Functional Study Of Mouse MiR-200s Which Are Related To Body Size Control

Body size is a fundamental, easily observed animal phenotype. The most obvious differencesamong different animals are differences of size. How does the individual sense the key size point andknow when to stop growing? Developmental scientist and zoologist have been puzzled by theseproblems for years and haven’t got satisfactory answers till now. So far, scientists can explainelementary size control mechanism of cell, organ and organism in model animals such as fruit fly andnematodes. However, no great progress has been made on mammals.miRNA is a class of single stranded noncoding RNAs with length about20-22nt, which was firstfound on nematodes. They could promote the degradation of target mRNA or inhibit their translationprocess by combination with the3’UTR of target genes. In2009, Seogang Hyun found a conservedmicroRNA miR-8and its target, USH, regulate body size in Drosophila. miR-8null flies are smaller insize and defective in insulin signaling in fat body which is the fly counterpart of liver and adipose tissue.Further studies have indicated that miR-8could regulate animal growth by participating in insulinsignaling pathway and affecting the TOR pathway.Based on the above finding, we proposed the plan studying the size control mechanism ofmammals from the insight of miRNA. We got the miR-200s expression profiling of mouse tissues andembryos at different developmental stages by Stemloop-RT PCR and QPCR and found they areubiquitously expressed in different tissues.miR-200b, miR-200c are highly expressed in male andfemale mice, respectively. Potential targets of miR-200s were predicted using Targetscan.We paid greatattention to Zfpm2gene and two binding sites were discovered in Zfpm3’UTR:224-230bp and763-769bp. We constructed a recombinant vector containing Zfpm2-3’UTR and dual luciferase reporterand verified the target via dual luciferase reporter assay system in mouse Hepa1-6cell, indicatingmiR-200s could interact with Zfpm2. Overexpression of miR-200s in Hepa1-6shown that miR-200could affect insulin signaling and increase the mRNA of Akt in cell. We constructed a conditionalknockout(CKO) vector for miR-429/200a/200b cluster and transfected it into Es cells. After positivecells were obtained, we injected them into pseudo pregnant mouse to get chimeric mice. Then, F1positive heterozygotes mice litters containing the CKO vector were breeding with mice expressing Crein adipose tissue only to get F2adipose tissue specific miR-429/200a/200b knockout heterozygotesmice (miR-429/200a/200b+/-). We analyzed the phenotype of F2mice and found knockout mice(miR-429/200a/200b+/-) have a significant body weight loss and reduced adipose tissue weightcompared with controls (miR-429/200a/200b+/flox). Meanwhile, the fat deposition is abnormal in liverand the heterozygotes knockout mice show patent fatty lesions and decreased Islands oflangerhansvolume. All these results could provide molecular basis for mammalian size control and give hope toshort people with high dream.