA (miR) 34a. Utilizing genetic loss- and gain- of function strategies, we show that miR34a inhibition ameliorates the pulmonary phenotype of BPD (including BPD-associated pulmonary hypertension), at least in part, via one of the downstream targets of miR34a, namely Angiopoietin1/Tie 2 signaling. In addition, we demonstrate translational clinical significance of our findings by showing increased miR34a and decreased Ang1 expression in 3 independent cohorts of human lung samples.

The effects of up-regulation of microRNA-148a on the expression of anti-oncogene preproenkephalin, p16 and ras association domain family 1A in pancreatic carcinoma AsPC-1 cells

Objective To investigate the effect of up-regulation of microRNA (miRNA, miR)-148a on the methylation and protein expression of anti-oncogene preproenkephalin (ppENK), p16, ras association domain family 1A (RASSF1A) and the proliferation of pancreatic cancer AsPC-1 cells. Methods MiR-148a mimics were transfected into AsPC-1 cells mediated by Lipofectamine 2000. AsPC-1 cells were devided into three groups, blank group (transfected Lipofectamine), mimincs-NC group (transfected negative mimics) and mimincs-148a group (transfected miR-148a mimics). At 72 h after transfection, the expression of miR-148a was analyzed by real-time quantitative polymerase chain reaction (Real-time PCR). The CpG island methylation status of anti-oncogene ppENK, p16, RASSF1A were measured by methylation specific polymerease chain reaction (MSP). The expression of DNA methy transferas 1 (DNMT1), ppENK, p16 and RASSF1A protein were detected with Western blotting. The cell proliferation activity was examined by methyl thiazol tetrazolium (MTT) assay. Results The expression of miR-148a in mimincs-148a group was significantly higher than those in blank group and mimincs-NC group (4.63±1.32 vs. 1.00±0.30 vs. 0.93±0.37, P=0.002), while the expression of DNMT1 protein in mimincs-148a group was significantly lower than those in blank group and mimincs-NC group (1.00±0.00 vs. 1.04±0.19 vs. 0.27±0.15, P=0.001). ppENK, p16 and RASSF1A genes were positive methylation in blank group and mimincs-NC group, while ppENK and p16 genes were negative methylation, and RASSF1A gene was partial methylation in mimincs-148a group. The expression of ppENK, p16 and RASSF1A protein in mimics-148a group were significantly higher than those in blank group and mimics-NC group (ppENK: 2.06±0.32 vs. 1.00±0.00 vs. 1.01±0.21, P=0.002; p16: 2.02±0.12 vs. 1.00±0.00 vs. 0.96±0.12, P=0.000; RASSF1A: 1.95±0.37 vs. 1.00±0.00 vs. 1.09±0.12, P=0.002). The cell proliferation activities in mimincs-148a group were significantly slower than those in in blank group and mimincs-NC group (P=0.026). Conclusion Up-regulation of miR-148a could lead to demethylation of ppENK, p16 and RASSF1A gene promoter CpG island and up-regulate its protein expression, and inhibit cell proliferation in pancreatic cancer AsPC-1 cells. The mechanism may be related to the inhibition of DNMT1 expression induced by miR-148a. Key words: Pancreatic cancer; MicroRNA-148a; DNA methy transferas 1; Anti-oncogene; Methylation; Cell proliferation

MicroRNA-93 regulate proliferation and apoptosis of renal cancer cells by targeting large tumor suppressor homolog 2

Objective To evaluate the effect of microRNA (miRNA, miR)-93 regulate proliferation and apoptosis of renal cancer cells by targeting large tumor suppressor homolog 2 (LATS2). Methods Chose renal cancer cells line GRC-1, and used dual-luciferase report gene experiment to verification the targeted relationship between miR-93 and LATS2. Respectively transfected Neg-miR, pre-miR-93 and anti-miR-93 to GRC-1 cells. The miR-93 expression was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The LATS2 protein expression was detected by Western blotting. The cell proliferation activity was detected by methyl thiazol tetrazolium (MTT) method. The cell apoptosis were detected by flow cytometry. Results Between miR-93 and LATS2 had some binding sites. Compared with transfection Neg-miR, co-transfection pre-miR-93 and LATS2-wt can made luciferase activity of GRC-1 significantly decreased (P=0.000). Compared with Neg-miR group, the miR-93 expression of pre-miR-93 group significantly increased (P=0.000), and the LATS2 protein expression significantly decreased (P=0.000). The miR-93 expression of anti-miR-93 group significantly decreased (P=0.000), and the LATS2 protein expression significantly increased (P=0.000). Compared with Neg-miR group, the proliferation activity of pre-miR-93 group significantly increased (P=0.000), and the apoptosis rate of significantly decreased(P=0.000). The proliferation activity of anti-miR-93 group significantly decreased(P=0.000), and the apoptosis rate of significantly increased (P=0.000). Conclusion miR-93 can regulate proliferation and apoptosis of renal cancer cells by targeting LATS2. Key words: MicroRNA-93; Large tumor suppressor homolog 2; Renal cancer; Proliferation; Apoptosis

Serum exosomal miR-210 as a noninvasive molecular diagnosis biomarker in clear cell renal cell carcinoma

Objective The aim of this study was to evaluate the value of serum exosomal miRNAs, originating from tumor tissue cells, could be used as noninvasive biomarker for distinguishing clear cell renal cell carcinoma (ccRCC). Methods 30 pairs of tissue samples and the corresponding serum samples were collected from 20 ccRCC male patients and 10 female ccRCC patients, operated in our department from June 2015 to June 2016. Their age ranged from 45 to 70 years old, mean 57 years old. Based on the miRNA microarray analysis of ccRCCs miRNA expression profiles, we picked up four miRNAs, including miR-210, miR-224, miR-452, and miR-34a, to confirm our hypothesis. Then the expression quantity of these four miRNAs in tissues, serums and serum exosomes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Sensitivity, specificity and area under curve (AUC) for serum miRNA levels were determined using receiver operator characteristic (ROC) analysis. Results Expression of miR-210, miR-224, and miR-452 were higher in tumor tissues compared to those in adjacent noncancerous tissues(P<0.05), increasing by 20.51-fold, 54.08-fold and 2.48-fold respectively. But only miR-210 was significantly higher in ccRCC patients compared to healthy controls (HCs) in serum and serum exosome (P<0.05), increasing by 2.45-fold and 2.32-fold respectively. ROC curve analysis indicated that the serum exosomal miR-210 level might serve as a useful biomarker for differentiating patients with ccRCC from those with HCs; the AUC was 0.8789 (95% CI 0.7803-0.9775) and the sensitivity and specificity was 92.1% and 80.0%, respectively. Conclusion The detection of miR-210 in the serum exosome is useful for early diagnosis of clear cell renal cell carcinoma. Key words: Clear cell renal cell carcinoma; Serum exosome; MicroRNA-210; Biomarker

Study on promotion of bone cancer pain via rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling by negative regulation of microRNA in mice

Objective To observe microRNA (miR-93) regulation in bone cancer pain(BCP) and explore the underlying mechanisms of miR-93 and Rho associated coiled-coil forming protein kinase (ROCK) signaling affecting the biological behaviors of BCP in mice. Methods Male mice were randomly divided into Control group, Sham group, bone cancer pain (BCP group), BCP+PBS group and BCP+Y-27632(ROCK inhibitor) group(n=13). The paw mechanical threshold and paw thermal latency were used to evaluate the behavioral changes on 1 day before surgery and 1, 3, 5, 7, 10, 14 after surgery. L4-L6 dorsal root ganglions were removed on postoperative day 14, followed by Real-time PCR to measure the expression of miR-93. The ROCK2 promoter activity regulated by miR-93 was evaluated by a dual luciferase reporter assay. Western blotting was used to evaluate the protein expression level of ROCK2, phospho-LIM domain kinase (LIMK) and phospho-Cofilin. Results Following intrafemoral carcinoma inoculation, robust mechanical allodynia[Sham group(6.81±1.04) g, BCP group(5.12±0.47) g]and thermal hyperalgesia[Sham group(11.75±1.46) s,BCP group (7.61±1.07) s]in C57BL/6J mice were respectively developed on day 5 and 7, and the symptoms grow progressively with time (P<0.05). The expression level miR-93 is decreased in a time dependent way in BCP group with progressively increased pain. However, the ROCK2 expression level was increased (P<0.05), and subsequently activated the LIMK/Cofilin pathways. Dual luciferase reporter assay demonstrated that miR-93 directly targeted the 3'-UTR of the ROCK2 gene, resulting in inhibition of the post-transcriptional translation of ROCK2. ROCK inhibitor significantly up-regulated paw withdrawal threshold[BCP+PBS group(3.43±0.89) g, BCP+Y-27632 group (5.74±1.02) g], paw withdrawal latency[BCP+PBS group (5.48±1.03) s, BCP+Y-27632 group (9.82±1.24) s]and reduced the protein expression level of ROCK2, phospho-LIMK and phospho-Cofilin (P<0.05). Conclusions Reduced expression of miR-93 can promote BCP through activating ROCK2/LIMK/cofilin pathway. Therefore, miR-93 or ROCK inhibitor may be novel targets for BCP. Key words: Bone cancer pain; Analgesia; MicroRNA-93; Rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling

Inhibitory effects of microRNA-375 on biological behaviour of human retinal capillary endothelial cells induced by hypoxia

Background Studies showed that microRNA (miR)-375 suppresses the growth, apoptosis, migration and adhesion of tumor cells, and it plays a regulation to the changes of vascular endothelial growth factor (VEGF) in tumor tissue to arrest neovascularization.However, whether miR-375 intervenes the formation of new blood vessel in eyes is unelucidated. Objective This study was to explore the effects of miR-375 on human retinal capillary endothelial cells (HRCECs) function induced by hypoxia. Methods HRCECs were cultured using IMDM and divided into normal control group, CoCl2 model group, CoCl2+ miR-375 mimic group, CoCl2+ miR-375 mimic control group, CoCl2+ miR-375 inhibitor group and CoCl2+ miR-375 inhibitor control group, and hypoxia cell models were created by adding 200 μmol/L CoCl2.MiR-375 and frizzled 4 (FZD4) small interfering RNA (siRNA) were transfected into the cells by 50 nmol/L miRNA lipidosome for 48 hours.The proliferation of the cells was detected by MTT assay; migrated number of the cells was examined by Transwell chamber assay; ELISA was employed to detect the concentrations of VEGF and VE-cadherin in the medium; the expression of β-catenin, cyclinD1, matrix metalloproteinase-2 (MMP2) and VEGF proteins were analyzed by Western blot; tube length of vessel formation was evaluated by Matrigel assay.Cultured cells were divided into normal control group, CoCl2 model group, CoCl2+ mock group and CoCl2+ FZD4 siRNA group, the relative expression of FZD4, a miR-375 targeted gene, was detected by luciferase reporter. Results The relative expression of miR-375 mRNA was significantly increased in the CoCl2+ miR-375 mimic group compared with the CoCl2+ miR-375 mimic control group and reduced in the CoCo2+ miR-375 inhibitor group compared with the CoCo2+ miR-375 inhibitor control group (t=-19.237, 8.764, both at P<0.01), with a higher transfected efficacy for miR-375.The cell proliferative fold, migrated cell number, VEGF and VE-Cadherin contents in the medium and the tube length were significantly different among the CoCl2 model group, CoCl2+ miR-375 mimic group, CoCl2+ miR-375 mimic control group, CoCl2+ miR-375 inhibitor group and CoCl2+ miR-375 inhibitor control group (F=24.324, 26.776, 14.113, 19.225, 15.040, all at P<0.001), and those in the CoCl2+ miR-375 mimic group were evidently reduced in the CoCl2+ miR-375 mimic group compared with the CoCl2+ miR-375 mimic control group, while those in the CoCl2+ miR-375 inhibitor group were considerably elevated in comparison with the CoCl2+ miR-375 inhibitor control group (all at P<0.01). The expressions of β-catenin, cyclinD1, MMP2 and VEGF protein were significantly different among the normal control group, CoCl2 model group, CoCl2+ miR-375 mimic group, CoCl2+ miR-375 mimic control group, CoCl2+ miR-375 inhibitor group and CoCl2+ miR-375 inhibitor control group (F=11.753, 13.283, 16.770, 10.334, all at P<0.001). In addition, the cell proliferative fold, migrated cell number and the tube length were significantly increased in the CoCl2 model group and CoCl2+ mock group, and those in the CoCl2+ FZD4 siRNA group were decreased in comparison with the CoCl2+ mock group (all at P<0.05). Conclusions MiR-375 inhibits the growth, migration and tube formation ability of HRCECs in hypoxic status probably by regulating the activation of Wnt pathway via directly targeting FZD4. Key words: MicroRNAs/physiology; Retina; Endothelium, vascular; Neovascularization, pathologic; Signal transduction/physiology; Wnt proteins/metabolism; Cell proliferation /drug effects; Gene expression regulation

Study on the correlation between microRNA-195 expression level and glioma patient’s prognosis

Objective To explore the correlations between microRNA-195 (miR-195) expression level and glioblastoma patient’s pathological grade and prognosis. Methods The expression of miR-195 was detected by real-time quantitative polymerase chain reaction (Real-time PCR) in 34 cases of formalin fixed and paraffin embedded (FFPE) tissues of glioma, overall survival time was recorded. Results MiR-195 expression level was negatively correlated with clinicpathological grades. The median survival time in patients with high miR-195 expression level was 56.53 months, 41.53 months longer than that with low expression level. The risk of death for the sufferers with high miR-195 expression level was 36.3% of whom with lower expression level. Conclusion The expression of miR-195 was related to clinicpathological grade and prognosis of glioma. MiR-195 may be considered as a new promising prognostic molecular marker. Key words: Glioma; MicroRNA-195; Prognosis; Clinic pathological grade

Expression and significance of microRNA-195 and Bcl-2 mRNA in the childhood acute lymphoblastic leukemia cells

Objective To investigate the expression and significance of microRNA-195 (miR-195) and Bcl-2 in different type of childhood acute lymphoblastic leukemia(ALL) cells. Methods A total of 110 patients diagnosed as ALL from the Pediatrics Department and Outpatient Department of the First Affiliated Hospital of Zhengzhou University from July 2010 to December 2016 were recruited.The patients included newly diagnosed group, remi-ssion group and relapse group in which the newly diagnosed group was divided into standard risk(SR) group, interme-diate risk (IR) group and high risk (HR) group.And another 14 gender-and age-matched children received bone orthopaedic surgery who excluded tumor and blood system diseases in the same hospital were recruited as control group during the same period.Bone marrow samples of 2-5 mL were drawn from all the subjects.The samples of miR-195 and the expression of Bcl-2 mRNA were detected by using quantitative real time polymerase chain reaction, while the expression of Bcl-2 protein was also detected by using Western blot. Results MiR-195 expression was lower in the newly diagnosis and relapse ALL children(SR: 1.25±0.15; IR: 1.63±0.54; HR: 0.78±0.35; relapse: 0.46±0.18), compared with the control group, and there were statistically significant differences (t=42.44, 28.63, 41.35, 42.65, all P=0.000), but the expression of Bcl-2 mRNA was higher in the newly diagnosis and relapse ALL children than that in the control group (SR: 2.56±0.91; IR: 4.52±0.43; HR: 5.49±1.28; relapse: 5.32±1.15), which had statistically significant differences (t=3.58, 9.86, 10.12, 8.81, all P=0.000). With the rising of the criticality of ALL, the expression of miR-195 reduced gradually, and the expression of Bcl-2 mRNA increased step by step.Determining the protein of Bcl-2 detected by Western blot showed the same result. Conclusions The lower expression of miR-195 and the higher expression of Bcl-2 may be a marker to distinguish the risk factors of ALL, which may be important for the diagnosis and the determination of the degree of risk for the ALL children. Key words: Acute lymphoblastic leukemia; microRNA-195; Bcl-2

MicroRNA 375 regulates proliferation and migration of colon cancer cells by suppressing the CTGF-EGFR signaling pathway.

MicroRNA 375 (MIR375) is significantly down regulated in human colorectal cancer (CRC) tissues; we have previously identified MIR375 as a colon cancer associated microRNA (miRNA). We identified putative MIR375 target genes by comparing the mRNA microarray analysis data of MIR375-overexpressing cells with the candidate MIR375 target genes predicted by public bioinformatic tools. We investigated that the connective tissue growth factor (CTGF) is a direct target gene of MIR375. Expression of CTGF, a ligand of epidermal growth factor receptor (EGFR), was markedly enhanced in human CRC tissues in comparison with the corresponding normal colon tissues. We demonstrated that the expression levels of molecules in EGFR signaling pathways were regulated by MIR375 in colorectal cells. Using immunohistochemistry and the xenograft of MIR375-overexpressing colorectal cells in mice, we showed that MIR375 regulates cell growth and proliferation, angiogenesis, cell migration, cell cycle arrest, apoptosis, and necrosis in colon cells. Furthermore, results of MIR375 overexpression and cetuximab treatment indicated that the apoptosis and necrosis in colon cells were synergistically enhanced. Our results suggest that the down-regulation of MIR375 modulates EGFR signaling pathways in human colorectal cells and tissues by increasing CTGF expression; therefore, MIR375 may have a therapeutic value in relation to human CRC.

MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPβ regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

Antagonistic Effects of p53 and HIF1A on microRNA-34a Regulation of PPP1R11 and STAT3 and Hypoxia-induced Epithelial to Mesenchymal Transition in Colorectal Cancer Cells

In colorectal tumors, hypoxia causes resistance to therapy and promotes metastasis. Loss of the tumor suppressor p53 (encoded by TP53) provides cancer cells with a selective advantage under conditions of hypoxia, but little is known about the mediators of this effect. Isogenic colorectal cancer (CRC) cell lines with different TP53 genotypes were placed under conditions of hypoxia. We examined the effects on levels and activity of microRNA-34a (MIR34A) in CRC cells. We determined the expression and localization of protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11, also called INH3, HCGV, IPP3, HCGV, TCTE5, TCTEX5, or CFAP255) in 82 human colon cancers. We analyzed data on human colorectal carcinomas from the Cancer Genome Atlas collection to determine whether expression of PPP1R11 was affected by altered level or activity of p53, markers of epithelial-to-mesenchymal transition (EMT), or MIR34A or was associated with metastasis. We determined the effects of disruption Mir34a, Mir34b, and Mir34c in ApcMin/+ mice. DLD-1 cells were transfected with small inhibitor RNAs against PPP1R1, injected into the tail veins of immune-compromised mice, and followed by noninvasive bioluminescence imaging. The hypoxia inducible factor 1 alpha subunit (HIF1A) directly repressed the MIR34A gene in p53-defective CRC cells, whereas expression of MIR34A was induced in p53-proficient CRC cells exposed to hypoxia. Down-regulation of MIR34A was required for hypoxia-induced EMT, invasion and migration, and activation of STAT3 in CRC cells. We identified PPP1R11, whose product inhibits PP1, as a target of MIR34A. PPP1R11 mediates phosphorylation (activation) of STAT3, so expression of MIR34A reduced activation of STAT3 in p53-deficient CRC cells. Ectopic expression of PPP1R11 in CRC cells induced EMT, invasion, and migration, which all required STAT3. Increased expression of PPP1R11 in p53-deficient CRC cells was required for hypoxia-induced EMT, invasion, migration, and resistance to 5-fluorouracil, as well as metastasis of xenograft tumors to lungs of mice. Adenomas and derived tumoroids of ApcMin/+ mice with disruption of Mir34a, Mir34b, and Mir34c had increased levels of PPP1R11. Colorectal tumors from patients had increased levels of PPP1R11 at areas of invasion, compared with other areas of the tumor; increased level PPP1R11 associated with TP53 mutations and metastasis to the liver. HIF1A represses, whereas p53 increases, expression of MIR34A in CRC cells. MIR34A reduces expression of PPP1R11 to prevent activation of STAT3 and inhibit the EMT and metastasis. Strategies to target this pathway might be developed to inhibit CRC metastasis and overcome resistance to therapy associated with hypoxia.

Expression of microRNA-210 in gastric cancer and its relationship with clinicopathological factors and prognosis

Expression of microRNA-210 in gastric cancer and its relationship with clinicopathological factors and prognosis

MiR-148a increases glioma cell migration and invasion by downregulating GADD45A in human gliomas with IDH1 R132H mutations.

High-grade gliomas are severe tumors with poor prognosis. An R132H mutation in the isocitrate dehydrogenase (IDH1) gene prolongs the life of glioma patients. In this study, we investigated which genes are differentially regulated in IDH1 wild type (IDH1WT) or IDH1 R132H mutation (IDH1R132H) glioblastoma cells. Growth arrest and DNA-damage-inducible protein (GADD45A) was downregulated and microRNA 148a (miR-148a) was upregulated in in IDH1R132H human glioblastomas tissues. The relationship between GADD45A and miR-148a is unknown. In vitro experiments showed that GADD45A negatively regulates IDH1R132H glioma cell proliferation, migration, and invasion, and neurosphere formation in IDH1R132H glioblastoma stem cells (GSC). In addition, a human orthotopic xenograft mouse model showed that GADD45A reduced tumorigenesis in vivo. Our findings demonstrated that miR-148a promotes glioma cell invasion and tumorigenesis by downregulating GADD45A. Our findings provide novel insights into how GADD45A is downregulated by miR-148a in IDH1R132H glioma and may help to identify therapeutic targets for the effective treatment of high-grade glioma.

Sustained expression of miR-26a promotes chromosomal instability and tumorigenesis through regulation of CHFR.

MicroRNA 26a (miR-26a) reduces cell viability in several cancers, indicating that miR-26a could be used as a therapeutic option in patients. We demonstrate that miR-26a not only inhibits G1-S cell cycle transition and promotes apoptosis, as previously described, but also regulates multiple cell cycle checkpoints. We show that sustained miR-26a over-expression in both breast cancer (BC) cell lines and mouse embryonic fibroblasts (MEFs) induces oversized cells containing either a single-large nucleus or two nuclei, indicating defects in mitosis and cytokinesis. Additionally, we demonstrate that miR-26a induces aneuploidy and centrosome defects and enhances tumorigenesis. Mechanistically, it acts by targeting G1-S transition genes as well as genes involved in mitosis and cytokinesis such as CHFR, LARP1 and YWHAE. Importantly, we show that only the re-expression of CHFR in miR-26a over-expressing cells partially rescues normal mitosis and impairs the tumorigenesis exerted by miR-26a, indicating that CHFR represents an important miR-26a target in the regulation of such phenotypes. We propose that miR-26a delivery might not be a viable therapeutic strategy due to the potential deleterious oncogenic activity of this miRNA.

MicroRNA-26a-interleukin (IL)-6-IL-17 axis regulates the development of non-alcoholic fatty liver disease in a murine model.

Non-alcoholic fatty liver disease (NAFLD) is a hepatic presentation of obesity and metabolic syndrome. MicroRNA 26a (Mir-26a) has been reported to play functions in cellular differentiation, cell growth, cell apoptosis and metastasis. A recent paper indicated that Mir-26a regulated insulin sensitivity and metabolism of glucose and lipids. However, the role of Mir-26a in NAFLD still needs to be investigated further. In our current study, vectors encoding pre-Mir-26a (LV-26a) and an empty lentiviral vector (LV-Con) delivered approximately 2 × 107 transforming units of recombinant lentivirus were injected into mice through the tail vein. LV-26a-infected mice were protected from glucose dysmetabolism and showed markedly decreased total liver weight, hepatic triglyceride deposition and serum alanine transaminase (ALT) concentration when compared with LV-Con-treated mice. LV-26a-treated mice also exhibited decreased infiltration of immune cells in the liver - something attributed to reduce infiltration of T cell receptor (TCR)-γδ+ , granulocyte-differentiation antigen-1 (Gr-1)+ cells and CD11b+ cells. Next, we found that Mir-26a inhibited the expression of interleukin (IL)-17 and IL-6 in vivo and in vitro. Furthermore, the decreased expression of IL-17 in the liver tissue induced by Mir-26a was abrogated completely by IL-6 overexpression. The decreased total liver weight, hepatic triglyceride deposition and serum ALT concentration induced by Mir-26a was also abrogated completely by IL-6 over-expression. In conclusion, the Mir-26a-IL-6-IL-17 axis regulates the development of NAFLD in a murine model.

MicroRNA 34a contributes to virus-mediated apoptosis through binding to its target gene Bax in influenza A virus infection

Influenza A virus is a cytolytic virus that induces apoptosis in numerous cell types. However, molecular mechanism of cell apoptosis induced by influenza A virus is not well understood. The present study was aimed to investigate the molecular mechanism of miR-34a involved in influenza A virus induced apoptosis. Firstly, microRNA array was used to select miRNAs involved in influenza A virus infection. Then, Luciferase reporter assay and western blot were carried to determine the target gene of miR-34a. Finally, miR-34a mimic was transfected in influenza A virus-infected A549 cells, and western blot was used to test the function of miR-34a and its target gene in in influenza A virus infection. miR-34a was found significantly down-regulated in influenza A virus-infected A549 cells and was further confirmed by qRT-PCR in vitro and in vivo (P<0.01). Next, transfection miR-34 mimic/inhibitor in influenza A virus-infected A549 cells showed that overexpression of miR-34a could inhibit influenza virus-induced apoptosis. Furthermore, target prediction analysis revealed that miR-34a complemented to the 3'-UTR of Bax mRNA. Luciferase reporter assay and western blot confirm that miR-34a directly targeted Bax. Moreover, we detected influenza virus infection enhanced the luciferase reporter translational activity in A549 cells transfected with the construct with Bax 3'-UTR encoding miR-34a binding site. However, western blot analysis showed that the up-regulation of Bax protein level was significantly reversed by overexpression miR-34a. It is, therefore, concluded that influenza virus infection regulated pro-apoptotic Bax expression through miR-34a downregulation, which might be the partial mechanism of influenza virus-induced cell apoptosis.

Expression and clinical significance of microRNA-148a in patients with non-small cell lung cancer

Objective To investigate the expressions of microRNA-148a (miR-148a) in non-small cell lung cancer (NSCLC) tissues and adjacent non-tumor tissues and the relationships with clinicopathologic features, to analyze the roles of miR-148a in evaluating prognosis. Methods Expression levels of miR-148a in 48 pairs of NSCLC and adjacent non-tumor tissues were detected by quantitative real-time PCR (RT-PCR). The patients were divided into 2 groups according to the median value of miR-148a. The relationships between the expression levels of miR-148a and clinicopathological features were analyzed. The survival rate was estimated by Kaplan-Meier method, and single factor analysis was performed. The relationship between the expression level of miR-148a and prognosis was analyzed by Log-rank test. Results miR-148a expression levels were significant down-regulated in NSCLC tissues compared with adjacent non-tumor tissues (1.052 ± 0.659 vs. 1.397 ± 0.667, t=2.549, P=0.013). miR-148a expression level was correlated with tumor size (χ2=4.594, P=0.030), lymph nodes metastasis (χ2=5.486, P=0.019) and clinical stage (χ2=4.090, P=0.043), while it was not correlated with age (χ2=0.354, P=0.551), gender (χ2=2.277, P=0.131), histological type (χ2=0.403, P=0.525) and smoking (χ2=0.444, P=0.505). Kaplan-Meier analysis revealed a significant correlation between low miR-148a expression level and poor overall survival (23.9 months vs. 38.7 months, χ2=4.941, P=0.026), and no significant correlation with progression-free survival (21.6 months vs. 29.4 months, χ2=2.168, P=0.141). Conclusion Down-regulation of miR-148a is correlated with worse clinicopathological features, which serves an independent marker for poor prognosis in patients with NSCLC. Key words: Carcinoma, non-small-cell lung; MicroRNAs; Pathological conditions, signs and symptoms; Survival analysis

Expression and clinical significance of microRNA-375 in the plasma of patients with hepatocellular carcinoma

Expression and clinical significance of microRNA-375 in the plasma of patients with hepatocellular carcinoma

Downregulation of B-cell lymphoma/leukemia-2 by overexpressed microRNA 34a enhanced titanium dioxide nanoparticle-induced autophagy in BEAS-2B cells.

Titanium dioxide (TiO2) nanoparticles (TNPs) are manufactured worldwide for a wide range of applications and the toxic effect of TNPs on biological systems is gaining attention. Autophagy is recognized as an emerging toxicity mechanism triggered by nanomaterials. MicroRNA 34a (miR34a) acts as a tumor suppressor gene by targeting many oncogenes, but how it affects autophagy induced by TNPs is not completely understood. Here, we observed the activation of TNP-induced autophagy through monodansylcadaverine staining and LC3-I/LC3-II conversion. Meanwhile, the transmission electron microscope ultrastructural analysis showed typical morphological characteristics in autophagy process. We detected the expression of miR34a and B-cell lymphoma/leukemia-2 (Bcl-2). In addition, the underlying mechanism of TNP-induced autophagy was performed using overexpression of miR34a by lentivirus vector transfection. Results showed that TNPs induced autophagy generation evidently. Typical morphological changes in the process of autophagy were observed by the transmission electron microscope ultrastructural analysis and LC3-I/LC3-II conversion increased significantly in TNP-treated cells. Meanwhile, TNPs induced the downregulation of miR34a and increased the expression of Bcl-2. Furthermore, overexpressed miR34a decreased the expression of Bcl-2 both in messenger RNA and protein level, following which the level of autophagy and cell death rate increased after the transfected cells were incubated with TNPs for 24 hours. These findings provide the first evidence that overexpressed miR34a enhanced TNP-induced autophagy and cell death through targeted downregulation of Bcl-2 in BEAS-2B cells.

Expression of MicroRNA‐148a and its Target Gene EPAS1 (Endothelial PAS domain‐containing protein 1) in Intestinal Cells in Response to Bifidobacterium bifidum MIMBb75

Bifidobacterium bifidum MIMBb75 is a probiotic strain isolated from healthy human feces. We have previously shown that this strain modulates the composition of the gut microbiota, a core probiotic benefit. Though, molecular mechanisms underlying effects specific to this strain are unknown, particularly at the transcriptional and post‐transcriptional (microRNA, miRNA) level. We found that the expression of intestinal miRNA, including miR‐148a, depends on the presence of the microbiota. MiR‐148a is a proposed biomarker of intestinal health. One of its target genes, EPAS1, also called HIF2α (Hypoxia‐Inducible Factor 2α) is a pro‐inflammatory and pro‐carcinogenic transcription factor.The aim of this study was to investigate the effect of B. bifidum MIMBb75 on the intestinal expression of miR‐148a and EPAS1 in vitro and in vivo.The expression levels of miR‐148a and EPAS1 were assessed by qPCR using RNA extracted from Caco‐2 cells incubated with B. bifidum MIMBb75, B. bifidum NCC390 or B. longum NCC 2705 (cell ratio of 10:1) or control vehicle for 1, 4 and 24 hours at 37°C. To prove that EPAS1 gene expression changes do indeed depend on miR‐148a modulation by B. bifidum MIMBb75, miRNA silencing was performed via transfection of miR‐148a inhibitor in Caco‐2 cells for 72h, followed by incubation of transfected cells with B. bifidum MIMBb75. In addition, C57BL/6J male mice were gavaged for 2 or 14 days with 108 colony forming units of B. bifidum MIMBb75/day or vehicle alone and sacrificed by cervical dislocation. Caecum was used for RNA extraction and gene expression analysis as described above.Of the three strains tested, only B. bifidum MIMBb75 showed a significant upregulation of miR‐148a after 1 and 4 hours of co‐incubation with Caco‐2 cells (P &lt;0.01), but not at 24h. Oral administration of B. bifidum MIMBb75 increased significantly miR‐148a expression in the caecum after 2 days (P&lt; 0.05) but not after 14 days of probiotic administration, which was accompanied by a decrease of EPAS1 expression (P&lt;0.01) at day 2. In vitro silencing of miR‐148a using miRNA inhibitor constructs reversed EPAS1 downregulation.This study brings a new concept of strain‐ and time‐dependent bifidobacteria‐host crosstalk via microRNA. Because early exposure to B. bifidum MIMBb75 repressed the expression of EPAS1, this probiotic may be helpful in intestinal inflammatory conditions associated with EPAS1 over‐expression, such as colitis. Probiotic‐dependent regulation of host gene expression via miRNA may open the way to new therapeutic avenues.Support or Funding InformationNSERC, JP Bickell Foundation, Lawson Family Chair in Microbiome Nutrition Research