| BackgroundFat is an important energy provider for our body,but excess fat often leads to obesity and other diseases and becomes "scrap".Obesity,atherosclerosis,insulin resistance and other diseases are all related to adipose tissue dysfunction.The growth and development of fat are closely related to the proliferation and differentiation of adipose-derived stem cells(ASCs).ASCs are a kind of multi-differential differentiation potential cells with self-replication ability,such as adipogenic differentiation,osteogenic differentiation,chondrogenic differentiation,hepatoblast differentiation,and neuroblast differentiation.ASCs have the characteristics of easy to fetch material,wide source,and suitable for large-scale culture.It is one of the most active adult stem cells in the field of tissue engineering.CCAAT/enhancer binding protein α(C/EBPa)and peroxisome proliferator-activated receptors γ(PPARy)are the major regulators during adipogenic differentiation in ASCs.They promote fat-specific gene expression and induce adipogenic differentiation.MicroRNAs(miRNAs)are a class of about 22 nt non-coding RNAs with regulatory functions.It can bind all or part of the target gene by means of complementary base pairing,degrade the target gene or repress the translation of the target gene,thereby regulating the cells.Recent studies have shown that miRNAs participate in the regulation of self-renewal,proliferation,and multi-directional differentiation of ASCs.In order to explore the relationship between transcription factor C/EBPa and miRNA,and to use miRNA to accurately control the directional differentiation ability of ASCs,it is necessary to screen and verify C/EBPα target miRNA and explore its influence on the differentiation of ASCs.This will lay the theoretical foundation for the study of adipose stem cells and the treatment of adipose related diseases.Methods1.Use the miRNA target gene prediction software(TargetScan,miRanda,miRDB,RNA22,PicTar)to determine the free energy of the binding site and the stability of the secondary structure by using the key gene C/EBPa in adipogenic differentiation of hASCs as a target.The scoring criteria were used to screen target miRNAs that can regulate C/EBPa.2.Extraction of adipose derived stem cells on day 0,1,3 and 6 of adipogenic differentiation.Real-time quantitative real-time PCR(qRT-PCR)was used to detect the expression of miRNAs in adipogenic differentiation of hASCs.Screening the negatively correlated miRNAs in adipogenic differentiation.3.Using PCR technology to amplify the C/EBPα 3’UTR fragment,mutate the hsa-miR-326 target site sequence on the C/EBPa 3’UTR,and subclon them to a dual luciferase reporter vector to construct psiCheck2[C/EBPα 3’ UTR-WT/MUT]vector.Target miRNA and wild-type and mutant target site vectors were co-transfected into 293T cell line to verify the role of target miRNAs in regulating transcription factor C/EBPa.4.Using lentiviral vector(purchased from Shanghai GK),to over-express or interfere miR-326 in adipose-derived stem cells,Western blot,qRT-PCR,and oil red O stainingwere used to detect C/EBPa and the effect in the adipogenic differentiation of transfected hASCs.Results1.Use multiple target gene prediction software to predict the potential target miRNA of C/EBPa.The preliminary results showed that 424,104,49,1194,and 9 miRNAs could be predicted,respectively.One miRNA that could be simultaneously predicted by 5 kinds of software is hsa-miR-326.Further analysis revealed C/EBPa 3’UTR has two miR-326 target sites.2.Using qRT-PCR to analysis C/EBPα 3’ UTR targeted miRNAs on days 1,3,and 6 of adipogenesis.As the adipogenic differentiation time increased,the expression of miR-326 gradually decreased.After 1 day of adipogenesis,the expression level of miR-326 was 0.5462±0.0655 times that of GM(P<0.0001);for 3 days of adipogenesis,the expression level of miR-326 was 0.4376±0.11417 times that of GM(P<0.0001);for 6 days of adipogenesis,the expression level of miR-326 was 0.241610.046 times that of GM(P<0.0001).However,the expression of miR-92a,miR-301b and miR-548h-5p did not change significantly.3.The psiCheck2[C/EBPα 3’ UTR-WT/MUT]vector was successfully constructed.The successfully constructed reporter gene vector and the miR-326 overexpression plasmid were co-transfected into HEK293T cells.When miR-326 overexpression plasmid and wild type 3’UTR were co-transfected,the luciferase activity was significantly downregulated(P<0.0001),whereas the miR-326 overexpression plasmid and the mutant 3’ UTR were co-transfected with the luciferase activity did not change significantly.There was no significant decrease in co-transfecting the anti-miR-326 plasmid.4.Lentivirus transfected adipose stem cells successfully overexpressed miR-326 but did not achieve the purpose of interference with miR-326 expression.The transfected stem cells were differentiated by adipogenic differentiation.Oil red O staining,qRT-PCR and Western Blot all showed that the overexpression of miR-326 decreased the ability of adipogenic differentiation,and the difference was statistically significant.Interfering with miR-326(anti-miR-326)group did not achieve the purpose of up-regulating adipose-derived stem cell adipogenic differentiation.Conclusion1.The miRNAs of C/EBPa,key factor of adipogenesis,were screened by bioinformatics prediction methods.The expression of miR-326 was decreased along with adipogenic differentiation,suggesting that miR-326 may closely related to it.2.The 3’UTR of C/EBPa wild-type and mutant-type plasmid were constructed.The dual luciferase reporter assay was used to further verify that miR-326 can bind to the C/EBPα-3’UTR.3.The expression of miR-326 was up-regulated or down-regulated in stem cells.The adipogenic differentiation ability of the stem cells after the up-regulation was weakened and the protein expression of C/EBPa was decreased.It further verified the combination of miR-326 and C/EBPα-3’ UTR,which blocked translation of C/EBPa,decreased its expression and in turn inhibited the adipogenic differentiation of hASCs. |
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