Identification Of MicroRNA Targets In Tumorigenic Regulatory Network By Photo-reactive Probes

Micro RNAs(miRNAs)are group of short(?22 nucleotides)endogenous,noncoding RNAs that induce RNA interference(RNAi).In regulatory network,miRNAs directly inhibit translational event of a specific group of target m RNAs and regulate a wide variety of physiological processes.Mature miRNA,upon loading to RNAinduced silencing complex(RISC),binds to a specific region of the target m RNAs.The results of such binding play a key role to explore biological functions of miRNA.Crosslinking immunoprecipitation(CLIP)is an effective and credible method to identify miRNA targets,compared to computer algorithms according to the complementarity-based methods and thermodynamic-based methods.However,CLIPbased method is tedious and time-consuming and needs complicated protocols.Thus,the identification of novel targets of miRNA with straightforward and robust methodology is highly desirable.OBJECTIVES:The objective of this thesis is to design and prepare a highly efficient photoreactive oligonucleotide-based miRNA probe,measure the target recognition efficiency as well as reactivity,and evaluate the possibility of the probe for fast and straightforward miRNA target identification.In this thesis,we first design and prepare psoralen modified miRNA probes(miR probe),which have photo-reactivity and bioactivity.Upon transfection,miR probe loads to RISC(miRISC)and binds to target m RNAs.A subsequent UVA(360 nm)treatment to the live cells produces covalently crosslinked miR probe-target m RNA complex inside cells,which is then isolated from cell lysate and subsequently subjected to the reverse transcription.As psoralen conjugation to m RNA is a definite stop signal to the reverse transcription(RT)reaction,the hybrid miRNA-m RNA does not yield c DNA,which results in decreased reads of target m RNAs in RNA sequencing(RNA-seq).Therefore,this method(based on photo-activity probes and RNA-seq technology)can highly efficiently identify multiple novel targets of distinct miRs.METHODS:(1)Chemical synthesis of miR probe: First chemically synthesize NHS activated psoralen derivatives,and conjugate the NHS-Psoralen in the specific nucleotide bearing free amino group in the seed sequence of miR-29 a and miR-34 a,respectively.Then,analyze and characterize the product probe using denaturing PAGE gel.(2)In vitro reactivity of miR probe: Anneal the psoralen modified miR with the sense sequence,treat with UVA(360 nm)for crosslinking,analyze and confirm the crosslinked double strand RNA on denaturing PAGE gel.(3)In vivo reactivity of miR probe: After transfecting A549 cells with the psoralen functionalized miRs(miR-29a-ps and miR-34a-ps),respectively,the m RNA level of well-established targets was analyzed by using RT-q PCR analysis,thereby evaluated the in vivo reactivity of the miR probes.(4)Targets identification via miR probe: After transfecting A549 cells(non-small cell lung cancer,NSCLC)cells with miR probes,treated the cells under UVA at various time points;then,performed RNA-seq analysis on the ploy(A)RNA extracted from transfected cells after intracellular UV crosslinking.(5)Validation of probe efficiency(miR-29a): Based on RNA-seq analysis,potential candidate targets of miR-29 a was further evaluated by RT-q PCR analysis;The entire 3? untranslated region(3? UTR)of candidate targets m RNAs were cloned into luciferase-expressing vector,respectively.The effective binding site(s)of miR-29 a in 3? UTR of candidates were validated by dual luciferase assay.To confirm the direct targets of miR-29 a,total protein obtained from A549 cells transfected with miR-29 a were analyzed by using western blotting assay.(6)Validation of probe efficiency(miR-34a): Next,RT-q PCR,dual luciferase assay,as well as western blotting were further utilized to validate and confirm the direct targets of miR-34 a from the RNA-seq results.RESULTS:(1)Analysis of two psoralen modified miR probes by denaturing PAGE gel confirmed the success of the chemical synthesis.Among the two miR probes,the one prepared from 4,5?,8-trimethylpsoralen showed an apparently retarded band,confirming the high reactivity of the miR probe.After transfecting A549 cells with psoralen functionalized miRs(miR-29a-ps and miR-34a-ps),the m RNA level of wellestablished targets was correspondingly downregulated to some extent,suggesting that psoralen modification does not alter the RNA interference activity of miRNA.(2)We transfected A549 cells with miR probes and conducted RNA-seq analysis on the ploy(A)RNA extracted from transfected cells after intracellular UV crosslinking.Quantitative analysis of RNA-seq reads revealed that,compared to the control,a total of 27 genes were downregulated(log2(fold change)<-0.2,-log10(p-value)> 0.4,FPKM > 100)in miR-29a-ps treated samples and a total of 34 genes were downregulated in miR-34a-ps treated samples.(3)Further confirmation revealed that 10 out of 27 genes were downregulated by miR-29a-ps.The entire 3? UTR of 10 candidate targets m RNAs were cloned into luciferase-expressing vector,respectively.After co-transfected HEK293 T cells with reporter vectors and wildtype miR-29 a,the reporters containing 3? UTR of PTTG1 and PTGR1 showed significantly decreased luciferase expression in miR-29 a treated cells,suggesting that miR-29 a has effective binding site(s)in 3? UTR of these genes.By using western blotting assay on total protein obtained from A549 cells transfected with miR-29 a,a considerably decreased expression of PTTG1 and PTGR1 was observed,thus confirming that both are direct targets of miR-29 a in A549 cells.(4)Validation of miR-34 a targets revealed that the expression of 10 out of 34 genes were downregulated by miR-34a-ps.After analysis by dual luciferase assay and western blotting,ILF2 was identified as a novel direct target of miR-34 a.The expression of luciferase in reporter containing 3? UTR of was significantly decreased,which suggested that ILF2 m RNA has direct binding site(s)of miR-34 a in 3? UTR.Further analysis by western blotting showed that ILF2 protein level significantly decreased in A549 cells after the transfection of miR-34 a,confirming that ILF2 is a direct target of miR-34 a.CONCLUSIONS:(1)Synthesis of 4,5?,8-trimethylpsoralen functionalized miR probes was smooth with high purity and high yield;miR probes have in vitro and in vivo reactivity and bioactivity,and suitable for analysis of gene expression in live cells.(2)The synergistic approach of miR probe and RNA-seq can identify miR targets in live cells;the methods developed in this thesis are straightforward and highly efficient,and can be applied to the probe design for any miRNA.(3)In the NSCLC cells,we identified two novel direct targets of miR-29a(PTTG1,PTGR1),one novel direct target of miR-34a(ILF2),thus providing valuable strategy for the study of miRNA biology in lung cancer development.