Effects Of MiR-204/211on Differentiation And Dedifferentiation In RPE

RPE (retinal pigment epithelium) will lead to many serious disease when it isdysfunction. Cell transplantation therapy have been adopted to cure RPE relateddiseases. The primary limitation of RPE transplantation therapy is the limited cellsource. Many studies were put effort to produce RPE in vitro. But the mechanisms ofRPE differentiation is still unclear, the in vitro production of RPE cells was restricted.Thus, understand the mechanism of RPE differentiation will be very helpful. Mystudy was rely on the previous work of RPE differentiation in our lab, and focus onthe effect of microRNAs in the differentiation and de-differentiation process of RPEcells.Two RPE cell lines: ARPE-19and hfRPE were selected as material in my study.I predicted the potential miRNA target sites of genes which play key roles in the RPEdifferentiation process. Then I choose microRNAs which predictively target to3ormore regulator of RPE differentiation as candidates. We proved the key role ofmiR-204/211in the maintainance of RPE cells via QPCR, immunostaining, flowcytometry cycle analysis and dual luciferase reporter assay. The results aresummarized as follows:1. We predicted some miRNAs which may regulate RPE differentiation andmaintainance through literature-mining and bioinformatics analysis. Highly expressedmiRNAs in2RPE cell lines were identified through QPCR method, includehas-miR-204/211/194.2. To investigate the role of miR-204/211in RPE differentiation and maintainance, wetransiently transfect miRNA mimics into two RPE cell lines, ARPE-19and hfRPE,and observed significantly upregulation of RPE65, CRALBP, and TYRP2.Corresponded, we observed significantly downregulation of RPE65, CRALBP, andTYRP2in cells which transfected with miR-204/211antagomirs immediately.However, RPE65, CRALBP, and TYRP2were restored in miR-204/211antagomirstransfected cells at10days after transfection.3. Further more, I investigate the cell cycle alteration of ARPE-19and hfRPE whichmiR-204/211were upregulated or downregulated. Via Flow cytometry, I found cellproliferation was affected by downregulate of miR-204/211rather than upregulatemiR-204/211.These results show that miR-204/211could effectively regulate thedifferentiation and dedifferentiation of RPE cells, and this effect may relevant to cell cycle regulation. These results confirmed the importance of miR-204/211in themaintainance of RPE cells. Further examine of functional roles of miR-204/211inRPE cells could reveal the internal regulation network of microRNAs and signalingpathways.