Investigation Of MicroRNA Associated With Serum Starvation

Objective:MicroRNAs (miRNA) are a new family of small RNA molecules (22nt) that control the expression levels of their target genes. Thousands of miRNAs have been identified in recent years and their functional identification has become one of the most active research fields in biology. To detect the regulating miRNA and to predict the target genes involved in serum starvation condition in human normal cells using miRNA microarray, and deeply study the biological functions which miRNA involved through regulating the expression of target genes.Methods:The expression level of miRNAs in serum starvation condition (without serum in culture) and normal condition (with10%serum in culture) from human normal hepatic cells was compared by using miRNA microarray. The function of miRNA which expression level was raised (miR-27a, miR-320, miR-338) was inhibited by specific antisense oligonucleotide (ASO) or which expression level was depressed (miR-181b1) was enhanced by specific small interference RNA (siRNA) oligonucleotide, after that the cell activities were examined through MTT experiments, respectively. Then the effect for apoptosis was examined though TUNEL experiment. The computational target prediction algorithm is combined with the mRNA microarray to predict the target genes of the human regulating miRNA involved in serum starvation condition. According to the known function of the target gene, we evaluated the biological function of the human regulating miRNA involved in serum starvation condition in the regulation process of survival activity.Results:The expression level of miR-27a, miR-320and miR-338was raised and the expression level of miR-181bl was depressed according to the analysis of miRNA microarray. Using functional assays of MTT experiments, we found that the activities of LO2cells were significantly inhibited when the function of miR-27a, miR-320or miR-338was inhibited, and enhanced when the function of miR-181b1was enhanced.The expression profile of genes in LO2cells cultured with10%serum or without serum conditions analyzed by the long oligomer array with human7267genes including apoptosis, cancer, signal transduction, liver enzyme metabolism and cytokine related genes. Adcy3, bc13, hlx1, et al were the putative target genes of hsa-miR-27a predicted by computational target prediction algorithm. Ctnnb1, ywhaq, adcy3, et al were the putative target genes of hsa-miR-320. Rps19, p4hb, atp5gl, et al were the putative target genes of hsa-miR-338. Bcl-9, ddit4, dusp10, et al were the putative target genes of hsa-miR-181b-1.Conclusion:We confirmed the regulating miRNA involved in serum starvation condition. We predicted the target genes of miR-27a, miR-320, miR-338and miR-181b1in human cells. MiR-27a, miR-320, miR-338and miR-181bl could induce the mRNA degradation of their target genes, respectively. The miR-27a, miR-320, miR-338and miR-181bl could indirectly inhibit the activities of cells by regulating the expression level of their target genes, and control the response of the cells in serum starvation. The study of regulation function and related mechanism were helpful for us to understand their biological characteristic and regulation mechanism.