Doxorubicin Induces Long-chain Non-coding RNA SNHG1 Nuclear Homeostasis Impairs HnRNPC Interaction With P53

Long non-coding RNA is a class of RNAs with more than 200 nucleotides in length,which do not have the ability to encode proteins and have a diversity of gene expression and regulation functions,regulating gene expression at the epigenetic modification,transcription and transcription at different levels.In cells and tissues long non-coding RNAs and their binding proteins required RNA-protein complexes formed so as to jointly play a role,then looking for long non-coding RNA specific binding proteins and protein specific binding long non-coding RNAs are of great importance in studies of long non-coding RNA function.At the same time,does lncRNA nuclear distribution in a specific stimulus conditions(such as DNA damage,etc.)will change? Does this change have biological significance? Does it depend on the binding of lncRNA to specific proteins? An in-depth study of these scientific issues will help to reveal the function of unknown lncRNAs and their new regulatory mechanisms.As an important transcription factor,p53 protein regulates the transcription of its target genes by binding specific DNA sequences of theirpromoter regions.Numerous studies show that,p53 gene is an important tumor suppressor gene,and plays an important rolein DNA damage repair,cell cycle arrest and apoptosis.Mutated p53 gene is promoting the development of tumor cells by regulating the cell cycle,apoptosis and cell proliferation process.When faced with a variety of stress signals,p53 protein is also regulated by various regulatory mechanisms,which both p53 expression and protein stability can be regulated by proteins,and there are RNAs involved as well.And a variety of long non-coding RNAs such as lincRNA-p21,PANDA,MALAT1 are also proved to be involved in the regulation of p53 regulating network in recent experiments,which affects the expression of the downstream genes.Is there any new lncRNAs involved in the p53 protein regulatory network? Does lncRNA mediate the direct interaction between p53 protein and other proteins? These scientific questions need further study.Heterogeneous nuclear ribonucleoprotein(hnRNP)is an important class of RNA binding proteins,which can interact with a variety of proteins and RNAs in cells,regulate alternative splicing of pre-mRNA and have important biological functions in mRNA transport,mRNA stability regulation and mRNA identification and classification.Some hnRNPs are involved in the p53 protein function regulation,such as hnRNPK can regulate p53 transcriptional activity,when DNA damage occurs hnRNPK levels increased significantly then further promote E3 ubiquitin ligase MDM2 ubiquitin degradation and co-regulate target genes transcription with p53.At the same time,some studies show that long non-coding RNA regulate functions of p53 by binding hnRNPs that have interactions with p53,for instance lncRNA Apela combines with hnRNPL protein leading the decreasing of binding capacity of hnRNPL and p53 protein,therefore,which increases p53 protein levels and the mitochondrial distribution of p53 protein.Preliminary work in our laboratory proved that hnRNPC could modulate the stability and transcriptional activity of p53 protein,and the two proteins have direct interaction,which can be repressed by RNAs.Based on this,we want to seek long non-coding RNAs that can prevent the interaction between p53 and hnRNPC,and the purpose is to reveal the role of long noncoding RNA in the regulation of p53 protein and their mutual network.HnRNPC is an important RNA binding protein that has very high expression level in cells,and its distribution restricts in the nucleus.Accordingly,we conclude that long non-coding RNA that we need to be screened out should bind hnRNPC and have a high level of expression,and distribute in the nucleus under certain conditions.We first applied a combination of hnRNPC CLIP data and expression data of long non-coding RNA in p53+/+ HCT116 cell line and sought for lncRNAs with high expression levels and with the ability to bind hnRNPC,within which SNHG1 has the highest expression level.Also we found SNHG1 retained in the nucleus induced by doxorubicin,therefore,it has been screened for functional verification.We also revealed that 498-509 bp motif of SNHG1 is essential for its nuclear retentioninduced by doxorubicin which is partly due to its affinity with NCL.By RNA immunoprecipitation of hnRNPC we indicated that lncRNA SNHG1 can combine with hnRNPC.By RNA pulldown experiments of SNHG1 we demonstrated that 657-667 bp region of SNHG1 is a key area for binding,and immunoprecipitation and GST pulldown experiments showed that SNHG1 could weaken the direct interaction between p53 and hnRNPC,but SNHG1 Del missing hnRNPC binding capacity would not affect combination of hnRNPC with p53.In order to study the biological effects caused by change of lncRNA SNHG1 nuclearcytoplasmic distributionunder conditions of doxorubicin treatment,we want to simulate the conditions of increased SNHG1 distribution levels within the nucleus under doxorubicin treatment and nuclear overexpression of SNHG1 came to our minds.In the case of conventional eukaryotic expression vector not capable of achieving our demands we constructedSNHG1 and deletion hnRNPC binding sites SNHG1 Del nuclear expression vector based on snoVector;in human colon cancer p53+/+ HCT116 cells,dual luciferase reporter experiments proved that nuclear overexpressing SNHG1 could increase the transcriptional activity of p53,whereas SNHG1 Del had no such function;p53 protein expression level and half-life could be further enhanced by nuclear overexpressing SNHG1 instead of SNHG1Del;CCK8 cell proliferation assay and flow cytometry apoptosis assay showed nuclear expression SNHG1 instead of SNHG1 Del could inhibit cell proliferation and promote p53-dependent apoptosis.It has been reported that SNHG1 in non-small cell lung cancer and liver cancer has the function of promoting cell proliferation.Does it imply that in the normal state,SNHG1 which is located in the cytoplasm also promotes the development of colon cancer function? By searching lncRNA expression database of tumor samples of human we found that the expression of SNHG1 in a variety of tumor tissues was higher than the level of the adjacent tissues,and colon cancer data from multiple samples also showed that SNHG1 expression level was higher than adjacent tissues.Thus we inferred that in normal cells SHNG1 may promote tumor development;in HCT116 cells we found that interfering SNHG1 could increase the transcriptional activity of p53 by dual luciferase reporter gene experiments;p53 protein expression level could be further enhanced by interfering SNHG1;flow cytometry apoptosis assay showed that interfering SNHG1 could promote p53-dependent apoptosis;RNA pulldown and mass spectrometry essay showed that SNHG1 may bind NCL and ILF3 that were p53 interactors and important regulatory proteins of p53,which may be an important part of molecular mechanisms regulating p53 of lncRNA SNHG1 under normal conditions.In summary,the innovation and conclusions of this study are as follows:1.It was confirmed for the first time that doxorubicin induced nuclear retention of SNHG1,and it was related to the affinity of SNHG1 with NCL.2.For the first time,it was confirmed that SNHG1 regulates protein-protein interactionbycompetitive binding whichis a new mechanism for protein interaction regulation anddiscover that SNHG1 can weaken the direct interaction of p53 with hnRNPC.3.Overexpressing SNHG1 in the nucleus by using the expression vector in the nucleus tosimulate the presence of doxorubicin-induced SNHG1 cell nucleus and it was proved thatnuclear-retained SNHG1 under doxorubicin can alter the p53 protein level,itstranscription activity and p53-dependent apoptosis.4.Without DNA damage,SNHG1 mainly localizes in the cytoplasm and downregulatingSNHG1 can also enhance p53 protein level and its transcriptional activity,whichindicates that SNHG1 may have different funtions with different nuclear-cytoplasmicdistributions.