Regulatory Mechanisms Of LncRNA HSP90AA1-IT1 On CDK2 Expression In Gliomas Through Mir-885-5p

Glial-derived gliomas account for the vast maj ority of malignant brain tumors.In the past 30 years,the incidence of glioma has increased at a rate of 1%-2%per year,with a high degree of malignancy and poor prognosis.Even with the most powerful comprehensive treatment the 5-year survival rate is only 0.1%-8.9%.Therefore,new more effective treatments have become the focus of glioma research.The occurrence and progression of glioma is a complex process involving multiple factors,and uncontrolled proliferation caused by uncontrolled cell cycle is one of the key process.Cell cycle play a key role in tumorgenesis,and cycle-dependent kinases(CDKs)are the core of cell cycle control system.CDK2 is an important member of the CDK family.It plays an important role in various aspects of cell cycle regulation,such as centrosome replication,DNA synthesis,G1-S transition and G2 phase progression.CDK2 is not only a rate-limiting enzyme that regulates cell cycle progression and apoptosis,but also a downstream regulator of many oncogenes/tumor suppressor genes.Therefore,the uncontrolled cell cycle progression caused by abnormal expression of CDK2 may play an important role in the development of glioma.Non-coding RNA plays an important role in the process of life.Its function can be extended to almost all biological processes,such as embryonic development,cell proliferation,differentiation,apoptosis,gene imprinting,stress response and canceration.Its characteristics include:(1).With high specificity,ncRNAs has characteristic expression in different tissue regions and disease processes.This feature suggests that glioma-specifi abnormal expression of ncRNAs may be closely related to dysregulation of CDK2 activity.(2)The regulatory relationship is complex.Each ncRNA corresponds to multiple target genes,and each target gene can also be regulated by multiple ncRNAs.(3)Regulatory mechanisms are diverse,ncRNAs affect on target genes at chromatin remodeling,DNA sequence,transcription and post-transcriptional levels.This makes the regulatory network of gene expression mediated by ncRNA extremely complex,and many problems are still unclear.MicroRNAs are a class of ncRNAs with a length of about 22 nt.They directly degrade or block the translation of the target gene by binding to the 3’UTR region of the target gene,and inhibit the expression of the gene at the post-transcriptional level.However,the regulatory capacity of microRNAs is still relatively limited,and only about 40%of all proteins are regulated by microRNAs.Another type of ncRNA is long non-coding RNA(LncRNA)whose transcript length exceeds 200 nt.With the development of molecular genetics and epigenetics,the role of long non-coding RNA in brain-related diseases has attracted more and more attention:its functions are extensive and complex,involving almost all biological processes such as embryonic development,gene imprinting,inflammation stress,tumors and so on.It has more characteristic expression patterns in tumors and many other diseases.Because of the diverse regulatory mechanisms of lncRNA,it can act on different target genes at multiple levels,which makes the regulatory network of gene expression mediated by IncRNA extremely complex.Current studies have found that microRNAs and LncRNAs are not isolated in gene expression regulation,but closely related.The interaction between them has attracted much attention.Especially the hypothesis of Competitive Endgenous RNA(CeRNA)provides a new way to elucidate the regulatory relationship between micro RNA and LncRNA.In 2011,Pandolfi first proposed in Cell:ceRNA molecules(mRNA,LncRNA,pseudogenes,etc.)exist widely in cells,which can competively bind to the same microRNA through microRNA response elements(MRE)to regulate the expression level of each other;for example,LncRNA can competively bind to one or more microRNAs as ceRNA molecules,thus rEdUcing the expression level of each other.Low microRNAs inhibit downstream target genes and promote"reactivation" of target gene expression.The CeRNA hypothesis represents a new model of gene expression regulation.It integrates a variety of ncRNA(such as microRNA,LncRNA)and RNA into a regulatory network.Compared with the microRNA regulatory network,ceRNA involves more RNA molecules,the regulatory network is larger,the regulatory mechanism is more elaborate and complex,and it can excavate gene functions and interconnections from a deeper level.However,the current research on ncRNA regulating CDK2 is limited to a few independent microRNAs,and the related research on LncRNA or CeRNA has not been reported.In order to find a ceRNA network targeting CDK2,this experiment mainly focus on the specific mechanism of HSP90AA1-IT1 regulating CDK2 expression through competitive binding of microRNA-885-5p.OBJECTIVE1.To study the expression of 1ncRNA HSP90AA1-IT1 in glioma.2.To study the biological role of 1ncRNA HSP90AA1-IT1 in glioma cells.3.To explore the molecular mechanism of 1ncRNA HSP90AA1-IT1 regulating CDK2 expression through miR-885-5p.METHODSPart Ⅰ:1.Detection of tissue expression level of lncRNA HSP90AA1-IT1 The expression of lncRNA HSP90AA1 in glioma tissues with different pathological grades was detected by qRT-PCR.2.Detection of epidemiological characteristics of lncRNA HSP90AA1-IT1 Through follow-up medical records,explore the relationship between IncRNA HSP90AA1-IT1 and clinical data,and compare the total survival time of patients with different expression levels of lncRNA HSP90AA1-IT1.Part Ⅱ:1.Establishment of a glioma cell model that knocks out lncRNA HSP90AA 1 The IncRNA HSP90AA1-IT1 knockdown U251 and U87MG cell lines was established infection of HSP90AA1-IT1 shRNA lentivirus.2.To study the effects of lncRNA HSP90AA1-IT1 on cell proliferation,migration and apoptosis.2.1 The effects of lncRNA HSP90AA1-IT1 on cell proliferation and cell cycle progression were detected by CCK8,EDU fluorescence staining and flow cytometry.2.2 Transwell cell culture and scratch test were used to observe the effects of lncRNA HSP90AA1-IT1 and microRNA-885-5p on cell invasion and migration ability.2.3 Annexin V-PI staining flow cytometry was used to detect the effect of 1ncRNA HSP90AA1-IT1 on apoptosis.2.4 Western-blot was used to detect the effect of lncRNA HSP90AA1-IT1 on the expression of markers related to epithelial-mesenchymal transition.Part Ⅲ:1.To clarify the interaction between microRNA-885-5p and 1ncRNA HSP90AA1-IT11.1 The expression of lncRNA HSP90AA1-IT1 was detected by qRT-PCR in miR-885-5p overexpressed cell lines compared with control group,and the expression of miR-885-5p was detected by qRT-PCR in lncRNA HSP90AA1-IT1 cell lines.1.2 The miR-885-5p overexpressed glioma cell lines were transfected 1ncRNA HSP90AA1-IT1 3’UTR-luciferase reporter gene plasmid.The regulation of miR-885-5p on lncRNA HSP90AA1-IT1 was analyzed by double luciferase activity assay.1.3 The possible binding site of lncRNA HSP90AA1-IT1 fragment on miR-885-5p was mutated by localized mutation technique.The above experiments were repeated to determine the target regulatory site of miR-885-5p on lncRNA HSP90AA1-IT1.2.To study the molecular mechanism of regulation of CDK2 expression by miR-885-5p.2.1 In the cell model of miR-885-5p overexpression,the expression of CDK2 was detected by qRT-PCR and Western blot.2.2 The miR-885-5p overexpressed glioma cell lines were transfected CDK2 3’UTR-luciferase reporter gene plasmid.The regulation of miR-885-5p on CDK2 was analyzed by double luciferase activity assay.2.3 The possible binding site of CDK2 mRNA 3’UTR was mutated by localized mutation technique.The above experiments were repeated to determine the target regulatory site of CDK2 by miR-885-5p.3.To explore whether lncRNA HSP90AA1-IT1 regulates the expression of CDK2 through competitive binding to miR-885-5p.3.1 In the lncRNA HSP90AA1-IT1 overexpressed/knockdown cell lines,the expression of CDK2 was detected by qRT-PCR and Western blot.3.2 MiR-885-5p was overexpressed in the lncRNA HSP90AA1-IT1 up-regulated cell lines;miR-885-5p was down-regulated in the lncRNA HSP90AA1-IT1 down-regulated cell lines.Observe whether the expression of CDK 2 was reversed compared with previous results.4.Eestablish a tumorigenic model of nude mice and verify the above results through in vivo experiments.RESULTSPart Ⅰ:lncRNA HSP90AA1-IT1 is highly expressed in glioma and indicate poor prognosis1.Detection of tissue expression level of lncRNA HSP90AA1-IT1 The results of qRT-PCR showed that the expression of lncRNA HSP90AA1-IT1 in glioma tissues was significantly higher than that in paratumoral brain tissues.2.Detection of epidemiological characteristics of lncRNA HSP90AA1-IT1 The expression of lncRNA HSP90AA1-IT1 was correlated with the expression of Ki-67 and WHO grade of glioma,and the high expression of lncRNA HSP90AA1-IT1 suggested poor prognosis.Part Ⅱ:lncRNA HSP90AA1-IT1 promotes glioma proliferation,migration and epithelial mesenchymal transition,and inhibits apoptosis1.Establishment of a glioma cell model that overexpresses or knocks out 1ncRNA HSP90AA The qRT-PCR results showed that the expression of lncRNA HSP90AA1-IT1 in infected cells was significantly lower than that of the control group.2.To study the effects of lncRNA HSP90AA1 on cell proliferation,migration and apoptosis.2.1 CCK8,EDU fluorescence staining and flow cytometry analysis showed that down-regulting expression of lncRNA HSP90AA1-IT1 could significantly inhibit the proliferation of glioma cells and block the cell cycle in G1 phase.2.2 Transwell assay showed that decreasing the expression of lncRNA HSP90AA1-IT1 could significantly inhibit the invasion and migration of glioma cells.2.3 Annexin V-PI flow cytometry showed that rEdUcing the expression of lncRNA HSP90AA1-IT1 could promote glioma cell apoptosis.2.4 Western blot showed that lncRNA HSP90AA1-IT1 promoted epithelial-mesenchymal transition.3.To study the effect of lncRNA HSP90AA1-IT1 on the growth of transplanted tumors in nude mice.Nude mice tumorigenesis experiment showed that rEdUcing the expression of lncRNA HSP90AA1-IT1 could inhibit the growth of glioma in vivo and the expression of CDK2.Part Ⅲ:To explore the molecular mechanism of lncRNA HSP90AA1-IT1 and miR-885-5p regLulating CDK2 expression.1.To stucdy the interaction between miR-885-5p and lncRNA HSP90AA1-IT11.1 qRT-PCR and Western blot showed that the expression of miR-885-5p was correlated with that of lncRNA HSP90AA1-IT1.1.2 Luciferase reporter gene experiment showed that the miR-885-5p inhibited expression of CDK2 by directly binding.2.To study the molecular mechanism of regulation of CDK2 expression by miRNA-885-5p.2.1 qRT-PCR and Western blot showed that over-expression of miRNA-885-5p could inhibit the expression of CDK2.2.2 Luciferase reporter gene experiment showed that the expression of miRNA-885-5p was inhibited by binding with CDK2.3.LncRNA HSP90AA1-IT1 regulates the expression of CDK2 through competitive binding to microRNA-885-5p3.1 LncRNA HSP90AA1-IT1 was positively correlated with CDK2 expression.3.2 LncRNAHSP90AA1-IT1 regulates the expression of CDK2 through competitively binding to miR-885-5p.4.The results of animal experiments are consistent with those of in vitro experiments.CONCLUSIONS1.In primary glioma samples,the expression of lncRNA HSP90AA1-IT1 was significantly up-regulated and correlated with poor prognosis.2.LncRNA HSP90AA1-IT1 can promote proliferation,migration and inhibit apoptosis of glioma cells.3.LncRNA HSP90AA1-IT1 is negatively correlated with the expression of microRNA-885-5p,and overexpression of lncRNA HSP90AA1-IT1 can weaken the anti-cancer effect of miR-885-5p.4.LncRNA HSP90AA1-IT1 regulates the expression of CDK2 through miR-885-5p.