The Expression And Function Of MIR-107 In Lamin Deficient Mice

LMNA mutations can cause a variety of diseases such as Hutchinson Gilford progeria syndrome, muscular dystrophies, familial partial lipodystrophy and cardiomyopathy syndrome, which are referred to as laminopathies. The A-type lamin deficient mice line (Lmna-/-) that exhibited similar clinical features to laminopathies, is one of the common used animal models to explore the function of A-type lamin. MicroRNAs (miRNAs) are critical post-transcriptional regulatory factors. A growing number of studies have established that miRNAs are involved in multiple physiological and pathological processes, including aging and cardiomyopathy, through regulating hundreds of targets. Nonetheless, the expression and function of miRNAs in laminopathies largely remain unclear. In this study, we have screened the differentially expressed miRNAs in Lmna deficient mice model, and further investigated the potential effects of the differentially expressed miRNAs in the laminopathies.PART 1 EXPRESSION PATTERN OF MIR-107 IN MEFS AND ORGANS OF LMNA-/- MICEObjective To verify the differentially expressed miRNAs screening by Illumina Next Gen Sequencing in Lmna-/- and Lmna+/+ mice fibroblast cells (MEFs), and explore its function. Methods Differential expression miRNAs (mmu-miR-1, miR-107, miR-671-5p, etc) screened by sequencing were validated by real time PCR. Expression pattern of miR-107 was analyzed in various organs of Lmna-/- and Lmna+/+ mice.Results Compared with Lmna+/+ MEFs, Lmna-/- mice MEFs exhibited lower expression levels of miR-107, about 47.9% of the wild type MEFs (P<0.05). Moreover, miR-107 was down-regulated in heart, liver and lung of Lmna-/- mice (P<0.05). Conclusion MiR-107 is differentially expressed in the MEFs and partial (heart, liver and lung) organs of Lmna-/- C57BL/6 mice.PART 2 MiR-107 AND ITS TARGET GENE CAV1 ARE INVOLVED IN THE SENESCENCE OF LMNA-/- MEFSObjective Use the MEFs of Lmna deficiency and wild type mice as modle to explore the role of miR-107 and its target gene CAV1 in ageing regulation. Methods Up-regulated or down-regulated the expression levels of miR-107 in MEFs, then analyzed the expression leveles of CAV1 and the activity of senescence-associated β-galactosidase. Results Compared with Lmna+/+ MEFs, the expression levels of Cav1 significantly increased by 1.7 times in Lmna-/- MEFs (P<0.05). When overexpression of miR-107 was in Lmna-/- MEFs (P4), the expression of CAV1 was inhibited, and the ratio of the senescence-associated β-galactosidase positive cells was decreased about 8%(P<0.05). When down-regulated the expression of miR-107 in the Lmna+/+ MEFs (P4), the CAV1 expression levels were increased, and the ratio of senescence-associated β-galactosidase positive cells was also increased about 10%(P<0.05). Conclusion The expression of Cav1 in Lmna-/- MEFs was significantly higher than in the Lmna+/+ MEFs. MiR-107 and its target gene CAV1 might be involved in the aging regulating in Lmna-/- MEFs.PART 3 CONFIRM THE BINDING SITES FOR MMU-MIR-107 IN CACNA2D1 GENE’S 3’UTRObjective:Predict and confirm the binding sites for mmu-miR-107 in Cacna2dl gene’s 3’UTR. Methods Target genes of mmu-miR-107 was predicted by TargetScan, miRanda, Clip-seq and miRDB, the 3’UTR of candidate target genes (Cacna2dl, Capza2, Celsr2, Csnklg2 and Tmem47) were inserted in to the plasmid pGL3. Bio-information assay revealed that the 3’UTR of Cacna2dl harbors three putative binding sites for miR-107. Then three mutant plasmids containing single binding site (Mut1 200-207, Mut2361-367 or Mut3902-908) and a mutant plasmid containing all the three binding sites (Mut4plus) were made by overlap extension PCR. Both recombinant plasmids and mimics miR-NC (or miR-107) were transfected into C2C12 cells, and the activity of luciferas was detected by luciferase reporter assay. Candidate Cacna2dl gene was verified by q-PCR and western bolt. Results The luciferase reporter plasmids of the five candidate target genes were successfully constructed. Compared with the C2C12 cells co-transfected with pGL3-3’UTR (containing candidate target gene’s 3’UTR) and miR-NC, the luciferase activity of C2C12 cells co-transfected with pGL3-Cacna2dl 3’UTR and miR-107 mimics was decreased 33.4%(P<0.01), the luciferase activity of C2C12 cells co-transfected with other plasmid and miR-107 mimics were not obviously decreased (P>0.05). Compared with the C2C12 cells co-transfected with mutant pGL3-Cacna2dl 3’UTR and miR-NC, the luciferase activity of C2C12 cells co-transfected with Mut2361-367 and miR-107 mimics, co-transfected with Mut3902-908 and miR-107 mimics were decreased (P<0.05); the luciferase activity of C2C12 cells co-transfected with Mutl200-207 and miR-107 mimics, co-transfected with Mut4plus and miR-107 mimics were not obviously decreased (P>0.05). Over-expression of miR-107 in C2C12 cells downregulates the mRNA and protein levels of Cacna2dl, meanwhile, down-expression of miR-107 in C2C12 cells upregulates the mRNA levels of Cacna2dl (P<0.05).Conclusion Cacna2dl might be a target gene of mmu-miR-107, and it 3’UTR harbors the binding sites is 200-207.