Study Of The Function Of An Osteoblast-specific Long Non-coding RNA On Osteoblasts Of Mice

Part one Study of lncRNA expression patterns and screening of osteoblast-specific lncRNAObjectiveTo detect the expression pattern of lncRNAs in different organs and cells of mice, search for new osteoblast-specific lncRNAsMethodsWe purchased several6-week-old male mice and got their heart, liver, lung, kidney, brain tissue under aseptic conditions and cultured osteoblasts followed by extraction of total RNA of tissue and cells. Then we purified the RNA and after that we made cDNA labeled and conducted PCR amplification. Fluorescence-labeled cRNA was purified and its concentration was measured. The cRNA fragment was hybridized with microarray. We analyzed multiple sets of data including the expression of lncRNAs in mice heart, liver, lung, kidney and brain, focusing on specific expression of lncRNAs in osteoblasts. The selected lncRNAs were Verified by Northern blot in different tissues and cells. Results1. lncRNAs in heart, liver, lung, kidney, brain and osteoblasts of mice showed different expression patterns.2. We found that17lncRNAs in osteoblasts were expressed and showed no or very low expression in other tissues.3. We found that one lncRNA, tentatively named lncRNA-OB, was highly expressed in osteoblasts while almost not expressed in other tissues. lncRNA-OB was located on chromosome. ConclusionWe discovered a new lncRNA preferentially expressed in osteoblasts. As the expression of lncRNAs in mice are related with their functions, lncRNA-OB may be related with osteoblasts differentiation.Part two Function of lncRNA-OB on osteoblasts ObjectiveTo and work on the function of lncRNA-OB on osteoblasts. MethodsFor the selected lncRNA, we inhibited its expression by siRNA and observed the expression of alkaline phosphatase and osteocalcin secretion in ST2cells before and after induction of osteogenic differentiation.We also detected the expression of RUNX2mRNA and Osterix mRNA. Searching for the upstream and downstream genes and analysis their functions. Results1. After inhibiting lncRNA-OB by siRNA, we found significantly reduction in the osteocalcin secretion and alkaline phosphatase activity.2. After inhibiting lncRNA-OB by siRNA, we also found significantly reduction in the expression of RUNX2mRNA and Osterix mRNA.3. The upstream genes and downstream genes.ConclusionlncRNA-OB promotes the osteogenic ability of osteoblasts.