LncRNA MALAT1 Promotes The Proliferation And Migration Of Schwann Cells By Elevating P2Y4R Through Sponging Mir-539-5p On Facial Nerve Regeneration

Facial nerve dysfunction seriously affects patients’ facial movement,sensation,mental health and social interaction.Even though the micro-nerve repair technology is relatively mature,there are still a considerable number of patients with unsatisfactory recovery of facial nerve function.Therefore,further studies on the mechanism of facial nerve injury repair are essential to improve the efficacy of nerve repair.In this thesis,supported by the National Natural Science Foundation of China(NSFC),we characterized the lncRNA Metastasis associated lung adenocarcinoma transcript 1(MALAT1)promotes P2Y4-receptor(P2Y4R)-mediated Schwann cells(SCs)migration and myelin sheath remodeling by regulating miR-539-5p,which provides a new candidate target for the clinical treatment of PNI in the future.Part Ⅰ Expression of lncRNA MALAT1,miR-539-5p and P2Y4R in facial nerve regeneration after injury in wild miceObjective:To investigate the upstream regulatory pathway of P2Y4R and the expression of P2Y4R,lncRNA MALAT1 and miR-539-5p in Facial nerve regeneration after Facial nerve injury(FNI)by high-throughput sequencing and bioinformatics.Methods:The functional recovery of each group was evaluated by facial nerve function score and electromyography.Transmission electron microscopy and toluidine blue staining were used to observe the structure of axons and myelin sheath,and the G-Ratio was calculated to evaluate the thickness of nerve myelin sheath.SCs specific marker S-100β/Fluorescence in situ hybridization,The localization and expression changes of lncRNA MALAT1,miR-539-5p and P2Y4R in FNI and regeneration were evaluated by FISH,FISH+immunofluorescence co-localization and fluorescence semi-quantitative analysis.The facial muscle MASSON staining was used to evaluate the morphological and structural changes of the target organs during facial nerve regeneration.High-throughput sequencing analysis was performed on the distal end of facial nerve crush at 0 and 14 days after injury to screen out the differentially expressed genes.P2Y4R regulated lncRNAs and endogenous competitive binding miRNAs were identified by constructing co-expression network and ceRNA interaction network.Starbase and Targetscan databases were used to predict the binding sites between them.Dual luciferase reporter gene was used to confirm the reliability of binding sites.Four groups of facial nerve regeneration models with different regeneration time(7d,14d,21d,28d)were established,and the normal control side was set up.Results:According to the facial nerve function score and the maximum amplitude of electromyography,it was found that the scores of the 7d,14d,21 d and 28d groups were gradually increased,and these four groups were smaller than the normal control side.However,the latency of 7d,14d,21d and 28d groups gradually became shorter,and these four groups were longer than the normal control side,and the differences were statistically significant(P<0.05).Toluidine blue staining showed that the deep blue myelin sheath gradually became dense with the time of regeneration after facial nerve injury.Transmission electron microscopy showed that the number of myelinated nerves in the 7d,14d,21 d and 28d groups gradually increased,but it was less than that in the normal control side.The G-Ratio values of the 7d,14d,21 d and 28d groups gradually decreased,and the G-Ratio values of the four groups were larger than those of the normal control side,and the differences were statistically significant(P<0.05).Masson staining showed that the number of muscle fibers per unit area and the diameter of muscle fibers in the four groups were smaller than those in the normal control side group,especially in the 14d group,and the differences were statistically significant(P<0.05).P2Y4R,lncRNA Malatl and miRNA-539-p were found to be located in SCs by Fish co-labeling.Fluorescence semi-quantitative analysis showed that the expression of P2Y4R and lncRNA Malatl increased sharply at the early stage of regeneration after FNI,reached the peak at 14 days,and gradually decreased at 21 days.However,the trend of miRNA-539-p expression was opposite.The differentially expressed mRNA,lncRNA and miRNA in the distal end of facial nerve crush were screened at 0 and 14 days after injury.There were 2119 up-regulated mRNAs,and the trend was consistent with the proliferation trend of SCs in regeneration after PNI.The upregulated P2Y4R gene was selected based on previous studies.Bioinformatics analysis showed that there were binding sites among differentially expressed lncRNA MALAT1,miR-539-5p and P2Y4R.Dual luciferase reporter gene showed that transfection with miR-539-5p mimic reduced the activities of wild-type P2Y4R 3’UTR and wild-type MALAT1 3’UTR,respectively,and the differences were statistically significant(P<0.05).Conclusion:In this experiment,the FNI model still did not recover completely after the completion of regeneration,indicating that once the facial nerve injury reaches the third degree,although there is a certain degree of self-regeneration,it cannot recover to normal level.Our group has previously confirmed that P2Y4R regulates SCs migration and promotes myelination in FNI regeneration,so the differential P2Y4R gene was selected as the study gene.lncRNA MALAT1 and miR-539-5p may be the regulatory genes upstream of P2Y4R,and the binding sites are reliable.lncRNA MALAT1 may mediate the increase of P2Y4R expression by binding to miR-539-5p,thereby participating in the process of facial nerve regeneration.Part Ⅱ Effects of lncRNA MALAT1、miRNA-539-5p 和 P2Y4R on the proliferation and migration of mouse primary SCs and its mechanismObjective:In vitro experiments confirmed the effects of lncRNA MALAT1,miRNA-539-5p and P2Y4R on the proliferation and migration of primary mouse SCs,and clarified that their effects were mediated by lncRNA MALAT1 to regulate the proliferation and migration of SCs via miR-539-5p/P2Y4R.Methods:Primary SCs were isolated from the Dorsalrootganglion(DRG)of neonatal mice and cultured.FISH and immunofluorescence were used to localize the location of lncRNA MALAT1,miR-539-5p and P2Y4R on primary SCs.Primary SCs were transfected with ①lncRNA MALAT1,②miR-539-5p and③P2Y4R overexpression and silencing lentivirus,respectively.Autofluorescence and RT-qPCR were used to evaluate the transfection efficiency of OE-target genome(overexpressed lentivirus),sh-target genome(silenced lentivirus),OE-NC group(empty lentivirus)and sh-NC group(inserted an unrelated sequence)into primary SCs.RT-qPCR was used to detect the expression levels of lncRNA MALAT1,miR-539-5p and P2Y4R in primary SCs.The expression of P2Y4R was detected by Western Blot.EdU and CCK8 were used to detect the proliferation of SCs.Cell scratch assay and Transwell assay were used to detect the migration ability of SCs.RNA pull down technique was used to verify the targeting binding between lncRNA MALAT1 and miR-539-5p,P2Y4R and miR-539-5p.Results:SCs were successfully isolated from the DRG of neonatal mice,and 95.70%of SCs were confirmed by S100β.P2Y4R,lncRNA MALAT1 and miR-539-5p were localized in primary SCs.The primary SCs were successfully transfected with lentivirus in all groups.①Overexpression/silencing of lncRNA MALAT1:In OE-MALAT1 group,the expression of P2Y4R was higher than,the expression of miR-539-5p was lower than,the cell proliferation was faster,the cell migration distance was longer,and the number of migrating cells was more than that in sh-MALAT1,OE-NC and sh-NC group(P<0.05).In sh-MALAT1 group,the expression of P2Y4R,the cell proliferation,cell migration distance and the number of migrating cells in OE-NC group were lower was lower than,while the expression of miR-539-5p was higher than that in OE-NC and sh-NC groups(P<0.05).② Overexpression/silencing of miR-539-5p:The sh-miRNA539-5p group had higher expression of P2Y4R faster cell proliferation,longer cell migration distance,and more migrated cells than OE-miRNA539-5p,OE-NC,and sh-NC groups.However,the expression of P2Y4R in OE-miRNA539-5p group was lower than that in OE-NC and sh-NC groups,the cell proliferation was slower,the cell migration distance was smaller,and the number of migrating cells was less than that in OE-NC and sh-NC groups(P<0.05).③Overexpression/silencing of P2Y4R:The proliferation of OE-P2Y4R group was faster and the number of migrating cells was more than that of sh-P2Y4R group,OE-NC group and sh-NC group,while the proliferation of sh-P2Y4R group was slower and the number of migrating cells was less than that of OE-NC group and sh-NC group,and the differences were statistically significant(P<0.05).RNA pull-down showed that P2Y4R significantly downregulated miR-539-5p,and lncRNA MALAT1 also significantly downregulated miR-539-5p,and the differences were statistically significant(P<0.05).Conclusions:lncRNA MALAT1 promotes the proliferation and migration of SCs by binding to miR-539-5p to mediate the increased expression of P2Y4R.PartⅢ lncRNA MALAT1 promotes facial nerve regeneration in mice and its mechanismObjective:In vivo experiments further confirmed that lncRNA MALAT1 mediated the increase of P2Y4R expression by binding to miR-539-5p,thereby promoting the proliferation and migration of SCs,thereby promoting facial nerve regeneration and target organ function recovery.Methods:The 28-day OE-MALAT1 group(lncRNA MALAT1 overexpression lentivirus),sh-MALAT1 group(lncRNA MALAT1 silencing lentivirus),Control group(empty lentivirus)and NC group(normal saline)were established FNI regeneration models,and the normal control side was set up.Facial nerve score and electromyography were used to evaluate the degree of muscle function recovery.Transmission electron microscopy and toluidine blue staining were used to observe the structure of regenerated axons and myelin sheathe,and the G-Ratio was calculated-FISH+immunofluorescence co-localization and fluorescence semi-quantitative were used to evaluate the localization and expression changes of lncRNA MALAT1,miR-539-5p and P2Y4R in the distal nerve of the injured side in each group.The morphological and structural changes of target organs during nerve injury and regeneration were evaluated by facial muscle MASSON staining.Results:At 28 days after surgery,the facial nerve function score and maximum amplitude of electromyography showed that OE-MALAT1 group was greater than sh-MALAT1,Control and NC groups.The sh-MALAT1 group was significantly lower than the Control and NC groups(P<0.05).Electromyography showed that the latency of OE-MALAT1 group was shorter than that of Control,NC and sh-MALAT1 groups.The latency of sh-MALAT1 group was longer than that of Control group and NC group,and the differences were statistically significant(P<0.05).However,there was no significant difference in facial nerve function score,maximum amplitude and latency between OE-MALAT1 group and normal control group(P>0.05).Toluidine blue staining showed that the myelin sheath with dark blue staining in the overexpression group was dense,while that in the silencing group was loose.Transmission electron microscopy showed that the G-Ratio value of OE-MALAT1 group was lower than that of Control,NC and sh-MALAT1 groups.The G-Ratio value of sh-MALAT1 group was higher than that of Control group and NC group,and the differences were statistically significant(P<0.05).However,there was no significant difference in G-Ratio between OE-MALAT1 group and normal control group(P>0.05).The number and diameter of muscle fibers per unit area in OE-MALAT1 group were significantly higher than those in Control,NC and sh-MALAT1 groups.Compared with the Control group and NC group,the sh-MALAT1 group significantly decreased(P<0.05).There was no significant difference in the number and diameter of muscle fibers per unit area between the OE-MALAT1 group and the normal control group(P>0.05).P2Y4R,lncRNA Malatl and miRNA-539-p were co-localized in SCs by FISH and immunofluorescence.The expression levels of P2Y4R and lncRNA Malatl in OE-MALAT1 group were significantly higher than those in Control,NC and sh-MALAT1 groups,while the expression levels of P2Y4R and lncrna MALAT1 in sh-MALAT1 group were significantly lower than those in Control and NC groups(P<0.05).The expression of miRNA-539-p in OE-MALAT1 group was lower than that in Control,NC and sh-MALAT1 groups,while the expression of miRNA-539-p in sh-MALAT1 group was higher than that in Control and NC groups,and the differences were statistically significant(P<0.05).Conclusions:During the regeneration of FNI,up-regulation of lncRNA MALAT1 inhibits the expression of miR-539-5p in the distal end of the injured nerve,thereby mediating the enhanced expression of P2Y4R,thereby promoting the proliferation and migration of SCs,myelination,nerve fiber regeneration,and muscle fiber structure recovery.