MicroRNAs And ARE Binding Proteins Regulate The Genes Which Play Important Roles In Cardiac Remodeling

ObjectiveNaturally occurring microRNAs (miRNAs) are small posttranscriptional regulatory noncoding RNAs that regulate gene expression by affecting the translation or stability of target mRNAs.Tissue inhibitors of matrix metalloproteinases (TIMPs) maintain a balance in the metabolism of the extracellular matrix.Disruption of this balance may result in diseases associated with uncontrolled turnover of matrix, such as arthritis, cancer, cardiovascular diseases, nephritis, and acute lung injury.Research in the past five years has put miRNA into a prominent role in development and disease. MiRNAs have been discovered to be involved in tumorigenesis, development, control of cell proliferation, cell death,,fat metabolism,neuronal patterning in nematode, control of leaf and flower development in plants, insulin secretion, B-cell development, and neural stem cell fate, and they may be important for proper immune function.As miRNAs naturally exist in plants and animals to investigate whether miRNA target the tumor suppressor gene and disease related gene will provide clue to cure these diseases. Currently only the function and target site of a few miRNA has been verified. The bioinformatics tool and molecular cloning biology were employed to investigate whether miRNAs regulate the posttranscriptional regulation of genes that play important role in cardiac remodeling. TIMP3 is expressed in the heart and downregulated in the heart failure. hsa-miR-1 was shown to be overexpressed in individuals with coronary artery disease, and that when overexpressed in normal or infarcted rat hearts, it exacerbates arrhythmogenesis. Elimination of hsa-miR-1 by an antisense inhibitor in infarcted rat hearts relieved arrhythmogenesis. Downregulation of hsa-miR-1 can induce cardiac hypertrophy dysregulation of miRNA expression contributes to heart disease. An important aim for the future will be to identify the mRNA targets of the miRNAs responsible for adverse cardiac remodeling. As miRNAs often have numerous target mRNAs, the effects of individual miRNAs on cardiac growth and function may reflect altered expression of the products of multiple mRNAs. Further analysis of the functions of the regulatory miRNAs described in this study during adverse cardiac remodeling, and during heart development, promises to provide insights into heart disease and potential therapeutic targets. The expression of TIMP3 and miR-1 are abbrerantly expressed in cancer, arthritis and heart disease. Transgenic TIMP3 has been found to induce the apoptosis of cancer cells. We hope we could provide a new tool for gene therapy by finding the naturally occurring miRNAs which regulate genes that play important role in cardiac remodeling.Methods1,bioinformatics prediction:The analysis was done using the three algorithms, TargetScan, PicTar, and microinspector. commonly used to predict human miRNA gene targets.2,cell culture3,reverse transfection with Ambion transfection reagent siPort NeoFx which can save one day4,Westernblot:5,RNA isolationTotal RNA was isolated using the SV Total RNA isolation system(Promega,UK), followed manufacturer's instruction.6,Reverse Transcription7,Real time PCR8,Polymerase Chain reaction (PCR)9,double digestion(Amersham) of pGL3 control vector10,Rapid ligation11,Transformate into competent E-coli cells 12,Separation and extraction of DNA fragments by agarose gel electrophoresis13,DNA sequencing14,QIAGEN Endotoxin-free Plasmid Maxiprep15,Dual Luciferase assayRemove the growth medium from the cultured cells, and gently apply lml PBS to wash the surface of the 24-well plate. Swirl the vessel briefly to remove detached cells and residual growth medium. Completely remove the rinse solution before applying PLB reagent. Dispense into each culture well 100μl 1X Passive lysis buffer (Promega, UK) which coverd the cell monolayer. Place the culture plates on a rocking platform with gentle rocking to ensure complete and even coverage of the cell monolayer with 1X PLB. Rock the culture plates at room temperature for 15 minutes. Transfer the lysate to a 1.5ml eppendorf tube for further handling and storage. Clear the lysate samples for 30 seconds by centrifugation at top speed in a refrigerated microcentrifuge. Transfer cleared lysates to a new tube prior to reporter enzyme analyses. Prepare Luciferase Assay Reagent II (LARⅡ) by resuspending the provided lyophilized Luciferase Assay Substrate in 10ml of the supplied Luciferase AssayBufferⅡ. Prepare 1X Stop & Glo Substrate by adding 1 volume of 50X Stop & Glo Substrate to 50 volumes of Stop & Glo Buffer in a glass tube. The assays for firefly luciferase activity and Renilla luciferase activity are performed sequentially in one well Predispense 100μl of LAR II into the appropriate number of luminometer tubes to complete the desired number of DLR. Assays. Carefully transfer up to 20μl of cell lysate into the luminometer tube containing LARⅡ; mix by pipetting 2 or 3 times. Do not vortex. Place the tube in the luminometer and initiate reading. add 100μl of Stop & Glo(?) Reagent and vortex briefly to mix. Replace the sample in the luminometer, and initiate reading. Firefly and Renilla luciferase activities were measured consecutively by using dual-luciferase assays (Promega) 36 h after transfection.16,Generating two inserts with mutations respectively by using the QuikChange Multi Site-Directed Mutagenesis Kit. 17,Immunoprecipatition(一)Preparation of mRNP lysate from culture cells1,Grow and harvest tissue culture cells by washing two times with ice-cold PBS and pellet by centrifugation at 4℃/2000rpm/5'. We need 2 big dishes of cells per sample. Loosen the final cell pellet by gently flicking the bottom of the tube and add an approximately equal pellet volume of ice-cold PLB buffer supplemented with RNAse inhibitors and protease inhibitors.2,Mix cells by pumping several times with a hand pipettor (no vortex!) and place on ice for 10 min.3,Spin 30 min at 14,000 rpm (20,000 x g)/4℃. Transfer supernatant to the fresh Ependorf tubes。Freeze and store at-80℃. (Its better to use lysate directly for IP).4,Preclear the supernatant with 15μg (30μl from stock 0.5μg/μl) of IgG1 control, for 30 min/4℃. Add 50μl PAS non-coated with Ab, incubate 30 min/4℃with rotation. (Note that preclearing is not required for IP followed by RT-PCR. Its required only for IP followed by microarray) 5,Spin down 14,000 rpm/4℃. Save supernatant. This is your pre-cleared lysate. Do Bradford to measure protein concentration (measure 2μl of a 1:100 dilution). We routinely get 15-30μg/μl concentration of lysates and you will need anywhere from 50-100μl per IP of lysate.(二)Antibody coating of protein A beads1,Use PAS beads from Sigma (P-3391) (or preswollen beads from Sigma). In a 50 ml Falcon, swell beads overnight in 5% BSA solution at 4℃. Add extra solution until it covers beads 3-4 volumes (until 15 ml mark). In the morning, pour off excess so that the beads are 50% (vol/vol) slurry. May do this in advance but add 0.1% Na azide and store at 4℃.2. Add 30μg (150μl from stock 200μg/ml) of antibody to 100μl volume of PAS beads using RNAse free Eppendorf tubes. Add about 100-200μl of NT-2 buffer. Bind overnight on rotator at 4℃. Note that the amount of Ab required for IP will depend on the protein. The optimal amount of Ab needed should be determined by doing IP with this protocol with 1,5,10 and 30 ug Ab.3,Next morning, wash (14K rpm/5') the beads with 1 ml aliquots of ice-cold of NT25 times. After last spin take out the NT2. The beads are now ready to be used. May also do this in advance but add 0.1% Na azide and store at 4℃.(三)Immunoprecipitation of mRNPs1,Use 1.5 ml Eppendorf tubes. To precoated PAS/Ab (around 50μl), first add about 700μl NT-2 buffer. Then add all the additives 10μl 0.1 M DTT (do not add the DTT to the pellet directly, as this will reduce you antibody and the IP will not work!), 10μl RNAout,33μl 0.5 M EDTA. Add 100μl lysate (even if concentration of protein is lower than 30ug/ul) and fill-up with NT-2 to 1 ml mark on tube. Incubate 1-2 hrs at 4℃, end-over-end. Spin down (5000 g,5 mins). Wash pellet 5 times with 1 ml aliquots of ice-cold NT-2 buffer (5000 g,5 mins)2,After last wash, add 100ul NT2 buffer having 5ul DNase I (2U/ul). Keep at 37 C (or 30 C) for 5-10 mins. Add 1 ml NT2 buffer, spin 5000 g,5 mins, discard supernatant. Then, add the following to the PAS pellet:5μl of Proteinase K (10mg/ml), 1μl 10%SDS and 100μl NT-2. If you have several samples, its good to make a mastermix of NT2 buffer, Proteinase K and SDS). Incubate at 55℃for 15-30 min, with mixing.3,Spin 5000 g,5 mins, collect supernatant (～100ul)4,To beads add 200ul NT2 buffer, spin 5000 g/2 mins, collect supernatant (-200ul), discard beads.5,Combine supernatants (100ul and 200ul) and add 300ul lower layer of acid phenol-CHCl3 (Ambion)6,Vortex,1'RT (or 37C in shaker), short spin at RT (imp)/1'/max speed.7,Collect 250ul of upper layer, add 25ul sodium acetate pH 5.2,625ul 100% ETOH and 5ul glycoblue, mix well, keep O/N-20C.8,Next day, mix the tubes by inversion 3-5 times, spin 14.000rpm/4C/30 mins and discard supernatant 9,To the blue pellet add lml of 70% ETOH and mix by inversion or vortexing, spin 14.000rpm/4C/2'10,Discard supernatant, spin pellet 1'/14.000rpm/4C, pipette any 70% ETOH, air dry pellet at RT for 5', resuspend in 20-40 ul of water.Results1,Hsa-miR-1 downregulates the expression of TIMP3 potein in HEK 293 cells.2,Hsa-miR-1 downregulates the expression of TIMP3 mRNA compared with the negative control groups in HEK 293 cells.3,It has been validated that hsa-miR-1 has target site in TIMP3 3'UTR by cloning in HEK 293 cells, mutagenesis and dual luciferase assay. The luciferase activity of the mutant constructs are significantly higher that those of wild type constructs for the first potential target site, wheareas the negative control group did not show the same effect. For the second potential target site, the difference between the mutant constructs and wild type constructs are not significant.4,It has been validated that pre-miR-21 significantly downregulated TIMP3 expression and upregulated MMP9 expression. And the antogomiR-21 significantly upregulated the TIMP3 expression and downregulated MMP9 expression human cardiac fibroblast cells. This suggested that miR-21 regulated the cardiac remodeling pathway by targeting the important regulators in the pathway. Since cadiac remodeling leads to heart failure. This suggests that knockdown miR-21 by antagomiR-21 might have therapeutical effect on heart failure. It has been verified that miR-21 is another trans-acting factor of TIMP3 gene.5,It has been confirmed that ARE binding protein HuR contributes the TIMP3 mRNA stability in human cardiac fibroblast cells.ConclusionThe experiment verified hsa-miR-1 directly regulates the posttranscriptional of TIMP3 gene in 293 cells. hsa-miR-1 downregulates mRNA and protein expression of TIMP3 gene. The target site in TIMP3 was confirmed. In addition to hsa-miR-1, this studay also confirmed another two trans-acting factors. miR-21 and ARE binding protein HuR. TIMP3 and miR-1 were aberrantly expressed in cancer, arthritis and heart diseases. It has been validated that pre-miR-21 significantly downregulated TIMP3 expression and upregulated MMP9 expression. And antogomiR-21 significantly upregulated the TIMP3 expression and downregulated MMP9 expression in the cadiac fibroblast. This suggested that miR-21 regulated the cardiac remodeling pathway by targeting the important regulators in the pathway. This suggests that knockdown miR-21 by antagomiR-21 might have therapeutical effect on heart failure since cadiac remodeling leads to heart failure. The experiment provides the clue for investigating the cause of the diseases and how to cure the diseases.