Research Of MiR-505-3p With Regulating Axonal Development Of Neuron And Tumorous Proliferation

Micro RNAs are small non-coding RNAs of 18-23 nt in length with the function of both m RNA degradation and translational repression.In this research,we focused on exploring multiple biological functions of the vital mi R-505-3p in participating axonal development and tumorous proliferation,indicating mi R-505-3p regulates complex biological events via targeting on various genes.Previously,mi R-505-3p has been identified as an indicator of tumor genesis and development.It is supposed as one of potential early diagnostic markers for cervical carcinogenesis,biomarker for local recurrence or distant metastasis of synovial sarcoma,and prognostic biomarker for breast cancer.Besides,mi R-505-3p is proved to be a tumor repressor.It inhibited cell proliferation by inducing apoptosis in a drug-resistant breast cancer cell line MCF7-ADR.It regulates oncogene PSMD10 functional variant to reduce gastric cancer risk.It partially affects senescence and apoptosis by targeting SRSF1 under control of leukemia/lymphoma related factor LRF in MEF cell line.However,almost all of these studies focus mi R-505-3p on tumor related physiological events,whether mi R-505-3p mediate other physiological effect is still elusive.Neurons are binary cells with structural and functional differences between the somato-dendritic compartment and the axon.The development of axon,including establishment of asymmetry,axonal growth and axonal branching,is a pivotal step for neuron to develop and form neural circuitry in the brain.Dissociated cortical neurons extend several neurites which are still indiscriminate before growing asymmetrically,then a single neurite experiences rapid outgrowth and becomes the axon.During axon development,one of several nascent neurites undergoes drastic changes in cytoskeletal structure,membrane composition,protein turnover and energy consumption.Disturbance of any of these processes is proved to cause neurodevelopmental disorders such as autism spectrum disorders.Recent studies have shown mi RNAs to be involved in C.elegans axon specification,X.laevis retinal axon pathfinding,and mouse early neurites extension during development.However,the detailed mechanism of mi RNAs in regulating axon development is unclear.In this research,we found that mi R-505-3p functions as a novel negative regulator of autophagy,and as a novel positive regulator of axonal development in specifically facilitating neural polarity establishment,axonal extension and branching in vitro and in vivo.First,we applied dissected cultured mouse cortical neuron model to determine whether mi R-505 is involved in neuron morphological development.We found that only mi R-505-3p,rather than mi R-505-5p affected neural development.We observed that mi R-505-3p specifically promoted axonal development but had no effects on dendritic development.The enhancement of axonal development included facilitating neuron polarity establishment,axonal extension and branching.Second,we utilized in utero electroporation assay to test whether mi R-505-3p promoted axonal development in vivo.We found that mi R-505-3p increase axonal length of projecting cortical neuron in P3 mice,and increase axonal branching both in ipsilateral and contralateral side in P8 mice.Third,we designed and generated mi R-505-3p-specific deletion gene engineering mice with applying CRISPR/Cas9 system.We predicted and tested off-target effects of this line and confirmed that it was with no off-target effect.We used hematoxylin-eosin staining and toluidine blue staining and observed that axonal development were retarded in mi R-505 knockout mice.We further confirmed this alteration by utilizing immunofluorescence staining.Thus,we showed mi R-505-3p was required in axonal development both in vitro and in vivo.Forth,we applied a bio-information screen to predict candidate target genes of mi R-505-3p.Together with mi RNA targets prediction database and targets prediction based on RNA sequencing,we generated a candidate target genes list.We utilized dual-luciferase assay to validate these candidates.Among them,Atg12 showed a most obvious decrease in normalized dual-luciferase value under inhibition of mi R-505-3p.Similarly,Atg12 decreased the most of the m RNA by testing with q PCR assays.We further confirmed this candidate target gene with western blot assay.Thus,Atg12 was selected as a direct target gene of mi R-505-3p in mouse cortical neuron.Fifth,to investigate whether Atg12 is involved in axonal development we over-expressed Atg12 with methods of both lipofection and electroporation in cultured cortical mouse neurons.We observed that Atg12 inhibited neural polarity established,axonal extension and axonal branching,which were rescued by over-expression of mi R-505-3p.We also found that Atg12 exhibited the same effects in vivo.Thus,Atg12 was considered to be required in axonal development.Sixth,we applied transmission electron microscope strategy to monitor autophagosome of cultured cortical neuron or cortical plate.We found that autophagy pathway was activated in mi R-505 KO mouse.Both size and number of autophagosome were increased.We confirmed these observations by western blot.Thus,we provide evidences to show that deletion of mi R-505-3p resulted in maintaining of Atg12 and activation of autophagy pathway.Seventh,we utilized Rapamycin to activate autophagy pathway and Chloroquine to block autophagy pathway,to mimic the effects caused by over-expression of Atg12,and to further illustrate mi R-505-3p function in modulating autophagy pathway.We found that over-expression of Atg12 resulted in acceleration of autophagy pathway,increase of autophagosome size and number and decrease of mitochondria number.Thus,we believed that mi R-505-3p generally regulated Atg12 and then inhibited autophagy pathway.During this part of research,we also optimized and published methods to trace autophagasome based on GFP-LC3?labeling.Last,we observed that mitochondria number and size were decreased in cultured neurons,cortices and MEF cells with down-regulation of mi R-505-3p,showing a negative correlation with that of autophagosome.Thus,we showed mi R-505-3p targets on Atg12,inhibiting autophagy,promoting mitochondria accumulation and axonal development.On the other hand,serine and arginine rich splicing factor 1(SRSF1),also known as alternative splicing factor/splicing factor 2(ASF/SF2),is a RNA-binding protein with functions in several aspects of RNA metabolism.It is initially found responsible for constitutive splicing and alternative splicing.Although it is originally defined as alternative splicing factor,SRSF1 has been reported with functions in modulating m RNA transcription,nonsense-mediated m RNA decay(NMD)and protein sumoylation.SRSF1 has also been proved to be an onco-protein.Its over-expression drives malignant proliferation,which make SRSF1 to be an attractive therapeutic target.Recently,SRSF1 is reported been precisely controlled to maintain normal cell physiology by either mi RNA depended negative feedback loop or auto-regulation through RNA recognition motif 2 domain(RRM2)binding to 3'UTR of Srsf1 m RNA.However,whether RNA interference and SRSF1 auto-regulation influence each other to co-modulate tumorous proliferation is unknown.In this research,we also found that the concentration of serum in micro-environment influenced mi R-505-3p targeting on Srsf1.Indeed,it was because that the normal concentration of serum induced high expression of SRSF1 protein,which binds to Srsf1 transcript to form steric hindrance.Thus,the mi RNA targeting was interfered,leading restrain of mi R-505-3p from targeting on Srsf1 and Srsf1 driven tumorous proliferation in serum rich condition.First,to verify that mi R-505-3p target on Srsf1,we applied bioinformatics prediction on all the candidate target sites.Supplemented with dual-luciferase assays and western blot assays,I found the real target site on Srsf1 transcript,which was extremely conserved in sequence.Second,to examine whether mi R-505-3p inhibits tumorous proliferation via targeting on Srsf1,we used immunofluorescent staining on ki67 to label positive proliferating cells.I found mi R-505-3p was unable to inhibit tumorous proliferation in serum-rich condition whereas was able to inhibit tumorous proliferation in serum-reduced condition.Third,we set up a gradient serum concentration to observe the impact of serum-rich condition on SRSF1 protein and proliferation.After immunofluorescent staining on ki67 and SRSF1,we found that both expression level of ki67 and SRSF1 were positively correlated to serum concentration.Thus,we inferred the highly expressed SRSF1 protein influenced mi R-505-3p targeting on Srsf1 m RNA in serum-rich condition.Forth,we over-expressed exogenous SRSF1 protein to mimic SRSF1 up-regulation after serum-rich induction.We co-transfected mi R-505-3p with various truncated 3'UTR regions of Srsf1 and exogenous SRSF1 protein,by using dual-luciferase assay,we found the over-expressed exogenous SRSF1 was sufficient to restrain mi R-505-3p targeting on Srsf1.Fifth,we found a SRSF1 protein self-binding motif proximal to mi R-505-3p target site.By co-transfecting mi R-505-3p with various truncated 3'UTR regions of Srsf1 and exogenous SRSF1 protein,we confirmed that it was a real SRSF1 self-binding site.Sixth,we came up with a novel mechanism diagram to show that the target protein of mi R-505-3p,SRSF1,influences tumorous proliferation via a protein-m RNA auto-regulation,interfering mi RNA-m RNA binding.Taken together,the novel functions of mi R-505-3p in neuron largely expand our knowledge of single mi RNA controlling neural development,further highlighting the importance of autophagy in modulating development of central neural system.We showed mi R-505-3p involves in a complex regulation on Srsf1,further illustrating that the tumorous proliferation is resulted from interaction of gene background and micro-environment.It provides new clue for anticancer drugs designing and therapic strategy application.