The Role And Mechanism Of MiR-322 In Differentiation Of Embryonic Stem Cells Into Cardiomyocytes

Objective:The aim of this research is trying to study the effect mechanisms of miR-322 during embryonic stem cell differentiation into cardiomyocytes,to explore the patterns that miR-322 regulates the cardiomyocytes differentiating from stem cells.We cautiously choose mouse embryonic stem cell as our subjects,which predicts that celf1 is the target gene of miR-322,to investigate the following response as differentiation goes on,and the interaction between miR-322 and celf1 by overexpressing and knocking down miR-322 and celf1 with lentviral.As the research follow on,we hope to make some countable contribution to the development of new ideas,new directions,and novel targets in the therapeutic strategies for myocardial infarction,to provide more theoretical evidence and therapeutic options for the using of miR-322 and celf1 in the cardiovascular diseases,and in other diseases.Methods:1.MEF and mES-D3 cell culture: MEF cells were cultured,then treated with mitomycin C at P3;mES-D3 cells were cultured in the conditions of existed feeder(use the MEF cells as the feeder),to ensure the mES-D3 cell stably passing.2.Lentivirus preparation and transfection: The plasmid was combined with target gene/miRNA,puromycin-resistant gene and EGFP fluorophore.After the test of “multiplicity of infection”(MOI),the cells were infected with the prepared lentiviruses.then purified by the puromycin-resistant.3.mES-D3 cell differentiation in 5 groups: Cells was assigned in 5 groups(1)miR-322 overexpression vs wild type and empty virus,(2)miR-322 knock-down vs wild type and empty virus,(3)celf1 overexpression vs wild type and empty virus,(4)celf1 knock down vs wild type and empty virus,(5)miR-322/celf1-co-overexpression vs wild type and empty virus.Then aggregated cells in hanging drops,a classic method “embryoid bodies” was used as the differentiation model.We collected those embryoid bodies as fully maturitied.Then samples were collected at correct days.4.Antibodies and immunoassays(for western blotting): membranes were incubated in phosphate-buffered saline,with the primary antibodies(rabbit monoclonal antibody to Celf1(CUG-BP1),Nkx-2.5,mouse antibody to GAPDH,cTnT).Then incubated with horseradish peroxidase-conjugated secondary.Bound antibodies were visualized with enhanced chemiluminescence reagents.Celf1 in every experimental group was tested to check the infection.Antibodies of GAPDH,Oct-4,Nkx-2.5,cTnT was assayed at day 0,5,10.5.Real-time quantitative RT-PCR analysis: Using the expressed genes of day0 EBs in every experimental groups as a constitutive control,the genes of Oct-4,Sox-2,Nkx-2.5,MLC-2v,?-MHC,cTnT were tested during mES-D3 cell differentiation,in extracted total RN A,for total cDN A reverse transcription,the protocol and reagents of reverse transcription system were used.After the reaction,amplification curve and calibration curves were produced to e nsure similar qPCR efficiencies,to calculate the?Ct data with 2-??Ct,then analysis with GraphPad Prism 5.6.Immunofluorescence.IF was used to detect the distribution of cardiomyocytes cell,embryoid bodys was planted on the coated dishes,then washed,fixed at the correct days.The expression of ?-actinin under fluorescence was detected.Result:1.miR-322 played an important role in the developing of cardiomyocytes.(1)Overexpress miR-322 significantly improved the cardiac differentiation.(2)Down-regulating miR-322 reduced the beating area noticeably,and influenced the cardiomyocytes maturity.2.To control the differentiation of cadiomyocytes,celf1 was an crucial regulator.(1)Overexpresing of Celf1 inhibited cardiac cell maturity and impaired differentiation.(2)Knocking down celf1 gene had a positive effect during the development of the heart.3.Celf1 was a downstream signal molecules and a target gene of miR-322.(1)In the experimental groups of miR-322 overexpression alone,miR-322 /celf1-co-overexpression,celf1 overexpression alone,the express of celf1(CUG BP1)was more or less down regulated.(2)In the experimental group celf1 overexpression alone and miR-322/celf1-cooverexpression,overall outcomes of the cadiomyocytes markers,however,were similar in the two groups.Conclusion:(1)miR-322 will significantly improve cardiomyocytes differentiation.(2)Celf1 has a negative effect during the process of embryonic stem celldifferentiation into cadiomyocytes.(3)Inhibitation Celf1 may be the routes that miR-322 regulats cardiomyocytesdifferentiation.