The Expression Analysis And Functional Study Of MiR-126

Background:Cardiovascular diseases are the most common human diseases,which have the high morbidity and mortality and aroused extensive concern in the international community.Its physiological and pathological mechanisms and treatment have also been hot spots all around the world.At present,the morbidity and mortality of cardiovascular disease are increasing every year,which have seriously affected on the people's health status and bring an immeasurable loss to the national economy.Howerer,the pathogenesis of cardiovascular disease has not yet very clear.MiRNAs(miRNAs) are single-stranded small molecule non-coding RNA containing about 21-26 bases,which are highly conservative in evolution and regulate the gene expression at the transcriptional level.Posttranscriptional regulation by miRNAs is important for many aspects of development,cell differentiation,proliferation and apoptosis, hormone secretion and tumor formation.MiRNAs also play an extremely important role in the cardiovascular development and cardiovascular diseases.In the past research contructing the miRNA gene cDNA library,we found that miR-126 represents about 20%-23%of the total clone.According to the findings of previous studies,the miRNA gene expression was positively correlated with the miRNA clone number.We thus consider miR-126 is a high expression miRNA in the human heart.Therefore,we select miR-126 for further research and investigate its function.This study is to study the following aspects:First,we intend to detect the miR-126 expression in different tissues and cells,and we want to make clear wether the miR-126 highly expressed in the heart;Secondly,we predicted the target gene of miR-126 using bioinformatics and preliminary investigated the relationship between miR-126 and VEGF 3'-UTR in 293 cells by exogenous expressing of miR-126 and carrying the VEGF 3'-UTR reporter gene in 293 cells and detecting the reporter gene.Thirdly,we constructed a miR-126 lentiviral expression vector,and packaged the recombinant lentiviral particles, detected the virus titer.Then we set up stable miR-126 over-expressed transgenic cell lines (endothelial cell and lung cancer cell) and use RPA and Westblot to test the VEGF mRNA and protein expression when the the expression of miR-126 has increased.In addition,we used the stable expressed lentiviral miR-126 cells inoculated into nude mice to detect the effect of miR-126 over-expression on the inhibition of tumor models in vivo.The significance of this study is:To detect the expression of miR-126 and to make clear that miR-126 is cardiac highly expressed.And then we use cell lines to investigate the effect of miR-126 on different potential targets.Accordingly,it provides theoretical gist for studying the heart physiological function,exploring the pathogenesis of cardiovascular disease,as well as the screening of new therapeutic targets.PartⅠAnalysis of expression pattern of miR-126Object:Analyze the distribution of miR-126 in various tissues and cells of human.Methods:(1) The use of RPA method:Designed the DNA fragment according to miR-126,and added the sequence complementary to 3' sequence of the T7 promoter of mirVana miRNA Probe Construction Kit,and synthesized the oligonucleotide. Resuspended the oligonucleotide template to a concentration of 100μmol/L,and hybridized the template with T7 Promoter Primer of the kit,and used Klenow DNA polymerase to fill in.Transcription by T7 RNA polymerase was performed to produce 32P labeled probe.DNase I was added to digest the DNA template.The probes were purified by denaturing polyacrylamide gel,and were eluted from acrylamide gel slice and precipitated and resuspended to detect miR-126 in ribonuclease protection assay(RPA). Total RNA was extracted using Trizol from various tissues,5μg of total RNA was hybridized with the probes at 52℃for 16 h.RNases A/T1 were added and the mixture was incubated at 37℃for 30 min.The remained RNA was precipitated by ethanol and then separated on a 15%denaturing PAGE.The probes were visualized by autoradiography.(2) The use of RT-PCR method:The sequence-specific primers of miR-126 are designed in accordance with miR-126's sequenceir,and then we extracted the total RNA from different tissues and cells.Using the RNA and the RNA reverse transcriptase enzyme to obtain cDNA,cDNA is used as the template of PCR.The PCR reaction system was added with fluorescent probe,so we use the real-time monitoring of fluorescent signal to measure the quantity of miR-126.Results:RPA and RT-PCR showed that miR-126 was expressed predominantly in lung,heart and stomach,and can be detected in spleen,but was absent in kidney,brain and skeletal muscle.The expression of miR-126 is high in HUVEC and very low in HeLa cell,293 cell,A549cell,and Y90cell.Conclusion:The expression of miR-126 is different in different kinds of tissues and cells.MiR-126 is mainly expressed in heart,lung,and endothelial cell which might give us the cue that miR-126 probably participate the pathological and physical mechanism. PartⅡThe prediction of miR-126 target gene and validationObject:Predict the target genes of miR-126 using bioinformatics and verify its role on target genes using reporter gene system.Methods:Firefly luciferase expression vector was constructed to verify the relationship between miR-126 and VEGF.3'-Untranslated region(3'-UTR) of VEGF was PCR-amplified from human cDNA library and the product was inserted into pMIR-REPORT^TM Luciferase vector(pLuc,Ambion),downstream of the firefly luciferase coding region to construct pLV-miR-126.PLuc-VEGF/miR-126BS and pLV-miR-126 were co-transfected into 293 cells with Lipofectamine reagent.After 48-hour incubation,the cells were harvested and subjected to Luciferase assay.Firefly luciferase activities were measured by double luciferase assays System(Promega),and Renilla luciferase was used as internal control.Point mutation was used to mutate the binding site of miR-126 in VEGF-3'UTR to detect the inhibition effect.Results:(1) There are several target genes of miR-126,and we choose VEGF which is associated with the cardiovascular disease to be the target of reaseach.(2) Co-transfection of pLV-miR-126 in 293 cells can inhibit the expression of luciferase of pLuc-VEGF/miR-126BS.When the 3'-UTR was mutated,the inhibition was eliminated.Conclusion:The prediction of bioinformatics suggests that VEGF is the corresponding target gene of miR-126 which probably repress the the mRNA translation by combining with the 3'UTR of VEGF.PartⅢProduct of miR-126 lentivirusObject:C,onstruct Lentivirus vector to express miR-126,produce pseudoviral particles and determine its titer.Methods:PCR primers were designed according to miR-126 sequence,restriction endonuclease digesting sequences and protection bases were added.Genomic DNA was extracted from A549 cells and used as template.The target PCR products were purified by recovery from agarose electrophoresis.The DNA fragment was digested by EcoRⅠand BamHⅠ,and purified.Expression vector,pCDH-CMV-MCS-EF1-copGFP^TM was digested by EcoR I and BamH I and recovered.And the fragment was inserted into the vector and transformed competent DH5a.The DH5a was plated to plating medium and were incubated overnight.PCR was conducted to determine positive clones.The positive clones were seeded to LB medium and were incubated overnight,and plasmids were isolated,and sequencing was conducted with the primer from upstream site of vector. The sequencing results were analyzed and the correct plasmid was amplified by corresponding strain culture.The plasmids were prepared by Endotoxin-Free Plasmid Midi Kit.293TN cells were grown and plated for 2-3 times.The cells were seeded to a fresh 10-cm plate.And Lipofectamine 2000 was used to co-transfect the miR-126 lentiviral expression Vector and Lentivirus Package plasmid mix into 293 TN cells.The transfection efficiency was measured.Medium displacement was performed 24 hours after transfection.The supernatant was harvested,passed through a 0.45μm syringe filter to remove cell debris 48 hours after transfection.Pseudoviral titer was determined by serial dilution method.Results:(1) miR-126 genomic sequence was amplified and lentiviral expression vector were constructed.(2) LV-miR-126 pseudovirus was packaged and the titer was determined.(3) 293 cell infected by LV-miR-126 can over-express miR-126.Conclusion:LV-miR-126 infection can enhance the expression level of miR-126 in 293 cell lines.PartⅣThe relationship between miR-126 and VEGF in endothelial cellObject:Analyze the relationship between the expression of miR-126 and VEGF in endothelial cells.Methods:(1)Human Umbilical Vein Endothelial Cell was infected by recombinant lentivirus LV-miR-126.Cells were harvested 72 hours after infection,and total RNA and protein was isolated,and RPA and west blot were used to confirm the relationship of miR-126 and VEGF.(2) EC was infected by recombinant lentivirus LV-miR-126.Cells were harvested 72 hours after infection,and re-seeded to 96-well plate and were incubated for 24,48 and 72 hours.10μl MMT reagent was added and the cells were incubated for another 4 hours.The supernatant was removed and 200μl DMSO was added to dissolve the formazan crystals.Optical density value(OD) of each sample was measured at a wavelength of 570nm(630 nm as a reference) on a microplate reader.Results:In vitro,infection of LV-miR-126 in HUVEC can inhibit the expression of VEGF and the proliferation of HUVECConclusion:Over-expression of miR-126 in HUVEC can inhibit the expression of VEGF and repress the proliferation of endothelial cells,which indicate that miR-126 may take part in the atherosclerosis by binding to the 3'UTR of VEGEPartⅤThe relationship between miR-126 and VEGF in vivoObject:Analyze the relationship between the expression of miR-126 and VEGF in vivo.Methods:(1) A549,Y-90 and SPC-A1 cell lines were infected by recombinant lentivirus LV-miR-126.RPA and west blot were used to confirm the relationship of miR-126 and VEGF.MMT was used to detect the cell proliferation.(2)A549,Y-90 and SPC-A1 cell lines were infected by recombinant lentivirus LV-miR-126.Cells were harvested 48 hours after infection,typsinized and fixed with 70%ethanol on ice for 1 hour.The cell suspension was stained by propidium idide(PI) and was measured by flow cytometry.(3)A549 cells were infected by Lv-miR-126 and LV-GFP,and optimal MOI were determined.A549 cells were infected with optimal MOI.When the GFP level of infected cells was stable in 24-hour observation,limiting dilution assay was used to screen monoclone.When clones were selected,RT-PCR and west blot were conducted to detect the expression of miR-126 and VEGF.(4)Viable 2×106 of stable A549/LV-miR-126 and A549/LV-GFP were injected in the left foreleg armpit of nude mouse to establish cancer model.25 days after injection,all the mice were euthanized. The tumors were excised and weighted to determine the inhibitory effect of over-expression of miR-126 on cancer in vivo.Results:(1) Over-expression of miR-126 in lung cancer lines A549,Y-90 and SPC-A1 can inhibit expression of VEGF and the proliferation of A549,Y-90 and SPC-A1 cell lines.(2) Infection of LV-miR-126 can increase the percentage of cells at G1 phase and decrease the percentage of cells at S phase in A549,Y-90 and SPC-A1 cell lines.(3) The weight of tumors formed by A549 cells with stable miR-126 expression in nude mice was lower than control group.Conclusion:Over-expression of miR-126 in lung cancer cell lines,can inhibit the expression of VEGF and repress the proliferation of cancer cells and modulate cell cycles and decrease its tumorigenicity in nude mice by binding to the 3'UTR of VEGF.