Dysregulation of MicroRNA-34a Expression in Colorectal Cancer Inhibits the Phosphorylation of FAK Via VEGF

Background

MicroRNAs (miRs) are small non-coding RNAs that play important roles in cancer development where they can act as oncogenes or as tumor-suppressors. MiR-34a is a tumor suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in colorectal cancer (CRC).

Aim

This study aims to show the dysregulation of miR-34a in CRC and to characterize the function and mechanism of miR-34a in CRC cell lines.

Methods

The expression of miR-34a was detected using real-time PCR on CRC tissues and adjacent non-tumorous tissues. The ELISA was used to assess vascular endothelial growth factor (VEGF). The focal adhesion kinase (FAK) and phosphorylated FAK Y397 (pY397FAK) were measured by Western blot. The functions of miR-34a in vivo were measured by migration, invasion, CCK-8 assay and flow cytometry.

Results

MiR-34a was significantly downregulated and pY397FAK was upregulated in CRC cancer tissues. It plays an important role in inhibiting migration and invasion and in increasing apoptosis of CRC cells. Bioinformatic analysis suggested that VEGF may be a target of miR-34a, and this hypothesis was proved by ELISA and RT-PCR. The level of pY397FAK that could be activated by VEGF was downregulated in miR-34a overexpression CRC cell lines. The phosphorylation of FAK at 397 sites in miR-34a-stable cell lines was completely rescued by extra VEGF treatment.

Conclusion

MiR-34a is frequently downregulated in CRC and modulates the phosphorylation of FAK by negatively regulating VEGF.     

miR-34a通过调控Sirt1/FoxO1通路影响血管内皮细胞凋亡

目的 探讨microRNA-34a(miR-34a)对氧化型低密度脂蛋白(ox-LDL)诱导人主动脉内皮细胞(HAEC)凋亡的影响及作用机制.方法 ox-LDL处理HAEC,实时荧光定量PCR(qRT-PCR)检测miR-34a的表达水平.Annexin V-FITC/PI双染流式细胞仪(FCM)和Caspase-3活...     

MicroRNA-29a and MicroRNA-124 as novel biomarkers for hepatocellular carcinoma

BackgroundNumerous microRNAs (miRNAs) have been observed to be abnormally expressed in cancer. Therefore, miRNA signatures could be potential noninvasive diagnostic and prognostic biomarkers for hepatocellular carcinoma (HCC).AimsTo correlate miRNA-29a and miRNA-124 expression levels with the clinical features and survival rates of HCC patients.MethodsSerum miRNA expression in 150 samples (50 patients with HCC, 50 patients with liver cirrhosis, and 50 healthy controls) were quantified using real-time qRT-PCR.ResultsThe expression levels of serum miRNA-29a were higher and the levels of miRNA-124 were lower in patients with HCC than in patients with liver cirrhosis and controls. ROC curve analysis showed promising accuracy for both miRNAs in distinguishing patients with HCC from those with liver cirrhosis. Levels of miRNA-29a were related to tumor number, size, stage, and outcome, whereas levels of miRNA-124 were related to vascular invasion. The overall survival rate of patients with low miRNA-29a expression was significantly higher than that of patients with high expression. Additionally, the multivariate analysis identified miRNA-29a as an independent prognostic variable.ConclusionsThe investigated miRNAs showed acceptable accuracy in the diagnosis of HCC; therefore, both could be utilized as diagnostic biomarkers. Additionally, miRNA-29a could be used as a prognostic biomarker.     

MicroRNA-21 in Cardiovascular Disease

MicroRNA-21 (miR-21) is a highly expressed microRNA (miRNA) in cardiovascular system. Recent studies have revealed that its expression is deregulated in heart and vasculature under cardiovascular disease conditions such as proliferative vascular disease, cardiac hypertrophy and heart failure, and ischemic heart disease. miR-21 is found to play important roles in vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions. Accordingly, miR-21 is proven to be involved in the pathogenesis of the above-mentioned cardiovascular diseases as demonstrated by both loss-of-function and gain-of-function approaches. Programmed cell death 4 (PDCD4), phosphatase and tensin homology deleted from chromosome 10 (PTEN), sprouty1 (SPRY1), and sprouty2 (SPRY2) are the current identified target genes of miR-21 that are involved in miR-21-mediated cardiovascular effects. miR-21 might be a novel therapeutic target in cardiovascular diseases. This review article summarizes the research progress regarding the roles of miR-21 in cardiovascular disease.     

微小RNA-144表达对大鼠心肌细胞凋亡的影响

目的 探讨微小RNA-144(miR-144)对心肌细胞凋亡的影响.方法运用转染的方法,使大鼠H9C2胚胎心肌细胞中高表达miR-144(miR-144组),实时定量PCR确定miR-144表达上调后,通过细胞计数试剂盒-8(CCK-8)、半胱天冬酶(Caspase)-3、流式细胞术等方法测定细胞凋亡水平,观察miR-144对细胞凋亡的影响.空白脂质体及随机微小RNA片段作为空白对照及阴性对照.结果实时定量PCR结果显示,miR-144组miR-144的表达(2178.84±838.52)明显高于空白对照组(1.00±0.00)和阴性对照组(2.06±0.73)(P均＜0.01).与阴性对照组和空白对照组比较,miR144组细胞增殖明显低、Caspase-3活性明显高、细胞凋亡率明显高,而阴性对照组与空白对照组以上数值比较差异均无统计学意义.结论合成的miR-144片段(miR-144 mimics)能选择性上调miR144的表达,并促进心肌细胞凋亡、抑制细胞增殖.

Abstract：

Objective To investigate the effects of microRNA-144 (miR-144) expression on H9C2(2-1) myocytes. Methods MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with LipofectamineTM 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. Results Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84±838.52) compared with negative (2.06±0.73) and blank (1.00±0.00) control group ( all P ＜0. 01 ). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group,while no significant difference was found between the latter 2 groups. Conclusion MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.     

微小RNA-144表达对大鼠心肌细胞凋亡的影响

Objective To investigate the effects of microRNA-144 (miR-144) expression on H9C2(2-1) myocytes. Methods MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with LipofectamineTM 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. Results Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84±838.52) compared with negative (2.06±0.73) and blank (1.00±0.00) control group ( all P ＜0. 01 ). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group,while no significant difference was found between the latter 2 groups. Conclusion MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.     

微小RNA-144表达对大鼠心肌细胞凋亡的影响

Objective To investigate the effects of microRNA-144 (miR-144) expression on H9C2(2-1) myocytes. Methods MiR-144 was up-regulated in primary cultured H9C2 (2-1) myocytes through transfection. Cells transfected with LipofectamineTM 2000 and its mixture with miRNA synthesized randomly as blank control and negative control respectively. The up-regulation of miR-144 was confirmed by real-time PCR. Cell apoptosis was evaluated by means of CCK-8, Caspase-3 and flow cytometry. Results Real-time PCR results showed that the miR-144 expression was obviously increased in miR-144 up-regulation group (2178.84±838.52) compared with negative (2.06±0.73) and blank (1.00±0.00) control group ( all P ＜0. 01 ). The proliferation was lower, the activity of Caspase-3 was elevated and the apoptosis rates were significantly increased in miR-144 up-regulation group compared with negative and blank control group,while no significant difference was found between the latter 2 groups. Conclusion MiR-144 mimics may selectively up-regulate the expression of miR-144 in myocardial cells and consequently promote apoptosis and inhibit proliferation in myocardial cells.     

"Pseudo-Abnormal" Hemoglobins

Three examples of artefactual "pseudo-abnormal" hemoglobins were observed in the course of starch block electrophoresis analysis of over 400 specimens. In fresh samples from the same patients, these components were no longer demonstrable. It is suggested that repeated examination with different bloodspecimens should be done before an unusual component is accepted as atrue hemoglobin variant.

Submitted on February 28, 1961 Accepted on April 7, 1961     

MicroRNA-155通过CEH影响巨噬细胞泡沫化过程

目的探讨microRNA-155(miR-155)通过干扰人THP-1单核巨噬细胞内胆固醇酯水解酶(CEH)的表达来影响巨噬细胞泡沫化形成的机制。方法通过油红O染色和和高效液相色谱法(HPLC)分析胆固醇酯(CE)含量,观察阳性细胞率和细胞内胆固醇酯(CE)含量的变化情况。Western blot检测上述各组细胞内CEH、三磷酸腺苷结合盒转运体A1(ABCA1)、清道夫受体(SR-A)的表达变化。结果转染miR-155 mimics的细胞阳性比率、胆固醇酯、游离胆固醇(FC)、总胆固醇(TC)含量均随着时间的延长明显下降(P0.05);miR-155 mimics组细胞阳性比率、胆固醇含量和SR-A的表达均被明显降低(P0.05),而CEH和ABCA1的表达被明显增强(P0.05);miR-155 mimics+siRNA-CEH和siRNA-CEH组细胞阳性比率、胆固醇含量和SR-A的表达均明显增加(P0.05),而CEH和ABCA1的表达被明显降低(P0.05)。结论高表达的miR-155可以明显抑制巨噬细胞泡沫化的形成,该作用可能是通过增强巨噬细胞内CEH的表达进而影响ABCA1和SR-A等相关基因的表达来得以实现。     

The Unitary Nature of "Complete" and "Incomplete" Pathologic Cold Hemagglutinins

Several human pathologic sera containing high titered cold agglutinins werestudied to determine whether the serologic activity ascribed to an "incompletecold antibody" could be separated from the "complete" cold agglutinin activity.Separation was not achieved by physicochemical methods, including zoneelectrophoresis, density gradient ultracentrifugation, and anion exchangechromatography. Both activities were susceptible to destruction by mercaptans.Neither activity could be differentially absorbed from the sera. Using "Bombay" and I-negative ("i") red cells, a difference in specificity of the two activities for the H or I antigen of human erythrocytes could not be demonstrated.The simplest interpretation of these findings is that there is only one antibodyinvolved, the cold agglutinin, and that the serologic manifestation usually attributed to an additional "incomplete cold antibody", i.e. the production of apositive antiglobulin reaction of the "non--globulin" type, results from aninteraction of complement components with the cold agglutinin-erythrocytecomplex.

Three of these cold agglutinating sera were unreactive with I-negativeerythrocytes, in keeping with the reported anti-I specificity of these antibodies. A fourth serum retained moderate, though greatly reduced, activityagainst these cells, and the interpretation of this finding is discussed.

The anti-H specificity of the incomplete cold antibodies in normal humansera was confirmed by their failure to sensitize "Bombay" erythrocytes. Thiswas in sharp contrast to the excellent reactivity of the pathologic sera withthese cells, demonstrating that pathologic cold agglutinins are unrelated tothe incomplete cold antibodies present in most normal sera.

Submitted on October 23, 1961 Accepted on December 2, 1961     

MicroRNA-223 is neuroprotective by targeting glutamate receptors

Stroke is a major cause of mortality and morbidity worldwide. Extracellular glutamate accumulation leading to overstimulation of the ionotropic glutamate receptors mediates neuronal injury in stroke and in neurodegenerative disorders. Here we show that miR-223 controls the response to neuronal injury by regulating the functional expression of the glutamate receptor subunits GluR2 and NR2B in brain. Overexpression of miR-223 lowers the levels of GluR2 and NR2B by targeting 3′-UTR target sites (TSs) in GluR2 and NR2B, inhibits NMDA-induced calcium influx in hippocampal neurons, and protects the brain from neuronal cell death following transient global ischemia and excitotoxic injury. MiR-223 deficiency results in higher levels of NR2B and GluR2, enhanced NMDA-induced calcium influx, and increased miniature excitatory postsynaptic currents in hippocampal neurons. In addition, the absence of MiR-223 leads to contextual, but not cued memory deficits and increased neuronal cell death following transient global ischemia and excitotoxicity. These data identify miR-223 as a major regulator of the expression of GluR2 and NR2B, and suggest a therapeutic role for miR-223 in stroke and other excitotoxic neuronal disorders.MicroRNAs (miRNAs) are small noncoding endogenous RNA molecules that repress their target mRNA through complementary binding in the message 3′-UTR (1). MiRNAs play important roles in multiple physiological processes such as cell death and survival, cellular response to stress, stem cell division, and pluripotency (2). MiRNAs also play important roles in disease processes including cancer (3), cardiovascular disease (4), and neurodegenerative diseases (5). Due to their small size, relative ease of delivery, and sequence specificity in recognizing their targets, miRNAs are promising therapeutic targets (6).Stroke is the second major killer and the leading cause of disability worldwide (7). Overstimulation of the glutamate receptor (glutamate excitotoxicity) is a major mechanism for neuronal cell death during stroke, central nervous system (CNS) trauma, and chronic neurodegenerative disorders. Excessive calcium influx through the N-methyl-d-aspartate receptors (NMDARs) results in abnormally high intracellular calcium concentrations leading to lethal consequences. This calcium influx through the NMDAR requires membrane depolarization induced by sodium influx through 2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) (8). Whereas accumulating evidence indicates that phosphorylation and trafficking play important roles in regulation of glutamate receptor signaling, the molecular mechanisms regulating glutamate receptor expression levels remain unexplored. Recent work suggests that the miRNA pathway regulates the AMPAR subunit GluR2 expression (9, 10). In hippocampal neurons, MiR-125b has been shown to regulate the NMDAR subunit NR2A (11). However, miRNA regulation of glutamate receptor expression remains poorly characterized. Identifying miRNAs that could regulate the glutamate receptor provide the opportunity for treating stroke and chronic neurodegenerative diseases. Recent success in therapeutic targeting of small RNAs in animal models and in humans emphasizes such treatment strategies (6).MiR-223 is highly expressed in bone marrow and neutrophils where it plays an important role in regulating granulopoeisis and neutrophil function (12, 13). MiR-223 is deregulated in acute myeloid leukemia (14, 15). We find that miR-223 is also expressed in the nervous system and we demonstrate that miR-223 controls the expression and function of GluR2 and NR2B subunits of the glutamate receptor. Using in vitro and in vivo models of ischemic reperfusion brain injury and excitotoxic neuronal death we show that miR-223 is a neuroprotective microRNA.     

Hypothesis: "Immunologic" Oncogenesis

The evidence that immunologic mechanisms may predispose to malignancyis reviewed, and an attempt is made to relate neoplasia possibly induced byimmunologic or other reactions involving the recognition of self and not-selfto known oncogenic mechanisms. It is suggested that "immunologic" oncogenesis as well as malignant transformation induced by known oncogenic agentsmay be mediated at least in part by chromosomal aberrations. These couldact directly to promote neoplasia, or the cell death and proliferation consequentto them could promote other mutational events. Alternatively, the chromosomal changes themselves may not be sufficient for oncogenesis, but they maypotentiate malignant transformation by other agents such as viruses.

Submitted on January 10, 1967 Accepted on May 31, 1967     

MicroRNA-328 as a regulator of cardiac hypertrophy

Cardiac hypertrophy is a primary predictor of progressive heart disease that often results in heart failure. Growing evidence has demonstrated that microRNAs (miRNAs) play a critical role in regulating cardiac hypertrophy. This study was designed to evaluate the effect of miR-328 on cardiac hypertrophy and the potential molecular mechanisms. We found that transgenic overexpression of miR-328 in the heart induced cardiac hypertrophy in mice, which was accompanied by reduced SERCA2a level increased intracellular calcium concentration and calcineurin protein level, and enhanced NFATc3 nuclear translocation. However, normalization of miR-328 level by its antisense chemically modified with locked nucleic acid (LNA-antimiR-328) reversed the changes. Forced expression of miR-328 resulted in cardiomyocyte hypertrophy in cultured neonatal rat ventricular cells, which was accompanied by downregulation of SERCA2a expression and activation of the calcineurin/NFATc3 signaling pathway. These changes were abolished by LNA-antimiR-328. We validated the SERCA2a as a direct target for miR-328. MiR-328 expression was upregulated in cardiomyocyte treated with isoproterenol (ISO) to induce hypertrophy; while knockdown of miR-328 attenuated the hypertrophic responses. The level of miR-328 was significantly elevated in a mouse model of hypertrophy by thoracic aortic banding (TAC). Consistently, SERCA2a was downregulated, whereas calcineurin were upregulated, and NFATc3 nuclear translocation was enhanced. In contrast, hypertrophy in these mice was significantly alleviated when treated with miR-328 antisense. MiR-328 promotes cardiac hypertrophy by targeting SERCA2a. Our study therefore uncovered a novel molecular mechanism for cardiac hypertrophy and indicated miR-328 as a potential therapeutic target for this cardiac condition.