Comprehensive analysis of phospholipid in milk and their biological roles as nutrients and biomarkers.

Phospholipids (PL) have garnered significant attention due to their physiological activities. Milk and other dairy products are important dietary sources for humans and have been extensively used to analyze the presence of PL by various analytical techniques. In this paper, the analysis techniques of PL were reviewed with the eight trigrams of phospholipidomics and a comprehensive fingerprint of 1295 PLs covering 8 subclasses in milk and other dairy products, especially. Technology is the primary productive force. Based on phospholipidomics technology, we further review the relationship between the composition of PL and factors that may be involved in processing and experimental operation, and emphasized the significance of the biological role played by PL in dietary supplements and biomarkers (production, processing and clinical research), and providing the future research directions.

AFB1 Triggers Lipid Metabolism Disorders through the PI3K/Akt Pathway and Mediates Apoptosis Leading to Hepatotoxicity.

As the most prevalent mycotoxin in agricultural products, aflatoxin B1 not only causes significant economic losses but also poses a substantial threat to human and animal health. AFB1 has been shown to increase the risk of hepatocellular carcinoma (HCC) but the underlying mechanism is not thoroughly researched. Here, we explored the toxicity mechanism of AFB1 on human hepatocytes following low-dose exposure based on transcriptomics and lipidomics. Apoptosis-related pathways were significantly upregulated after AFB1 exposure in all three hES-Hep, HepaRG, and HepG2 hepatogenic cell lines. By conducting a comparative analysis with the TCGA-LIHC database, four biomarkers (MTCH1, PPM1D, TP53I3, and UBC) shared by AFB1 and HCC were identified (hazard ratio > 1), which can be used to monitor the degree of AFB1-induced hepatotoxicity. Simultaneously, AFB1 induced abnormal metabolism of glycerolipids, sphingolipids, and glycerophospholipids in HepG2 cells (FDR < 0.05, impact > 0.1). Furthermore, combined analysis revealed strong regulatory effects between PIK3R1 and sphingolipids (correlation coefficient > 0.9), suggesting potential mediation by the phosphatidylinositol 3 kinase (PI3K) /protein kinase B (AKT) signaling pathway within mitochondria. This study revealed the dysregulation of lipid metabolism induced by AFB1 and found novel target genes associated with AFB-induced HCC development, providing reliable evidence for elucidating the hepatotoxicity of AFB as well as assessing food safety risks.

[Distribution Characteristics and Risk Assessment of Tetracycline Antibiotics (TCs) in Soil-Vegetable System with Soil Fertilized with Animal Manure].

The effects of antibiotic contamination on vegetable safety and the ecological risks of soil after returning livestock and poultry manure to the land require sufficient future attention. Tetracycline antibiotics (TCs) are often detected at high concentrations in livestock manure and vegetable production soils. Recently, pot experiments and field investigation methods have often been used to understand the effects of TCs contamination on the vegetable safety and ecological risks of soil, whereas field experiments are employed less frequently. This study investigated the distribution characteristics of TCs in the soil-vegetable system following manure application using a combination of pot and field experiments. The human health risks of the edible parts of Chinese flowing cabbage were assessed using the health risk quotient method based on the acceptable daily intake (ADI-HQ), and the ecological risks of TCs-contaminated soils were evaluated using the risk quotient method associated with the species sensitivity distribution model (SSD-RQ). The results showed that oxytetracycline (OTC) was the major type of TCs in Chinese flowering cabbage based on both the pot and field experiments. The maximum contents (dry weight) of OTC in the aboveground parts of the Chinese flowering cabbage for the pot and field experiments were 29.25 µg·kg-1 and 45.03 µg·kg-1, respectively, whereas those of their underground parts were 87.32 µg·kg-1 and 135.44 µg·kg-1, respectively. Meanwhile, higher contents of TCs were detected in Chinese flowering cabbage collected from the field experiment than those from the pot experiment. OTC was also the major type of TCs in soil from both the pot and field experiments, with their contents up to 604.30 µg·kg-1 and 1013.68 µg·kg-1, respectively. Higher residual contents of three TCs were detected in soils collected from the field experiment than those from the pot experiment. Under the experimental conditions, with the except that OTC in Chinese flowering cabbage from the field experiment would pose medium health risks (HQ>0.1) to children, the contents of three TCs in other treated Chinese flowering cabbage would pose low health risks (HQ ≤ 0.1) to adults and children. In the pot experiments, three TCs present in Chinese flowering cabbage would pose low health risks (HQ ≤ 0.1) to adults and children. Additionally, the TCs in soils with manure application from the pot and field experiments may have posed both moderate or high levels of ecological risks (HQ>0.1 or HQ>1). Therefore, the effects of antibiotic contamination on vegetable safety and their potential ecological risks on soil following manure fertilization need to be given special attention.

Apically extruded debris, canal transportation, and shaping ability of nickel-titanium instruments on contracted endodontic cavities in molar teeth.

PURPOSE: Apically extruded debris, canal transportation and shaping ability were compared between contracted endodontic cavities (CECs) and traditional endodontic cavities (TECs) after instrumentation with XP-endo Shaper (XPS), ProTaper Gold (PTG), ProTaper for hand-use (HPT) and Hero Shaper. METHODS: The CECs or TECs groups were sub-divided into 24 groups according to root canal morphology and nickel-titanium (Ni-Ti) instruments. The weight of apically extruded debris was calculated using the Myers and Montgomery model. Pre- and postoperative images of teeth were scanned using micro-CT and the three-dimensional models were constructed and compared. RESULTS: Under CECs or TECs, XPS and PTG produced less apical debris and formed less canal transportation than HPT and Hero Shaper (P < 0.05). XPS group under CECs extruded less apical debris than that under TCEs for round canals with curvature of 20°-35° (P < 0.05). The centering ratios of four tested instruments were higher under TECs than those under CECs (P < 0.05). The HPT and Hero Shaper had more transportation under CECs than that under TCEs (P < 0.05). No statistical difference was found regarding shaping ability among all the groups. CONCLUSION: Under CECs, XPS preserves the original root canal anatomy, meanwhile it produces less apical debris than the other instruments.

Lipidomic profiling study on neurobehavior toxicity in zebrafish treated with aflatoxin B1.

Mycotoxin aflatoxin B1 (AFB1) has been proven to cause neurotoxicity, but its potential interference with the normal function of brain tissue is not fully defined. As the indispensable role of lipids in maintaining the normal function of brain tissue, the aim of this study is to clarify the effect of AFB1 short-term (7 days) exposure on brain tissue from the perspective of lipid metabolism. In this study, zebrafish were exposed to two concentrations (5, 20 µg/L). Through quantitative analysis of AFB1, the detection of AFB1 in zebrafish brain tissue was discovered for the first time, combined with the changes in zebrafish neurobehavior, the occurrence of brain injury was deduced. Subsequently, 1734 lipids in zebrafish brain tissue were mapped using ion mobility time-of-flight mass spectrometry (UPLC-QTOF-IMS-MS), which has great advantages in lipid detection. Comparative analysis of the abnormal lipid metabolism in zebrafish brain revealed 114 significantly changed lipids, mainly involving two pathways of sphingolipid metabolism and fatty acid degradation. This study discovered the detection of AFB1 in the brain and revealed a potential link between AFB1-induced behavioral abnormalities and lipid metabolism disorders in brain tissue, providing reliable evidence for elucidating the neurotoxicity of AFB1.

Porphyromonas gingivalis­derived lipopolysaccharide inhibits brown adipocyte differentiation via lncRNA­BATE10.

Epidemiological studies have suggested an association between obesity and periodontal disease. Brown adipose tissue (BAT) has an anti-obesity effect. However, the effects of periodontitis on obesity and BAT remain unclear. Therefore, the present study aimed to determine the effects of lipopolysaccharide derived from Porphyromonas gingivalis (P. gingivalis LPS) on brown adipocytes. For this purpose, the present study examined the effects of the intravenous administration of Porphyromonas gingivalis (P. gingivalis) in mice, the treatment of brown adipocytes with P. gingivalis LPS during differentiation, and the administration of small interfering RNA targeting interferon on brown preadipocytes by assessing the expression of genes involved in differentiation, using a long non-coding (lnc)RNA, and pro-inflammatory factors using reverse transcription-quantitative PCR. In addition, the accumulation of lipid droplets was examined using Oil Red O staining. P. gingivalis LPS reduced the expression of uncoupling protein 1 (UCP1) and lncRNA-BATE10 in brown adipocytes during differentiation. Consistent with this finding, P. gingivalis reduced UCP1 and lncRNA-BATE10 expression in the BAT of mice. lncRNA-BATE10 may thus be involved in the regulation of UCP1 expression that occurs during the differentiation of brown adipocytes treated with P. gingivalis LPS. Thus, P. gingivalis LPS may inhibit BAT differentiation by reducing lncRNA-BATE10 expression.

Field study on loss of tetracycline antibiotics from manure-applied soil and their risk assessment in regional water environment of Guangzhou, China.

Tetracycline antibiotics (TCs) introduced into agricultural fields via manure application tend to accumulate in soils and further reach water environments via surface runoff and leachate, posing potential risks to regional water environment. This study investigated the loss of tetracycline (TC), oxytetracycline (OTC), and chlortetracycline (CTC) in surface runoff and leachate samples collected from a vegetable farmland with manure application in Guangzhou, South China. A risk assessment method was constructed for evaluating the ecological and health risks of manure-associated antibiotics released from soil into water environment. The results showed that the concentrations of three TCs in surface runoff, 30-cm leachate, and 60-cm leachate after the first rainfall event were 2.79-35.97, 1.71-18.44, and 0.4-2.66 µg/L, respectively, which all decreased with sampling depth and the time after rainfall events. Up to 0.13% of TCs were transported into the surface water through surface runoff, while less than 0.01% of TCs were transported into the groundwater through leachate at 60 cm. OTC had a higher total mass percentage (0.13%) into surface water via runoff than CTC (0.11%) and TC (0.07%) likely due to its smallest Kd value and largest input mass. Based on loss percentages, their predicted environmental concentrations (PEC) ranged from 4.87 (TC) to 16.91 (OTC) ng/L in regional surface water and 1.42 (TC) to 5.20 (CTC) ng/L in regional groundwater. The risk assessment based on PEC results suggested non-negligible health risk (HQ > 1.0 × 10-6) and low ecological risk (RQ < 0.1) in both regional surface water and groundwater, drawing concerns on the potential hazards of TCs released from manure-amended soil into water environments.

Ultrasonic-assisted extraction of polysaccharides from Auricularia auricula and effects of its acid hydrolysate on the biological function of Caenorhabditis elegans.

The present study was designed to explore the in vivo-antioxidant capacity and the probable mechanism of AAPs-H, prepared from Auricularia auricula polysaccharides with the optimal extraction conditions by Box-Behnken design and acid hydrolysis, using Caenorhabditis elegans as a model organism. The effects of AAPs-H on the locomotion behavior, life span, antioxidant-related enzymes activities, and antioxidants levels in C. elegans were studied. Furthermore, the potentials of AAPs-H in up-regulating the expression of antioxidant-related genes in C. elegans, such as skn-1, sod-3 and sir-2.1, were also discussed. AAPs-H demonstrated a highly significant protective effect against the damage caused by paraquat, could significantly increase U-Turn frequency of worms (p < 0.01), extend their lifespan, enhance antioxidant systems including GR by 63.96% (p < 0.05), GSH-Px by 71.16% (p < 0.01), SOD by 78.65% (p < 0.01) and CAT by 98.52% (p < 0.01), increase the level of GSH by 28.12% (p < 0.05), and decrease the level of MDA by 39.29% (p < 0.01). The qRT-PCR results showed that AAPs-H could up regulate mRNA expression levels of skn-1, sod-1, sod-2, sod-3 and sir-2.1 in wild-type C. elegans (>1.6 fold) when treated with the concentration of 0.4 mg/mL (p < 0.05 or p < 0.01). Our studies provide evidence that AAPs-H improves antioxidant defense system, and up-regulation of oxidative stress related genes for prevention of stress damage in C. elegans.

In vitro and in vivo evaluation of the antifungal activity of fluoxetine combined with antifungals against Candida albicans biofilms and oral candidiasis.

Candida albicans biofilms are responsible for oral candidiasis. Fluoxetine is a widely used antidepressant, with certain anti-Candida activities. The antifungal activity of fluoxetine combined with various antifungals against C. albicans biofilms and oral candidiasis was evaluated in this study. The morphological change in the inhibition of fluoxetine on C. albicans biofilms was observed using SEM. The interactions between fluoxetine and antifungals against C. albicans biofilms were evaluated using microdilution checkerboard methods, FICI and the ΔE model. The synergistic combination was tested in vivo on the mice model of oral candidiasis. SEM imaging showed fluoxetine inhibited hyphal growth and biofilm formation. Fluoxetine combined with caspofungin exhibited synergistic effects against C. albicans biofilms. Antagonistic effects occurred when fluoxetine was combined with amphotericin B or terbinafine. Further, the fluoxetine combined with caspofungin significantly reduced the lesion score and CFU of C. albicans on the murine tongue (p < 0.05), and relieved oral candidiasis of the infected mice.

A new dicyanoisophorone-based ratiometric and colorimetric near-infrared fluorescent probe for specifically detecting hypochlorite and its bioimaging on a model of acute inflammation.

To explore how hypochlorous acid (HClO) affects human health, a highly sensitive, selective, and trace detection method for hypochlorite (ClO-) is crucial for determining its non-negligible function in both environment and living systems. Herein, a dicyanoisophorone-phenylboronic acid-based novel ratiometric near-infrared fluorescent probe (Probe 1) was designed for the rapid and specific detection of ClO- based on the intramolecular charge transfer (ICT) mechanism. Excess addition of HClO to the Probe 1 solution, 186-times ratio (I652/I582) augment were gained. And this probe provided a colorimetric and ratiometric fluorescence response to ClO- with a high selectivity, a rapid response (within 30 s), and had an extremely low detection limit (15.7 nM). In addition, owing to the good sensing properties and low cytotoxicity of Probe 1, it can be used to expediently visualize exogenous ClO- in HepG2 cells and endogenous ClO- in RAW264.7 macrophage cells. Furthermore, the probe was successfully used for the bioimaging of zebrafish with an acute inflammation. Thus, Probe 1 is a promising vehicle to identify the level of HClO in animals with associated diseases.

A Ratiometric and Colorimetric Hemicyanine Fluorescent Probe for Detection of SO2 Derivatives and Its Applications in Bioimaging.

Based upon the intramolecular charge transfer (ICT) mechanism, a novel ratiometric fluorescent probe EB was developed to detect SO32-/HSO3-. The probe displayed both colorimetric and ratiometric responses toward SO32-/HSO3-. It displayed a quick response (within 60 s), good selectivity and high sensitivity (a detection limit of 28 nM) towards SO32-/HSO3-. The SO32-/HSO3- sensing mechanism was confirmed as the Michael addition reaction by ESI-MS. Moreover, the probe could be applied to measure the level of sulfite in real samples, like sugar and chrysanthemum, and it could also be used to detect SO32-/HSO3- in HepG2 cells through confocal fluorescence microscopy, which proved its practical application in clinical diagnosis.

Pumpkin polysaccharide preparation, simulated gastrointestinal digestion, and in vivo biodistribution.

We employed a single factor and response surface methodology based on Box-Behnken design (BBD) to optimize the extraction of pumpkin polysaccharides. We then simulated pumpkin polysaccharide gastrointestinal digestion in vitro and investigated their biodistribution in mice. The optimal extraction conditions, with a yield of 7.38 ±â€¯0.21%, were as follows: a concentration of NaOH 1.20%, a ratio of material to liquid of 1:11, and an extraction time of 2.1 h, respectively, according to single factor and BBD experiments. In the gastrointestinal experiment in vitro, the molecular weight of the polysaccharides markedly decreased after gastric digestion for 30 min, suggesting the decline is due to the breakdown of polysaccharide glycosidic bonds. The simulated intestinal fluid had little effect on polysaccharides digestion within 240 min. Analysis of the biodistribution in mice indicated that the polysaccharides distribute in the duodenum, jejunum, and ileum 30 to 60 min after intragastrical administration, and are absorbed in the jejunum and ileum after 60 to 360 min. These results provide information on the digestion and biodistribution of pumpkin polysaccharides and offer a theoretical basis for further understanding the absorption mechanisms in vivo.

Evaluation of the antioxidant effects of acid hydrolysates from Auricularia auricular polysaccharides using a Caenorhabditis elegans model.

Caenorhabditis elegans is an important model organism for studying stress response mechanisms and identifying genetic pathways that influence longevity. The present study was designed to explore the in vivo-antioxidant potential and the probable mechanism of acid hydrolysates prepared from A. auricula polysaccharides (AAPHs-F) with the optimal acid hydrolysis conditions using Box-Behnken design, and C. elegans was used as a model organism. The effects of AAPHs-F on the locomotory behavior, lifespan, activities of antioxidant-related enzymes and levels of antioxidants in C. elegans were studied. In addition, the potential of AAPHs-F in up-regulating the expression of antioxidant-related genes in C. elegans, such as daf-16, skn-1, sod-1, sod-2 and sir-2.1, and the inhibition of cell apoptosis of C. elegans were also discussed. The results indicated that AAPHs-F could significantly increase the U-Turn frequency of nematodes, extend their lifespan, enhance antioxidant systems including superoxide dismutase (SOD) by 70.60%, catalase (CAT) by 73.45% and glutathione reductase (GR) by 258.68% (p < 0.01), increase the level of glutathione (GSH) by 110.22% (p < 0.01), and decrease the level of reactive oxygen species (ROS) and malondialdehyde (MDA) by 31.86% and 46.16% (p < 0.01), respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that AAPHs-F could up-regulate mRNA expression levels of daf-16, skn-1, sir, sod-1 and sod-2 in wild-type C. elegans (>1.3 fold) when treated at a concentration of 0.1 mg mL-1 (p < 0.05 or p < 0.01). AAPHs-F was concluded to be heteropolysaccharides composed of mannose, glucose and galactose with a molar ratio of 12.7 : 3.25 : 1. The molecular weight of AAPHs-F was determined to be 885.37 Da. Furthermore, AAPHs-F is mainly formed of (1 → 3)-linked-α-d-glucopyranose, and carboxyl or acetamide is present in the molecule. In summary, our studies provide evidence that AAPHs-F helps improve the antioxidant defense system, and up-regulation of stress and longevity related genes suggests the possible involvement of these genes in the prevention of stress damage in C. elegans.

Perception of the speed of self-motion vs. object-motion: Another example of two modes of vision?

We investigated the effect of reduced contrast on speed perception for two types of tasks: (a) the speed of a rotating image, an example of "object-motion," and (b) speed of travel when viewing wide-screen videos recorded from inside a car, an example of "self-motion." Both types of stimuli were presented over a range of spatial contrasts. The results showed that reduced contrast caused significant decreases of perceived speed for the rotating disk, replicating the well known Thompson Effect. Reduced contrast had inconsistent effects on perceived speed of self-motion, however, resulting in perception of faster self-motion at the lowest speed, slower self-motion at higher speeds, and no effect at intermediate speed. Although further research is needed, the differential effects of reduced contrast on perceived speed of object-motion vs. self-motion are consistent with evidence for two modes of vision.

Integrative analysis of differential miRNA and functional study of miR-21 by seed-targeting inhibition in multiple myeloma cells in response to berberine.

BACKGROUND: Berberine is a natural alkaloid derived from a traditional Chinese herbal medicine. It is known to modulate microRNA (miRNA) levels, although the mechanism for this action is unknown. Here, we previously demonstrate that the expression of 87 miRNAs is differentially affected by berberine in multiple myeloma cells. Among 49 miRNAs that are down-regulated, nine act as oncomirs, including miR-21. Integrative analysis showed that 28 of the down-regulated miRNAs participate in tumor protein p53 (TP53) signaling and other cancer pathways. miR-21 is involved in all these pathways, and is one of the most important oncomirs to be affected by berberine in multiple myeloma cells. RESULTS: We confirmed that berberine down-regulated miRNA-21 expression and significantly up-regulated the expression of programmed cell death 4 (PDCD4), a predicted miR-21 target. Luciferase reporter assays confirmed that PDCD4 was directly regulated by miR-21. Bioinformatic analysis revealed that the miR-21 promoter can be targeted by signal transducer and activator of transcription 3 (STAT3). Down-regulation of interleukin 6 (IL6) by berberine might lead to inhibition of miR-21 transcription through STAT3 down-regulation in multiple myeloma. Furthermore, both berberine and seed-targeting anti-miR-21 oligonucleotide induced apoptosis, G2-phase cell cycle arrest and colony inhibition in multiple myeloma cell lines. Depletion of PDCD4 by short interfering RNA could rescue berberine-induced cytotoxicity in multiple myeloma cells. CONCLUSIONS: Our results suggest that berberine suppresses multiple myeloma cell growth, at least in part, by down-regulating miR-21 levels possibly through IL6/STAT3. This led to increased PDCD4 expression, which is likely to result in suppression of the p53 signaling pathway. These findings may also provide new mechanistic insight into the anti-cancer effects of certain compounds in traditional Chinese herbal medicines.

miRNA-21 regulates arsenic-induced anti-leukemia activity in myelogenous cell lines.

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules involved in modulation of cellular sensitivity to anti-cancer drugs. miRNA-21 (miR-21), one of the most prominent miRNAs in the genesis and progression of many human cancers, has been rarely characterized in myelogenous leukemia. Arsenic trioxide (ATO) was successfully used in the treatment of acute promyelocytic leukemia (APL) etc. However, cytotoxicity or insensitivity is a major concern in the successful treatment of leukemia. Here, we used a specific precursor miRNA-21 (pre-miR-21) or anti-miRNA-21 oligonucleotide (AMO-miR-21) to study sensitivity of HL60 and K562 cells to ATO. Cell viability and cell cycle were evaluated by MTT assay and PI assay using flow cytometry, respectively. Levels of miR-21 and its target PDCD4 were quantified by real-time PCR and/or western blot. AMO-miR-21 or ATO alone led to growth inhibition, apoptosis and G1 phase arrest of cell cycle. Apoptotic cells were confirmed morphologically with Hoechst staining. Moreover, there was somewhat synergistic effect of AMO-miR-21 and ATO in growth inhibition and apoptosis promotion. Meanwhile, enforced pre-miR-21 expression increased resistance to ATO, nevertheless not affecting cell growth alone. Dual-luciferase reporter vector containing two tandem PDCD4 3' UTR validated that PDCD4 was directly up-regulated by miR-21. Therefore, miRNA-21 by targeting PDCD4 may play a functional role in modulating ATO-induced cell death, and strategy using AMO-miR-21 and its combination with ATO may be useful as a myelogenous leukemia therapy.

Anti-miR-21 oligonucleotide enhances chemosensitivity of leukemic HL60 cells to arabinosylcytosine by inducing apoptosis.

Drug insensitivity or resistance is a major obstacle for successful treatment of acute myeloid leukemia. MicroRNAs (miRNAs) are small non-coding RNA molecules. Increasing evidence suggests that miRNAs modulate cellular sensitivity to anticancer drugs. We used a specific anti-miR-21 oligonucleotide (AMO-miR-21) to sensitize leukemic HL60 cells to arabinosylcytosine (Ara-C) by down-regulating miR-21. AMO-miR-21 alone effectively inhibited HL60 cell viability as measured by MTT assays and induced apoptosis as evaluated by flow cytometry, whereas AMO-miR-21 in combination with Ara-C enhanced HL60 cells to Ara-C-sensitivity and promoted Ara-C-induced apoptosis. Levels of miR-21 and its target PDCD4, quantified by real-time PCR, showed that expression of miR-21 was significantly decreased after AMO-miR-21 treatment. PDCD4 as a direct target of miR-21 in leukemic HL60 cells was confirmed by the dual-luciferase reporter assay. Our study suggests that AMO-miR-21 significantly sensitizes HL60 cells to Ara-C by inducing apoptosis and these effects of AMO-miR-21 may be partially due to its up-regulation of PDCD4. Therefore, exploiting synergistic effects between AMO-miR-21 and Ara-C might be an effective clinical strategy for leukemia chemotherapy.

[Study on the sensitivity of leukemic cells to arsenic trioxide enhanced by targeted suppression of mIRNA-21].

OBJECTIVE: To study the effect of antisense oligonucleotide targeted on miRNA-21 (AMO-miR-21) for enhancing the arsenic trioxide (As2O3) sensitivity of leukemic K562 cells and its possible acting mechanism. METHODS: Chemosynthetic AMO-miR-21 was transfected to K562 cells using Lipofectamine TM 2000. The inhibitory effects of As2O3 and AMO-miR-21, used singly or in combining, on cell proliferation were detected by MTT, their inhibition rate and IC50 were calculated. Cell cycle and apoptosis were assessed with PI stain; expression of miRNA-21 in cells was detected quantitatively by real-time PCR, and the potential target gene PDCD, protein expression was detected by immuno-fluorimetry. RESULTS: Used in combining with AMO-miR-21, the IC50 of As2O3, could be lowered from 2.1 micromol/L to 1.23 micromol/L, and the sensitivity of cells to As2O3 increased to 1.78-fold; with the amount of apoptotic cells increased significantly. Transfection with AMO-miR-21 alone could downregulate the expression of miRNA-21 in cells (P < 0.01), and up-regulate PDCD, protein expression level significantly. CONCLUSIONS: Combined use of AMO-miR-21 and As2O3 could increase the sensitivity of K562 cells to As2O3, which provides a novel potential approach for treatment of leukemia. AMO-miR-21 realizes it anti-tumor action by way of targeted inhibition on miRNA-21, and further up-regulates the expression of anti-tumor gene PDCD4.

Anti-miR-21 oligonucleotide sensitizes leukemic K562 cells to arsenic trioxide by inducing apoptosis.

Arsenic trioxide (ATO), an ancient traditional Chinese medicine, has been successfully used as a therapeutic agent for leukemia. Drug resistance and toxicity are major concerns with the treatment. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that might modulate cellular sensitivity to anticancer drugs. miRNA-21 (miR-21) is one of the most prominent miRNAs involved in various aspects of human cancers. However, miR-21 has been rarely characterized in chronic myelogenous leukemia (CML). Here, we used a specific anti-miR-21 oligonucleotide (AMO-miR-21) to sensitize K562 cells to ATO by degradation of miR-21. The results showed that both AMO-miR-21 and ATO caused growth inhibition, apoptosis, and G1-phase arrest in K562 cells. Meanwhile, AMO-miR-21 significantly promoted ATO-mediated growth inhibition and apotosis without affecting the G1 phase. Apoptotic cells were confirmed morphologically with Giemsa's staining. Furthermore, dual-luciferase reporter vector, containing two tandem miR-21 binding sites from PDCD4 3'UTR, validated that PDCD4 was directly regulated by miR-21. Therefore, AMO-miR-21 sensitized leukemic K562 cells to ATO by inducing apoptosis partially due to its up-regulation of PDCD4 protein level. The combination of ATO and AMO-miR-21 present therapeutic potential for CML.

Antisense oligonucleotide against miR-21 inhibits migration and induces apoptosis in leukemic K562 cells.

MicroRNAs (miRNAs) are small non-coding RNA molecules that are widely involved in cancer-related processes. The microRNA-21 (miR-21) has been identified as the only miRNA overexpressed in a variety of cancers, including leukemia. However, the function of miR-21 is yet unknown in chronic myelogenous leukemia (CML). Antisense oligonucleotides (ASOs), as inhibitors of miRNAs, have already been applied to therapeutic development and functional identification in miRNA research. In this study, we found that the antisense inhibition of miR-21 in K562 cells suppressed cell migration, promoted cell apoptosis, and inhibited cell growth, and up-regulated the expression of the tumor suppressor gene PDCD4. Meanwhile, pre-miRNA-21 increased migration and decreased cell apoptosis without affecting proliferation. We also validated that PDCD4 is a functional target of miR-21 in K562 cells. These effects of miR-21 might be partially due to its regulation of PDCD4. Our data suggest that miR-21 may play an oncogenic role in the cellular processes of CML, and antisense inhibition of miR-21 may therefore be useful as CML therapy.