The Molecular Mechanisms Of Non-coding RNAs Regulating DRG Neuron During Nerve Regeneration

After peripheral nerve injury, the proximal nerve stump will spontaneouslyregenerate due to the activation of intrinsic growth capacity of neurons and a favorableenvironment produced by Schwann cells. The sciatic nerve, comprising a mixedpopulation of motor and sensory axons, is a commonly used model in nerveregeneration studies. The sensory neurons extending into the sciatic nerve are located inthe L4-L6dorsal root ganglia (DRGs). The activation of transcription factors and themodulation of regeneration-associated genes responsible for peripheral neuronalsurvival and regrowth are still to be explored.Non-coding RNAs (ncRNAs) are featured by lack of protein-coding sequence, yetcan participate in many processes that coordinate gene expression particularly duringdevelopment. The ncRNAs can be grouped into two major classes: the small ncRNAs,and long noncoding RNAs (lncRNAs). microRNAs (miRNAs), a class of approximately22nucleotide non-coding RNA molecules, negatively regulate the expression of a widevariety of genes mainly through a direct interaction with the3‘-untranslated regions(3‘-UTRs) of their corresponding mRNA targets. The number of putative mRNA targetsthat can be regulated by miRNAs at the post-transcriptional level is estimated to exceed60%of total human genes, suggesting the pivotal roles of miRNAs in a diverse array ofphysiological and pathological processes. lncRNAs, containing nucleotides rangingfrom200to more than100,000. Although lncRNAs are once thought to be the darkmatter‘of the genome, they couple bioenergetic properties with information storage andprocessing and orchestrate a wide array of biological processes. For example, they areknown to be able to serve as mediators of mRNA decay, as structural scaffolds fornuclear substructures, as host genes for miRNAs, and as regulators of chromatinremodeling. The functions of the vast majority of lncRNAs are not clear. In our work,we want to screen out some key miRNAs and lncRNAs by microarray, which could trigger the intrinsic growth capacity of neurons.Part I miR-21and miR-222inhibit apoptosis of adult dorsal root ganglionneurons by repressing TIMP3following sciatic nerve injuryObjectives:To search for the miRNA that regulate the apopotosis of DRG, and explore itsaction mechanism.Methods:1. Using microarray analysis, we investigated the time-dependent expressionpatterns of microRNAs in the L4-L6DRGs following rat sciatic nerve injury.2. Searching for the putative targets of the differentially expressed miRNAs usingTargetScan and miRanda database. In order to know credible biological functions, weconducted the Gene Ontology (GO) analyses regarding the intersected genes.3. Verifing the expression of the differentially expressed miRNAs by qRT-PCR.4. Overexpressing the selected miRNAs in the cultured DRG neurons, and thenanalyzing the roles of miR-21mimic (miR-21) and miR-222mimic (miR-222) on theapopotosis of DRG neurons by PE-conjugated annexin V and7-aminoactinomycin D(7-AAD) kit.5. Overexpressing miR-21and miR-222in the cultured DRG neurons, and thenanalyzing their roles on the survival of DRG neurons by CCK-8kit.6. Overexpressing miR-21and miR-222in the cultured DRG neurons, and thenanalyzing their roles on the activation of Caspase-3by Western blot.7. Integrating miRNA targets with differentially expressed mRNAs to obtainpotential targets, and finding that TIMP3is a potential target of miR-222.8. Overexpressing miR-21and miR-222in the cultured DRG neurons, andconfirming the change of TIMP3mRNA and protein expression by qRT-PCR andWestern blot.9. Searching for the upstream molecules regulating the expression of miR-21.We treated DRG neurons with IL-6, and then determined the miR-21and TIMP3expression levels by qRT-PCR. Results:1. In the L4-L6DRGs, we found that13known miRNAs were differentiallyexpressed at0,1,4,7d post injury.2. Constructing the network between13miRNAs and45mRNA targets.3. qRT-PCR demonstrated the up-regulation of miR-21and miR-222.4. FCM-based assay showed that miR-21and miR-222inhibit apoptosis of adultdorsal root ganglion neurons.5. Cell viability assays showed that overexpressing miR-21and miR-222enhanced the survival of DRG neurons.6. Western blot showed that overexpressing miR-21and miR-222inhibited theactivation of Caspase-3in DRG neurons.7. qRT-PCR and Western blot showed that overexpressing miR-21and miR-222inhibited the expresssion of TIMP3mRNA and protein.Conclusions:On the basis of microarray profiling and functional analysis, we showed thatmiR-21and miR-222inhibited cell apoptosis and enhanced cell viability in culturedDRG neurons. TIMP3, a pro-apoptotic protein in various cancer cells, was identified asa common target of miR-21and miR-222. Additionally, IL-6could induce up-regulationof miR-21expression. Our systematic research confirmed the role of miRNAs inregulating peripheral nerve injury and regeneration, thus offering a new approach toperipheral nerve repair. Part II miRNA-222targeting PTEN promotes neurite outgrowth from DRGneurons following sciatic nerve transectionObjectives:To search for the miRNA that regulate the neurite outgrowth from DRG, andexplore its action mechanism.Methods:1. Using microarray analysis, we investigated the time-dependent expressionpatterns of microRNAs in the L4-L6DRGs following rat sciatic nerve injury. 2. Searching for the putative targets of the differentially expressed miRNAs usingTargetScan and miRanda database. In order to know credible biological functions, weconducted the Gene Ontology (GO) analyses regarding the intersected genes.3. Verifing the expression of the differentially expressed miRNAs by qRT-PCRand in situ hybridization.4. Overexpressing the selected miRNAs in the cultured DRG neurons, and thenanalyzing the roles of miR-222mimic (miR-222), or miR-222inhibitor (anti-miR-222)on the neurite outgrowth of DRG neurons by the analysis of neurite length.5. Integrating miRNA targets with differentially expressed mRNAs to obtainpotential targets, and finding that PTEN is a potential target of miR-222.6. Overexpressing miR-222, or anti-miR-222in the cultured DRG neurons, andconfirming the change of PTEN mRNA and protein expression by qRT-PCR andWestern blot.7. To explore the function of PTEN, two specific small interfering RNAs (siRNAs)against PTEN were synthesized.8. To test this assumption that PTEN was a direct target of miR-222, weperformed transfection of miR-222inhibitor into DRG neurons in the presence orabsence of siRNA-2against PTEN.9. Searching for the putative targets interactive PTEN with using Co-IP.10. Searching for the upstream molecules regulating the expression of miR-222.We treated DRG neurons with anisomycin, an antibiotic routinely used to activatejun-N-terminal kinase that is responsible for c-Jun activation, and then determined themiR-222and PTEN expression levels by qRT-PCR or Western blot.11. Investigating the coordination control of diffenrent pathways on the neuriteoutgrowth of DRG neurons. down-regulating PTEN by either miR-222or siRNA-2against PTEN for12h plus the ensuing20min stimulation of dibutyryl-cAMP(Db-cAMP), and then analyzing the neurite outgrowth from DRG neurons.Results:1. In the L4-L6DRGs, we found that26known miRNAs were differentiallyexpressed at0,1,4,7,14d post injury.2. Bioinformatics show that the638potential targets of the miRNAs wereinvolved in nerve regeneration.3. qRT-PCR and in situ hybridization experiments demonstrated the up-regulation of miR-222.4. Identifing that miR-222promotes neurite outgrowth from DRG neuron bytargeting PTEN.5. miR-222might regulate the phosphorylation of cAMP response elementbinding protein (CREB) through PTEN, and c-Jun activation might enhance themiR-222expression.6. Knockdown of PTEN by either miR-222or siRNA-2against PTEN plus thestimulation of Db-cAMP promoted neurite outgrowth from DRG neurons as comparedto the above-mentioned12-h knockdown of PTEN or the stimulation of Db-cAMPalone.Conclusions:On the basis of microarray profiling and functional analysis, we showed thatmiR-222, as one of the26differentiated miRNAs in DRG neurons after nerve injury,could regulate neurite growth by targeting PTEN, a major inhibitor of nerveregeneration. We also noted that knockdown of PTEN by miR-222might be synergisticwith cAMP/CREB-based signaling to improve the regenerative ability of neurons, atleast in vitro, thus offering a new approach to peripheral nerve repair. Part III LncRNA BC089918promotes neurite outgrowth from DRG neuronsObjectives:To search for the lncRNA that regulate the neurite outgrowth from DRG neurons,and explore its action mechanism.Methods:1. Using Arraystar4×44K Rat LncRNA, we investigated time-dependentexpression patterns of lncRNAs and mRNAs in the L4-L6DRGs following rat sciaticnerve injury.2. Searching for the putative targets of the differentially expressed lncRNAs usingcoexpression network. In order to know credible biological functions, we conducted thegene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses regarding the correlated targets.3. Confirming the expression of differentially expressed lncRNAs by qRT-PCRand in situ hybridization.4. Overexpressing siRNAs of the selected lncRNA in the cultured DRG neurons,and then analyzing the roles of lncRNA on the neurite outgrowth of DRG neurons bythe analysis of neurite length.Results:1. In the L4-L6DRGs, we found that105lncRNAs were differentially expressedat0,1,4,7d post injury.2. Bioinformatic analyses show that the115correlated targets of24differentiallyexpressed lncRNAs were involved in nerve regeneration.3. qRT-PCR and in situ hybridization experiments demonstrated thedown-regulation of lncRNA BC089918.4. Promotion of neurite outgrowth of DRG neurons by lncRNA BC089918silencing.Conclusions:On the basis of microarray profiling and functional analysis, we showed thatlncRNA BC089918, as one of the105differentiated lncRNAs in DRG neurons afternerve injury, could regulate neurite growth. Our systematic research confirmed the roleof lncRNAs in regulating peripheral nerve injury and regeneration, thus offering a newapproach to peripheral nerve repair.