Identification The New Targets Of MiR-138, MiR-30Family And Primary Study Of Their Biological Functions

MicroRNA (miRNA) is a group of~22nt length non-coding small RNA molecules. Theprimary precursor, pri-miRNA, is encoded from the introns in genome DNA and transcript byRNA polymerase Ⅱ. The molecular configuration of pri-miRNA contains stem-loopstructures and the mature miRNA locate in the3-or5-terminal of stem-loops. In differentspecies, Dorsha interacts with its coenzyme factor DGCR8or Pasha and act as a “molecularruler” which determining the cleavage site of pri-miRNA to generate a~70bp lengthpre-miRNA. The pre-miRNAs were transfered from nucleus to cytoplasm with the help oftransport protein Exportin-5. Then another molecular ruler, Dicer, cleavage thepre-microRNA into5-terminal phosphorate and3-terminal ends of2nt overhanged doublestrands RNA. This double strands RNA is a heterodimer constitute of mature miRNA and itscomplementary nucleotides chain. At last, the dimer unchained into single strand maturemiRNA through the interaction of helicase enzyme, and mature miRNA composing RISCswith some other molecules binding to the3UTR of target mRNA. In this procession，RNAinterfering happens.MiRNA has only~22nucleotides but regulates the expression of thousands of genes invivo. It is closely related to many physiological and pathological processes involved in cellproliferation, differentiation, metabolism and apoptosis, et al. There are many researches against miRNA in all kinds of aspects, but it’s still a difficult problem to get a thoroughunderstanding of miRNA. The first and the key procedure to investigate a miRNA functionsis to find out its functional target gene accurately. Different from the plant, the sequence ofmammalian miRNA and its target mRNA may not be completely complementary, a miRNAcan target a lot of genes and some miRNA also act as one functional cluster effect on onesingle gene. In addition, expression of miRNA is affected by temporal specificity, cellspecificity and development stage specificity. These problems make identify functional targetgene of miRNA being the biggest bottleneck of the miRNA research.In our former study, we discovered that the distribution ratio of the target mRNA incytoplasm or nucleus (cytoplasm/nucleus ratio, C/N) may affects the miRNA targeting thefunctional gene: For the multiple target genes of singe miRNA, the higher cytoplasm/nucleusratio，the more possibility to be the target. At the meantime, we found that functional target ofmiRNA in vivo is dynamic, it means the target of a miRNA may be inconsistent in differentcells or different states of the same cell. In conclusion, how to determine functional targets ofa miRNA in a particular state has a significant meaning on the interpretation of the miRNAfunction.There are two parts in this thesis.Part1: Dynamic change of functional targets of miR-138is consistent with themRNA cytoplasmic/nucleic Ratio in H1299cell.To verify the accuracy of target strategy that identified target genes based on C/N. Wecompared the bioinformatic prediction in online target gene database (miRanda, TargetScan,PicTar)with the H1299cell RNA microarray analysis. Fifty four genes had been predicted asthe targets of miR-138, and the C/N ratio of these genes were found greater than1. Then wetransfected miR-138mimics in vitro and detected target gene expression level by real-timePCR,87%of the genes were significantly down-regulated. This result showed that our targetgene screen method based on C/N has a relatively high accuracy.To verify the dynamic changes of miRNA functional target in different cell lines, wecompare the changes of miR-138target genes between transiently transfected and stableoverexpression of miR-138in H1299cell line. Compare transient transfection and stableoverexpression of miR-138target genes change. We find that the dynamic changes of miRNA functional target relate closely to the C/N of target mRNA, the higher C/N, the morepossibility to be down regulated.To verify the dynamic changes of miRNA functional target in different cell states, weculture H1299cell line stable expression of miR-138by starvation. In the procession,16mRNA expression level has changed (31.5%), including14genes has no significantdifferences at first while their expression levels began to fall after24h of starvation.Compared to0h,14genes C/N is significantly higher at24h.Among the above identified target genes, we chose Arognaute4(AGO4) as a newfunction target of miR-138to conduct further research. After we confirmed AGO4as thefunctional target of miR-138, we found that AGO4may be as a bridge protein to regulateTRIM21which could degrade HSPA5. Inhibition of miR-138could promote AGO4expression, while promoted the TRIM21degrading HSPA5and then induced the apoptosis ofH1299cells.Part2: MiR-30family regulates siRNA and miRNA functions by targeting GW182.To investigate the biological function of GW182which is a novel target of miR-30family in human HeLa cells. We analyze the potential target sites of miR-30family in the3UTR of GW182gene sequence by information biology. Luciferase assay, quantitativereal-time polymerase chain reaction and Western Blot confirm the target sites of miR-30family in GW182. We find that miR-30a、30b repressed GW182expression on both mRNAand protein levels. And results showed that miR-30a、30b could down-regulate the effect ofmiR-200b targeting ZEB1, miR-138targeting its target gene and GFP siRNA silencing theGFP expression by targeting GW182. These data suggested that miR-30family may play animportant role in the regulation of miRNA or siRNA function by targeting GW182.