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Objective To screen the expression profile of circular RNA associated with chemo-resistance in osteosarcoma,and to analyze and identify its possible molecular functions.Methods Three pairs of matched drug-resistant and sensitive human osteosarcoma cell lines (MG63/DXR vs MG63,U2OS/DXR vs U2OS,KHOS/DXR vs KHOS) were first tested by CCK-8 assay for three commonly used osteosarcoma chemotherapy drugs (doxorubicin,cisplatin,and methotrexate);then,using next-generation high-throughput RNA sequencing technology,comparative analysis of circRNA expression was performed in three pairs of matched multidrug-resistant and sensitive human osteosarcoma cell lines;followed by real-time quantitative PCR (qRT-PCR) to confirm the reliability and accuracy of sequencing data in chemotherapy-resistant osteosarcoma cell lines and tissues.In addition,a variety of bioinformatics analyses,including GO,KEGG pathway analysis and the construction of circRNA-miRNA networks,were performed to predict potential molecular functions of differentially expressed circRNAs and to construct relevant regulatory pathways or networks.Results Three osteosarcoma-resistant cells (MG63/DXR,U2OS/DXR,KHOS/DXR) were significantly resistant to the three common osteosarcoma chemotherapy drugs compared with control cells (MG63,U2OS,KHOS),which laid a solid foundation for the further experiments.RNA sequencing revealed a total of 80 circRNAs differentially expressed between the two groups.Compared with drug-sensitive osteosarcoma cells,57 circRNAs were upregulated and 23 circRNAs weredownregulated in the drug-resistant osteosarcoma cell lines (fold change≥ 2 or ≤0.5,P ＜ 0.05).Tenrandomly selected circRNAs were verified by qRT-PCR and the results showed that 9 of the 10 circRNAswere consistent with the sequencing results.In addition,KEGG pathway analysis showed that 56 pathways were significantly enriched in differentially expressed circRNAs,including Glycolysis/gluconeogenesis,ABC transporter,VEGF signaling pathway,and so on.Moreover,thedifferently expressedcircRNA-hsa_circ_0004674 with the highestfold change was highly expressedin the chemotherapy-resistant osteosarcoma cells and tissues,associated with poor prognosis in osteosarcoma patients.Some potential endogenous competitive RNA (ceRNA) regulatory pathways associated with hsa_circ_0004674,such as hsa_circ_0004674-miR-490-3p-ABCC2 and hsa_circ_0004674-miR-1254-EGFR,were constructed by use of the authoritative databases (target scan and miRanda) and literature searching and the miRNA response element sequences (MREs) between miRNAs that have a potential ceRNA regulatory relationship with hsa_circ_0004674 were alsopredicted.Conclusion CircRNA is closely related to tumor progression and may play a role in ostoosarcoma chemo-resistance.Besides,hsa_circ_0004674 may be a potential candidate for reversing drug resistance of osteosarcoma.
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Weighted gene co-expression network analysis (WGCNA) technology is a high-throughput gene expression data mining algorithm, which uses the idea of system biology to find the correlation of gene expression and to construct gene modules, so as to further discovery a high-throughput data mining algorithm with biological significance modules. In recent years, with the deep understanding of human diseases to gene level, WGCNA has been used increasingly in the researches of various diseases, especially in mining the highthroughput data about tumor-related genes. Moreover, with the continuous improvement of this technology, the research of this technology in disease pathogenesis, development and treatment etc has been developed from a single co-expression network analysis to the multiple technologies [such as genome-wide association study (GWAS) and support vector machine (SVM)] combined WGCNA or the innovative applicated WGCNA. As a high-throughput research method based on gene level, WGCNA is playing an important role.
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OBJECTIVE To study the cytotoxic characteristics of nitrogen mustard HN-3 in different cell. METHODS Human epidermal keratinocytes-fetal (HEKf), human dermal fibroblasts-adult (HDFa) and human lung fibroblasts (HLF) cell lines were treated with HN-3100, 300 and 450μmol·L-1 for 0.5, 2, 4, 6, 12, 24 and 48 h, respectively. Multi-parameter analysis technology based on cell imaging was used to examine the effects of HN-3 on cell survival, cell cycle arrest, apoptosis, autophagy and oxidative stress, along with parameters concerning nucleus, cytoskeleton (actin and tubulin), lysosome, nuclear membrane permeability (NMP), mitochondrial membrane potential (MMP) and phosphohistone H 2AX (pH2AX). RESULTS HN-3 caused irreversible cellular damage by significantly decreasing the number of HEKf, HDFa and HLF cells in a time-dependent manner (P<0.01). Before the cell number was reduced robustly, the content of reactive oxygen species and pH2AX significantly increased, but the glutathione content decreased after cells were exposed to HN-3 for 0.5 h (P<0.01). In addition, the content of lyso-some was reduced in HEKf cells at 0.5 h, but increased in HDFa and HLF cells at 0.5 and 2 h respec-tively, accompanied by the increase in microtubule-associated protein 1 light chain 3B (LC3B) puncta.With the significant reduction of the cell number in HEKf cell line, the nuclear intensity increased, nuclear area decreased, the intensity and area of F-actin and α-tubulin decreased, MMP decreased (P<0.01) and lysosomal intensity increased. But the effects of HN-3 on HDFa and HLF cell lines were quite different. The nuclear area increased, the intensity and area of F-actin and a-tubulin increased, MMP increased (P<0.01) and the intensity of lysosome increased. In HLF cells, there was an increase in LC3B puncta (P<0.01). In all the three cell lines, NMP and manganese superoxide dismutase content were increased, and cell cycle arrested at G2 phase. HN-3 Induced early apoptosis in HDFa cells but late apoptosis in HEKf cells. CONCLUSION HN-3 causes DNA damage, oxidative stress and lysosome damage at an early stage, whereas at the late stage, the imbalance of MMP, increase in NMP, and G2 phage arrest are the major cytotoxic effects. Moreover, HN-3 specifically induces nuclear condensation, cytoskeleton protein aggregation and apoptosis in HEKf cell. HN-3 Induces nuclear swelling, and loose cytoskeleton in HDFa cells and HLF cells, eventually inducing early apoptosis in HDFa cells and autophagic death in HLF cells.
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OBJECTIVE To evaluate the effect of single traditional Chinese medicine(TCM) herb extracts on hepatoma and normal fibroblast cells using high-throughput screening in order to obtain extracts with specific anti-hepatoma effect. METHODS 242 commonly used TCM herbs were extracted by petroleum ether,ethanol and water,respectively. The total number of TCM extracts was 554. The cyto?toxicity of samples was evaluated by MTT in human hepatoma cells Bel7402 and mice normal fibroblasts NIH3T3. RESULTS 7.4%of the total extracts had an inhibitory effect greater than 50%for Bel7402,but 14.8% for fibroblasts NIH3T3 cells. Extracts with an inhibitory effect above 50% on both Bel7402 and NIH3T3 cells accounted for 4.4%of the total extracts. Our results showed that the sample DF173 had preferable cytotoxicity effect on hepatoma carcinoma cells in a good dose-effect relationship. DF173 is an ethanol extract from Stephania tetrandra,which is a commonly used herb in TCM. The cytotoxic IC50 of DF173 against Bel7402 was 8.27 mg·L-1,and 19.48 mg·L-1 on NIH3T3. CONCLUSION The components of TCM herbs are highly complicated. The combination of tumor cells with normal fibroblast cells to evaluate the cytotoxicity effect during anti-tumor drug screening will contribute much to the discovery of TCM drugs with high anti-tumor efficiency and lower toxicity.
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Objective To investigate the changes of circulating miRNAs expression profiles in different subtypes of ischemic stroke according to the Trial of Org 10 172 in Acute Stroke Treatment.Methods We selected 16 patients diagnosed as acute ischemic stroke at first time in the Department of Neurology,the Affiliated Hospital of Medical College Qingdao University from November to December in 2012.They were divided into large artery atherosclerosis (LAA) stroke group (n =8) and small artery occlusive (SAO) stroke group (n =8).At the same time,8 healthy checkup subjects were selected as control.High-throughput sequencing was used to detect the expression profiles of miRNAs in the plasma,and the high-throughput sequencing results were validated by quantitative real-time polymerase chain reaction.We performed the miRNAs variance analysis and associated bioinformatics analysis.Results Thirty hundred and sixty-nine miRNAs were detected in LAA group,SAO group and the control group.We found remarkable differences (fold change ＞2,P ＜0.01) in the expression of 12 miRNAs,including let-7a-5p,miR-744-5p,etc.,between any two groups of the three groups.Thirty-four miRNAs,containing miR-126-5p,miR-23a-3p,miR-143-3p,etc.,had a lower expression (fold change ＞2,P ＜0.01)in LAA group than that in the control group.In comparison with the control group,miR-1304-3p and miR-451a were significantly down-regulated (fold change ＞ 2,P ＜ O.01),while 27 miRNAs were significantly up-regulated (fold change ＞2,P ＜0.01) in SAO group.The expression levels of miR-146b-5p,miR-23a-3p and miR-451 a were validated in accordance with the results of real-time PCR.Target gene prediction and functional analysis revealed that target genes regulated by differential expression of miRNAs were mainly associated with cell proliferation,adhesion,metabolism and other biological functions.Conclusion miRNAs are differently expressed in the plasma of LAA group,SAO group and healthy control group,which suggest that miRNAs might play different roles in the pathogenesis of LAA stroke and SAO stroke by regulating downstream target genes.
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Growing numbers of studies employ cell line-based systematic short interfering RNA (siRNA) screens to study gene functions and to identify drug targets. As multiple sources of variations that are unique to siRNA screens exist, there is a growing demand for a computational tool that generates normalized values and standardized scores. However, only a few tools have been available so far with limited usability. Here, we present siMacro, a fast and easy-to-use Microsoft Office Excel-based tool with a graphic user interface, designed to process single-condition or two-condition synthetic screen datasets. siMacro normalizes position and batch effects, censors outlier samples, and calculates Z-scores and robust Z-scores, with a spreadsheet output of >120,000 samples in under 1 minute.
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High-Throughput Screening Assays , RNA Interference , RNA, Small InterferingABSTRACT
A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.