ABSTRACT
A growing number of studies have suggested microRNAs (miRNAs) are involved in the modulation of myocardial ischemia-reperfusion (MI/R) injury; however, the role of endogenous miRNAs targeting endothelial cells (ECs) and its interaction with ICAM-1 in the setting of MI/R remain poorly understood. Our microarray results showed that miR-146a, miR-146b-5p, miR-155*, miR-155, miR-497, and miR-451 were significantly upregulated, whereas, miR-141 and miR-564 were significantly downregulated in the ECs challenged with TNF-α for 6 h. Real-time PCR analyses additionally validated that the expression levels of miR-146a, miR-155*, and miR-141 were consistent with the microarray results. Then, ICAM-1 was identified as a novel target of miR-141 by Target Scan software and the reporter gene system. Further functional experiments showed that elevated levels of miR-141 inhibited ICAM-1 expression and diminished leukocytes adhesion to ECs in vitro. In an in vivo murine model of MI/R injury, pretreatment with miR-141 mimics through the tail vein downregulated the expression level of ICAM-1 in heart and attenuated MI/R injury as evidenced by decreased infarct size and decline of serum cardial troponin I (cTnI) and lactate dehydrogenase (LDH) concentration. The cardioprotective effects of miR-141 mimics may be attributed to the decreased infiltration of CD11b(+) cells and F4/80(+) macrophages into ischemic myocardium tissue. In conclusion, our results demonstrate that miR-141, as a novel repressor of ICAM-1, is involved in the attenuation of MI/R injury via antithetical regulation of ICAM-1 and inflammatory cells infiltration. Thus miR-141 may constitute a new therapeutic target in the setting of ischemic heart disease.
Subject(s)
Endothelial Cells/metabolism , Genetic Therapy/methods , Intercellular Adhesion Molecule-1/metabolism , MicroRNAs/metabolism , Myocardial Infarction/prevention & control , Myocardial Reperfusion Injury/prevention & control , Myocardium/metabolism , 3' Untranslated Regions , Animals , Cell Adhesion , Coculture Techniques , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/pathology , Female , Gene Expression Regulation , HL-60 Cells , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Intercellular Adhesion Molecule-1/genetics , Leukocytes/metabolism , Macrophages/metabolism , Mice, Inbred BALB C , MicroRNAs/genetics , Myocardial Infarction/genetics , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/metabolism , Myocardial Reperfusion Injury/pathology , Myocardium/pathology , RNA, Messenger/metabolism , Time Factors , Transfection , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
AIM: To obtain the variable region gene sequence of heavy and light chain of mouse anti-human BAFF monoclonal antibody (mAb) on base of BAFF mAb which was cloned in our laboratory. METHODS: The total RNA was extracted from mouse anti-human BAFF mAb hybridoma cell line FMMUB(4);, and then the RNA was reverse transcribed into cDNA. Specific primers were designed to amplify the targeted gene. The targeted gene fragments were inserted into vectors to construct the clone vectors. The gene sequences were analyzed after identified by positive clones screening and restrictive enzyme digestion. RESULTS: The variable region gene sequences of mouse anti-human BAFF mAb were obtained. CONCLUSION: The variable region gene sequences of mouse anti-human BAFF mAb will provide experimental basis for further study on constructing engineered antibodies.
Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , B-Cell Activating Factor/immunology , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Animals , Base Sequence , Cloning, Molecular , Humans , Hybridomas/metabolism , Mice , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
AIM: To construct a prokaryotic expression vector for the expression of VEGFR2 D3.4/GST fusion protein, Express and purify the fusion protein. METHODS: The coding sequence of the third and fourth extracellular domain of human VEGFR2 gene fragment was synthesized and subcloned into pGEX4T-1 vector downstream of the GST fragment, an E.coli expression vector, to construct a recombinant plasmid pGEX4T-VEGFR D3.4. Then the plasmid was transformed into E.coli BL21 (DE3) pLysS and induced to express fusion protein VEGFR2 D3.4/GST with IPTG. The expressed protein was purified by washing in urea and detected by SDS-PAGE and Western blot. RESULTS: SDS-PAGE analysis showed that a novel protein with the expected molecular mass (M(r);) about 46 000 was expressed with the inducement of IPTG. And it existed mostly in the form of inclusion body. Grayscale scanning showed that the expressed VEGFR2 D3.4/GST fusion protein accounted for 38.6% of the total bacterium protein. After the purified product was washed by urea, its purity reached 87.1%. Western blot confirmed the recombinant protein was VEGFR2 D3.4/GST fusion protein. CONCLUSION: High purification VEGFR2 D3.4/GST fusion protein is obtained through the E.coli expression system.