miR-195-5p Tough Decoy Alleviates Hypertrophic Markers in Micromass Model

Background and Aim: Chondrocyte hypertrophy is a multi-stageevolutional process containing mineralization and differentiation. Withregard to our knowledge of signaling pathways and, in particular, thedetermination of the important regulatory role of miRNAs in signalingpathways, one of the goals that can be studied in helping to achieve amethod of a cop with cartilage hypertrophy is to study the effect of miRNAon the cartilage differentiation pathways. In this study, a miRNA195-5p tough decoy expresses an artificial transcript with a competitiveinhibitory effect so, by absorbing the desired miRNA.Methods: miR-195-5p tough decoy designed base on seed sequenceswith a bulge, In order to prevent the target from being captured byRNAi. Since we are working in the non-coding region, so firstly weput TAA as stop codon after emGFP then our construct cloned, and tomake desire space, sequentially ACGCGU sequence inserted to makedesired distance, Then the sequence put into a Tough Decoy frame andThe DNA synthesized and cloned in the PLVX vector, after analyzingby miRNAsong, plasmid gets ready to transfect by lipofectamin 2000into c28/i2 cell line. C28/i2 cell line was followed by a relativelydifferent transfection protocol due to its hard transfection and low rateof transfection and for the first time on the research, c28/i2 gets stabled.On 80% density, cells trypsinized and 1.7×104 cells seeded into six-wellplates, after overnight incubation cells transfected by lipofectamine 2000 (Invitrogen, Life Technologies, NY), then followed by Micromass pelletculture with StemPro® chondrogenesis complete mediumResults: The miR-195 has a confirmed hypertrophy signaling pathwaythat interferes with the pathway of Col2a1, Col10a1, MMP13, DLK1,Aggrecan, VEGF. In fact, the miR-195 has direct targets on the 3 UTR ofthe targets that bind them and results in reduced expression, In normalmode. Also, the expression level of miR-195 is inversely related to theexpression level of CCND1 and in case of high expression of miR-195,this gene expression reduced and vice versa. Another important targetof miR-195, which has a very important function of differentiation andevolution, is the WNT3a gene that has some seed for miR-195 in the3 UTR region. The high level of miR-195 reduces the expression of theWNT3a gene that leads to cell cycles stopping at G1 steps. Real-timePCR analyses showed Col2a1, CCND1, DLK1, Aggrecan, and ALP geneswere up-regulated in Tough decoy stabled cell line compared to controlcells during micromass pellet culture, while Col10a1, MMP13, Runx2were downregulated.Conclusion: In this study, we used a TUD containing cassette formiR195-5p, which is expressed by the CMV promoter. The expression ofthis construct leads to cytoplasm occupation with artificial miR-195-5ptargets, this will result in all of the miR-195-5p absorbed by constructand main targets remain clear. By comparing the results of real-timePCR and also, Alician blue and Alirazin red tissue stain, showed that thechondrocytes cells retained their cartilage shape in TUD stabled cellsmore than the control cells.

کلیدواژه ها:

Hypertrophy ، miR-195-5p ، Chondrogenesis ، miRNA Inhibitor ، Tough Decoy