Study On The Mechanism Of Long Non-coding RNA ENST00000444488.1 Regulating The Migration Of Vascular Smooth Muscle Cells

Background and Objective:Although long noncoding RNAs(lncRNAs)are reported in a wide range of physiological functions,their roles and underly mechanism in the pathogenesis of atherosclerosis(AS)and coronary artery disease(CAD)are poorly known.In our previous research,we analyzed the expression profiles of lncRNAs and mRNAs in the samples of the peripheral blood mononuclear cells(PBMCs)of 93 CAD and 48 healthy controls matched by region,age,and gender,and replicated the expression spectrum of IncRNA in another larger cases and controls,and finally,we identified differentially expressed IncRNAs.ENST00000444488.1 is one of the differentially expressed lncRNAs in CAD patients,and it can be used as a new biomarker for the diagnosis of CAD.But the role of ENST00000444488.1 in the pathogenesis of AS and CAD remains unclear.In this study,we aimed to explore the role and molecular mechanism of ENST00000444488.1 in AS.Methods:5' and 3'-rapid amplification of cDNA ends(RACE)assay and subsequent Sanger sequencing were performed to obtain the full-length sequence of ENST00000444488.1 transcript,and the size of ENST00000444488.1 transcript in vascular smooth muscle cells(VSMCs)and human umbical vein endothelial cells(HUVECs)was determined by Northern blot;RNA Fluorescence In Situ Hybridization(FISH)was used to detect the subcellular localization of ENST00000444488.1 in VSMCs;PhyloCSF was used to analyzed the conservatism of ENST00000444488.1 among species;BLAST software was used to predict the protein encoding ability,and transcription and translation experiments in vitro was used to prove the protein coding ability of ENST00000444488.1;Loss-of-function and gain-of-function strategies were used to explore the effects of ENST00000444488.1 on migration and proliferation of VSMCs through transwell and CCK-8 assays,respectively;RNA pull down,mass spectrometry analysis and Western blot were used to screen and identify the proteins interacting with ENST00000444488.1,and furthermore,RNA RIP was used to validate the interaction between ENST00000444488.1 and its binding protein in VSMCs.Immunofluorescence was used to detect the subcellular co-localization of ENST00000444488.1 and MYH9 protein;F-actin fluorescence staining was used to explore the effect of ENST00000444488.1 binding to MYH9 protein on F-actin rearrangement;CRISPR/Cas9 technique was used to construct the smooth muscle cell-specific ENST00000444488.1 transgenic mice.Results:Using known sequence of ENST00000444488.1,we designed primers and performed 5' and 3'-RACE to determine the full length of ENST00000444488.1.The 5' and 3'-RACE and subsequent Sanger sequencing showed that the full length of ENST00000444488.1 was 1279nt.Northern blot analysis further confirmed the size of the ENST00000444488.1 transcript in VSMCs and without other transcript isoforms.Because IncRNA function is dependent on subcellular localization,we performed RNA FISH assay to determine the subcellular localization of ENST00000444488.1,and endogenous ENST00000444488.1 localized in both the cytoplasm and the nuclear of VSMCs,but mainly in cytoplasmic and peri-nuclear regions.PhyloCSF analysis showed that ENST00000444488.1 with low conservatism among more than 100 vertebrate species,and BLAST software analysis predicted that ENST00000444488.1 had no ability to encode proteins.Transcription and translation experiments in vitro showed that no protein products were encoded.Thus,ENST00000444488.1 was indeed a long noncoding RNA.Endogenous ENST00000444488.1 mRNA was successfully knocked down to 20%in VSMCs by siRNA against ENST00000444488.1,and ENST00000444488.1 was efficiently over expressed in VSMCs infected with Ad-ENST00000444488.1.CCK-8 proliferation experiment showed the knockdown or overexpression of ENST00000444488.1 did not significantly affect VSMCs proliferation.Transwell migration assay showed that the knockdown of ENST00000444488.1 significantly promoted VSMCs migration,while the overexpression of ENST00000444488.1 had an opposite effect.In response to PDGFBB,a growth factor known to promote VSMC phenotype switching,ENST00000444488.1 expression was significantly down regulated in VSMCs.Furthermore,the overexpression of ENST00000444488.1 also significantly inhibited VSMC migration induced by PDGF-BB,suggesting that ENST00000444488.1 participated in VSMCs phenotype transition induced by PDGFBB.To further unravel the underlying mechanism by which ENST00000444488.1 inhibited VSMCs migration,we performed RNA pull-down assay with protein lysate from VSMCs followed by LC-MS analysis to identify ENST00000444488.1-interacting proteins.The full-length of ENST00000444488.1 was used as baits by generating both sense and antisense(negative control)RNA probes.Mass spectrometry identified several potential ENST00000444488.1-interacted proteins,including myosin heavy chain 9(MYH9),actin,and actin cross-linking protein.MYH9 was detected by subsequent Western blot from RNA pull-down assays in cell extracts from VSMCs.The interaction between MYH9 and ENST00000444488.1 was further confirmed by RNA immunoprecipitation(RIP)assay in VSMCs.Consistent with RNA pull-down results,ENST00000444488.1 was significantly enriched by MYH9 antibody compared with IgG-negative controls,indicating a specific interaction between ENST00000444488.1 and MYH9.Furthermore,FISH assay of ENST00000444488.1 and fluorescence histochemistry of MYH19 in the same slice confirmed the colocalization of ENST00000444488.1 and MYH9.Together,these results demonstrated that ENST00000444488.1 directly interacted with MYH9 protein and might mediate some molecular functions of MYH9.Next,we sought to determine the functional significance of the interaction between ENST00000444488.1 and MYH9.The overexpression of ENST00000444488.1 significantly down regulated MYH9 protein level,while the knockdown of ENST00000444488.1 significantly up regulated MYH9 protein level.The overexpression or knockdown of ENST00000444488.1 did not significantly affect MYH9 mRNA level.But the underlying mechanism of effect of the binding of ENST000004488.1 to MYH9 protein in affecting MYH9 protein level remains to be fully explored.The overexpression of MYH9 gene significantly promoted VSMCs migration,and the knockdown of MYH9 gene in VSMCs significantly inhibited migration,and moreover,MYH9 gene knockdown significantly inhibited the migration of VSMCs induced by PDGFBB.We sought to determine the effect of the interaction between ENST00000444488.1 and MYH9 protein on VSMCs migration.Functionally,the increase of VSMCs migration induced by the knockdown of ENST00000444488.1 could be reversed by the knockdown of MYH9 gene,demonstrating that the interaction between ENST00000444488.1 and MYH9 protein affected VSMCs migration.The contractile actomysin cytoskeleton is critical for control of cell shape and migration.Being an actin motor,MYH9 protein is involved in maintaining cytoskeleton structure by binding to actin filaments,thus we deduced that the interaction between ENST00000444488.1 with MYH9 protein might affect actin cytoskeleton reorganization.Staining for F-actin showed strong F-actin stress fiber bundles in VSMCs treated with PDGFBB or in VSMCs transfected with siRNA against ENST00000444488.1.In accordance with the migration phenotype,the overexpression of ENST00000444488.1 could inhibited F-actin cytoskeleton rearrangement in VSMCs induced by PDGFBB,and increased F-actin stress fiber bundles in VSMCs induced by the knockdown of ENST00000444488.1 could be reversed by the knockdown of MYH9.These results demonstrated that the interaction between ENST00000444488.1 and MYH9 affected actin cytoskeleton rearrangement,thus leading to inhibition of VSMCs migration induced by PDGFBB.CRISPR/Cas9 technique was used to contruct smooth muscle cell specific ENST00000444488.1 transgenic mice,and F1 generation mice with fl/+genotypes have been obtained.Conclusion:The full-length of ENST00000444488.1 is 1279nt.ENST00000444488.1 is mainly locate in cytoplasm of VSMCs with low conservativeness among 100 vertebrate species and does not have the ability to encode proteins.ENST00000444488.1 regulated the migration of VSMCs induced by PDGFBB,but did not affect the proliferation of VSMCs.ENST00000444488.1 interacted with MYH9 protein,and the interaction between ENST00000444488.1 and MYH9 protein affected MYH9 protein level,the rearrangement of VSMCs F-actin skeleton,and thus VSMCs migration.