The MicroRNA200b/a-TET3-REST Axis Is Required For Olfactory Globose Basal Cell Proliferation And Differentiation And Olfactory Behaviors

Smell plays an important role in the life activities of animals.As an important part of the mammalian olfactory system,the role of the main olfactory epithelium?MOE?is to sense external odor molecules.The MOE is an active organization and has the ability to regenerate throughout an organism's lifespan.Because the MOE is exposed to the nasal cavity and is in direct contact with the external environment,epigenetic factors,including micro RNAs?miRNAs?,have important regulatory effects on the differentiation of OSNs in the MOE.Therefore,the MOE is a good peripheral organ for studying the relationship between the epigenetic regulation and the neural differentiation,but its mechanism is still unclear.To explore the correlation between olfactory function and the epigenetic factors,especially miRNA expression,the small RNAs sequencing was preformed from the MOE tissues dissected from adult male adenylate cyclase 3 knockout AC3^-/-mice and wild-type(AC3^+/+)mice.The result from small RNA sequencing showed that the expression of miR-200b/a in the MOE of AC3^-/-mice was significantly down-regulated,and quantitative PCR?q PCR?analysis shown the expression of miR-200b/a in the the MOE of AC3^-/-mice was significantly down-regulated and in the MOE of AC3 knockin mice was significantly up-regulated,indicating that AC3 regulates the miR-200b/a expression.For analysis of their physiologically significant roles,miR-200b and miR-200a were simultaneously knocked down in the MOE of adult mice by a combination of CRISPR-Cas9and intranasal delivery of adeno-associated virus?AAV?.The experiments of behaviors,q PCR,Western Blot,and immunofluorescent staining were used to investigate the olfactory behavior and neural differentiation of OSNs in the MOE of the miR-200b/a knockdown mice?miR-200b/a KD mice?.The results showed that,compared with their control mice,the miR-200b/a KD mice showed severe defects in olfactory detection ability,male-male aggressive and male-female mating behaviors;Cas9 infecting Mash1-marked GBCs after AAV perfusion,enhanced proliferation capacity,prolonged S-phase of the cell cycle,decreased ability to exit the cell cycle of Mash1-marked globose basal cells?GBCs?in the MOE.Another,diminished ability to differentiate into olfactory neurons for GBCs led to a reduction in the number of olfactory neurons,accompanied by a large number of apoptosis in the MOE of the miR-200b/a KD mice;however,the number of horizontal basal cells,Sox2-marked GBCs,and sustentacular cells remained unchanged.In order to find the specific molecular mechanism of miR-200b/a involved in olfactory-related behaviors and neural differentiation in the MOE,the transcriptome sequencing analysis of the MOE tissue of miR-200b/a KD mice and its control mice was performed.The expression of almost all olfactory receptor in the MOE of the miR-200b/a KD mice was significantly down-regulated,which is consistent with the transcriptome sequencing of MOE tissue in DNA demethylase TET3 transgenic mice,and TET3 participates in neural differentiation process.Therefore,it is speculated that miR-200b/a may regulate the olfactory behavior and neurogenesis of the MOE in mice through the TET3.We proved that TET3 is a target gene of miR-200b/a and its expression was regulated by miR-200b/a through bioinformatics,the experiments of luciferin reporter and the experiments of the mimic and inhibitor in 3T3-L1 cell and the MOE tissues.The recovery experiment,that is,the AAV nasal perfusion method,was used to construct miR-200b/a+TET3 double knockdown mice?miR-200b/a+TET3 DKD mice?.The level of TET3 in the MOE of the miR-200b/a+TET3DKD mice was restored the level of the negative control?NC?mice.The olfactory behaviors and neural differentiation of the MOE were investigated.The results showed that the recovery of TET3 expression in the MOE could partially restore the olfactory-related behavioral defects and the neural differentiation defects caused by miR-200b/a knockdown,indicating that TET3 participates in olfactory-related behaviors and neurogenesis in the MOE.Previous studies have shown that TET3 forms a protein–protein complex with the RE1-silencing transcription factor?REST?to cooperatively function in the mouse retina and hypothalamus.We speculate that the interaction of TET3 and REST regulates the proliferation and differentiation of GBCs in the MOE.To test this postulate,we demonstrated that TET3interacts with the REST in mouse MOE by immunofluorescence and co-immunoprecipitation.Subsequently,we also used AAV nasal perfusion to construct miR-200b/a+REST double knockdown mice?miR-200b/a+REST DKD mice?to restore the expression of REST in the MOE of the miR-200b/a KD mice.The olfactory behavior and the MOE neural differentiation in the miR-200b/a+REST DKD mice were investigated,and the results showed that the restoration of REST expression in the MOE could restore olfactory-related behavioral defects and neural differentiation defects caused by miR-200b/a knockdown,indicating that REST is involved in olfactory-related behaviors in mice and neurogenesis in the MOE.In conclusion,our study demonstrated that a miR-200b/a/TET3/REST regulatory axis at the right time and stage of development is critical for a balance between proliferation and neuronal differentiation of GBCs in the MOE,as well as olfactory-mediated behaviors.Because miR-200b/a,TET3 and REST are abundantly expressed in many other tissues,the mechanical insights made in this study should have broad implications beyond MOE tissue.