Micrornas In Pc12 Cells Of Cocl <sub> 2 </ Sub>-induced Differential Expression Of The Differentiation Process

MicroRNAs(miRNAs) are evolutionarily conserved,endogenous, small,noncoding RNA molecules of about 22 nucleotides in length that function as posttranscriptional gene regulators.Some of them are seemed to play a crucial role in the process of nerve cells proliferation and differentiation.The rat pheochromocytoma cells(pcl2 cell) are derived from neural crest.When treated by CoCl₂ for 48 hours,pcl2 cells can generate protuberance like nervous axon.PCl2 cells are widely used as nerve stem cell differentiation model.In this article we analyzed the microRNA expression level in the process of PCl2 cell differentiation induced by CoCl2,we also observe the biological effect upon pcl2 cells via introduce exogenic mature and antisense microRNA in order to predict the potential function of microRNA.First of all,we built up the hypoxia-nerve differentiation cell model. After treating PCl2 cell with 150uM Cocl2 for 24h and 48h,expression of HIF-1αand its down-stream gene VEGF are up-regulated,reflecting that hypoxia treatment is efficient.Meanwhile,we can observe the differentiation phenomenon of PCl2 cell.And then,depending on microarray technique,we detected expression difference of microRNA in the process of CoCl2 inducing PCl2 cell differentiation.The results reveal that after treating for 24 hours,22 microRNAs are upregulated while 21 microRNAs are downregulated; after treating for 48 hours,45 microRNAs are upregulated while 30 microRNAs are downregulated.We chose two microRNAs as presentative-miR27b(down regulated) and miR325-3p(up regulated) as our research targets.Then we detect the expression difference via RNA reverse transcription and Real Time PCR.We found that expression level of miR27b is gradually declined while that of miR325-3p is ascending, which are accordant to the microarray results.On this basis,we transfect PCl2 cells with artifact mature miR27b oligonucleotides,mediated by liposome.48 Hours after transfection,the result of Real Time PCR indicates that the concertration of miR27b in transfected PCl2 cells is obviously higher than that of normal pcl2 cells, however,up-regulated miR27b did not affect PCl2 cell differentiation process.Meanwhile,we utilize liposome transfection method to introduce exogenic antisense miR325-3p in PCl2 cells induced by CoCl2.We found the knock-down of miR325-3p impose no obvious influence on PCl2 cell differentiation process,either.So we concluded that chosen microRNA-miR27b and miR325-3p are not essential roles in the process of CoCl2 inducing PCl2 cell differentiation.