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Objective:To investigate the effect of miR-27b-3p on radiation resistance of breast cancer cells.Methods:The relative expression levels of miR-27b-3p in normal tissues and breast cancer tissues were analyzed through GEO database and verified by the qRT-PCR assay. Cloning formation, immunofluorescence, and EDU assay were used to assess the functions of miR-27b-3p on radioresistance of breast cancer cells. The luciferase reporter assay was used to verify whether miR-27b-3p directly targeted PLK2 mRNA. A rescue experiment was performed to identify the influence of PLK2 overexpression on miR-27b-3p regulated radioresistance.Results:The expression level of miR-27b-3p in both breast cancer tissues ( t=2.99, P<0.01) and breast cancer cells ( t=21.21, 32.88, P<0.05) was significantly higher than those in normal breast tissues and cells, especially, it was elevated in radioresistant MCF-7R cells ( t=25.63, P<0.05). Overexpression of miR-27b-3p enhanced the cloning efficiency of MCF-7 cells ( t=10.32, P<0.05), and had a protective effect on the proliferation of irradiated MCF-7 cells ( t=8.77, 8.26, 8.03, P<0.05). But interference of miR-27b-3p reduced the cloning efficiency and proliferation of MCF-7R cells ( t=40.00, P<0.05) after irradiation with different doses( t=8.54, 8.32, 8.23, P<0.05). Moreover, PLK2 was verified to be a direct target of miR-27b-3p, and overexpression of PLK2 inhibited miR-27b-3p-mediated radioresistance of breast cancer cells (MCF-7: t=9.66, P<0.05; MCF-7R: t=6.42, P<0.05). Conclusions:miR-27b-3p contributes to the radioresistance of breast cancer cells by targeting PLK2.
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Tissue inhibitor of metalloproteinases-2 (TIMP-2) inhibits tumor migration and invasion. Obtaining TIMP-2 protein is conducive to a comprehensive and in-depth study of its function and mechanism in tumorigenesis and development. We collected human TIMP-2 protein through prokaryotic expression in vitro. We expressed, purified and characterized human TIMP-2 protein. First, the human TIMP-2 gene was cloned from the cDNA obtained by reverse transcription of total RNA of human lung cancer A549 cells, and constructed to pET28a vector. The recombinant plasmid pET28a-TIMP-2 was transformed into Escherichia coli BL21(DE3) after restriction endonuclease digestion and sequencing analysis. The expression of TIMP-2 protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and the expression conditions were optimized. After purification by nickel affinity column, the fusion protein His-TIMP-2 was identified by Western blotting method and its biological activity was detected by gelatin zymography. The fusion protein His-TIMP-2 existed in the form of inclusion body in E. coli. In a certain range, the concentration of IPTG had no significant effect on the expression amount of His-TIMP-2. But in this expression system, induction temperature and time were the key parameters, and the expression amount of His-TIMP-2 in E. coli increased with the increase of induction temperature. The purified and refolded fusion protein could effectively inhibit the activity of matrix metalloproteinases expressed by human lung cancer A549 cells. The acquisition of active fusion protein lays a foundation for further study of the function and mechanism of human TIMP-2, and is of great significance for tumor therapy.
Subject(s)
Humans , Cloning, Molecular , Escherichia coli/genetics , Recombinant Fusion Proteins/genetics , Recombinant Proteins , Tissue Inhibitor of Metalloproteinase-2/geneticsABSTRACT
Objective To screen radiotherapy resistance related microRNAs (miRNAs) in breast cancer and provide experimental basis for basic researches and clinical solutions of radiotherapy resistance in breast cancer patients.Methods The miRNA microarray dataset GSE107743 related to breast cancer radiotherapy patients was downloaded from the Gene Expression Omnibus (GEO).The GEO2R analysis tool was used to screen differentially expressed miRNAs in patients with local recurrence after radiotherapy.The target genes of differentially expressed miRNAs were predicted by the mirDIP database.GO enrichment analysis and KEGG pathway analysis on the target genes were performed by DAVID dataset.Finally,differential expression verification was performed in human breast cancer cell line MCF-7 by real-time fluorescent quantitative PCR.Results A total of 9 differentially expressed miRNAs related to radiotherapy resistance were screened by the GEO2R analysis tool,in which three miRNAs (hsa-miR-600,hsa-miR-525-3p and hsa-miR-591) were up-regulated and 6 miRNAs (hsa-miR-488-5p,hsa-miR-582-3p,hsa-miR-520h,hsa-miR-488-3p,hsa-miR-744-3p and hsa-miR-103b) were down-regulated.Target gene prediction results showed that there were 134 potential target genes in these nine differentially expressed miRNAs.These target genes were significantly enriched in related biological processes such as apoptosis and stem cell differentiation (all P＜0.05) and signal transduction pathways such as transforming growth factor-β and phosphatidylinositol 3-kinase-protein kinase B signaling pathway (all P＜0.05).The results of real-time PCR showed that the differential expression of six miRNAs,i.e.hsa-miR-600,hsa-miR-525-3p,hsa-miR-591,hsa-miR488-5p,hsa-miR-582-3p and hsa-miR-520h,was detected in the MCF-7 cells irradiated by 5 Gy 137Cs γ-rays,and this result was consistent with the results of GEO2R analysis.Conclusion The differentially expressed miRNAs screened from clinical samples of breast cancer patients with local recurrence using bioinformatics may be closely associated with the radiotherapy resistance of these patients.These miRNAs are expected to become new biomarkers for the therapy of radiotherapy resistance.
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Objective To investigate the in vivo radioprotective effect of adenosine triphosphate (ATP) on radiation damage induced by high dose γ-ray ionizing radiation(IR) in mice.Methods Specific pathogen-free C57BL/6 female mice were randomly divided into IR group and IR+ATP group by body weight,with 10 mice in each group.All the mice were treated with a 8 Gy one-time and high-dose whole body γ-ray irradiation.Within 6 h after irradiation,mice were injected intramuscular injection of 150 pl sodium chloride solution (9 g/L) for IR group,and 150 μl ATP solution (6 mg/kg) for IR+ATP group,respectively.The drug was administered once a day until the death of the animal.The mean survival days,survival rate,body weight and major organ coefficients in both groups were measured.Results The average survival days of mice in IR group and IR +ATP group were 6.5 d and 9.6 d,respectively.The survival rate of the mice in IR+ATP group was higher than that in IR group (P＜0.01).The body weight values of the mice in IR+ATP group was higher than that in IR group on the after the 4th day post-irradiation,and the differences were statistically significant (all P＜0.05).Except for heart and stomach,the organ coefficients of liver,spleen,lung,and kidney in IR +ATP mice were higher than those in IR group,and the differences were statistically significant (all P＜0.05).Conclusion ATP has certain radiation protection effect,and it can reduce the radiation damage of mice induced by high-dose (8 Gy) γ-ray IR so as to increase the survival rate.
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The TV arthroscope signal is turned into digital video signal through a video capture card,and then the digital signal is recorded and stored through a computer.Edge detection and tracking algorithm for image are used to process and analyze the acquired digital image of arthrosis.Based on the image processing,the shape parameters of the position with pathological changes can be measured automatically.Results show that these methods are very effective.This study provides some valuable materials for the further autorecognition research of endoscopic image.