The MicroRNA Expression Profiles In Human Cancer Cell Lines And The Regulation Of MiR-17-5p On Growth Of ES-2 Cells And K562 Cells

Objective:MicroRNAs are a large new class of tiny regulatory RNAs and function as modulators of gene expression by targeting mRNAs for cleavage or translational repression.In vertebrates microRNAs have been implicated as regulators of developmental timing,cell proliferation,programmed cell death and fat metabolism,and recently they are found to be abnormally expressed in several types of cancer.It is highly likely that the expression levels of individual miRNA molecules play an important role in cancers.The purpose of this experiment is to develop a microarray method to detect global miRNA expression levels in human cancer cell lines and study the potential function in the cancerigenisis.Methods:1.Using a high-density oligonucleotide array which we fabricated,the expression profiles of miRNAs were detected in human cancer cell lines including HeLa,K562,ES-2,MCF-7,HT-2 andA549.Results were confirmed by RT-PCR and Northern blot analyses.2.miR-17-5p was knockdown in ES-2 cells and K562 cells by transfecting the antisense oligonucleotide,and the activity and the number of the cells were examined by MTT assay and cell counting.3.The level of apoptosis in the transfected cells was determined by TUNEL and the cell cycle distribution was analyzed by FACS.4.To investigate the influence of miRNAs on cells growth,we used microarrays to examine changes in the messenger RNA profile and preidict the putative targets of miR-17-5p.Results: 1.Different expression profiles were detected in the six cancer cells and 115 miRNAs were differentially expressed significantly,among which miR-21 expressed in high level,while miR-125b,let-7 expressed in low level in all six cells,miR-17-5p and miR-20a expressed in cluster and more higher level in ES-2 than other cells.HeLa and MCF-7 were located on a single branch of the dendrogram in cluster analysis.2.The results we obtained with microarrays are consistent with our results from Northern blot and RT-PCR.Additionally,high expression of miR-17-5p in K562 cells was detected.3.We detect the apoptosis in ES-2 cells and an arrest in S phase of the cell cycle in K562 cells after the inhibition of miR-17-5p.4.Microarrays analysis shows that many changes in mRNA in ES-2 cells transfected by antisense-oligonucleocide to miR-17-5p.The potential targeting genes predicted by bioinformatics including E2F1,CHAF1A,COX7A2L,HECA,VPS26,YES1 have changes in expression level.5.MiR-17-5p may be involved in apoptosis and cell cycle process by negative regulating E2F1.Conclusions:1.miRNAs were differentially expressed significantly among types of cancer cells.2.miR- 17-5p may play an important role in progress of cancer through regulating cell cycle process and apoptosis.