| Glioma is one of the refractory types of tumours in the central nervous system,among which glioblastoma（GBM）has the highest degree of malignancy.Due to its growth characteristics,it has no clear boundary with normal brain tissue,which can easily lead to incomplete surgical resection,postoperative recurrence,and obvious resistance to radiotherapy and chemotherapy.Therefore,seeking new treatment strategies is a key issue that urgently needs to be addressed in clinical GBM treatment.In recent years,with the in-depth exploration of tumour epigenetics by researchers,many studies have confirmed that circularRNA（circRNA）not only exhibits significant dysregulation of expression in GBM tissue/cell malignant progression but also plays an important role in regulating malignant biological phenotypes such as tumour invasion,autophagy,chemotherapy resistance,and epithelial mesenchymal transition.The epigenetic regulation mediated by circRNA has become a hot research direction in basic oncology research,and circRNA has also become an emerging research target in the field of GBM targeted therapy,with great potential for application.In summary,targeting these naturally occurring circRNAs is a very promising method for treating tumours,which is of great significance for improving the current clinical treatment status of GBM.Nucleic acid drugs such as small interferingRNA（siRNA）,as a research hotspot in the field of biomedicine,have been widely used to explore gene function and nucleic acid drug therapy for malignant tumours.As an effector molecule ofRNA interference（RNAi）technology,siRNA has been proven to be an effective method for specifically blocking gene expression in circRNAs with special circular structures.It can design highly specific siRNA molecules based on the reverse splicing site sequence or intron region sequence of circRNAs to prevent knockdown of parental genes.AlthoughRNAi therapy has made breakthrough progress in GBM basic research,there are still many challenges to truly transform into clinically safe medication（such as blood stability,blood-brain barrier penetration,and effective release of in situ lesions）.Therefore,safe and efficient in vivo delivery remains the biggest obstacle to the application ofRNAi therapy.Based on this,constructing a nucleic acid drug delivery system using nanotechnology will be an important means and approach to solve this problem.These nanoscale carriers with unique characteristics can not only prevent the degradation of therapeutic nucleic acid molecules,allowing them to penetrate the blood-brain barrier（BBB）or cellular biological barrier from the systemic circulation but also achieve targeted transportation by surface modification of the carrier and binding to receptors on the cell membrane,enabling precise action of nucleic acid therapeutic molecules on target cells.Showing significant advantages in GBM treatment,it provides a new solution to overcome the shortcomings of nucleic acid drugs,such as short cycle time and inability to target tumour cells.This study takes the two popular research directions of"circRNA-mediated epigenetic regulation"and"RNAi tumour therapy"as the entry points and combines important molecular characteristics,such as stability,biocompatibility,biodegradability,low cytotoxicity,and targeting of the BBB and GBM by Angiopep-2-modified polymer nanomicelles.The scientific hypothesis is proposed thatRNAi nanonucleic acid drugs exert an antitumour effect in GBM targeted therapy through the circRNA/ceRNA pathway.Research Objectives:As a common and fatal malignant tumour,the expression disorder of tumour suppressor genes and oncogenes in GBM is the fundamental cause of changes in its biological characteristics.Based on previous full transcriptome sequencing combined with bioinformatics analysis by the research group,the interaction network of GBM epigenetic regulation has been further enriched,providing effective targets for GBM targeted therapy and screening and synthesizing nucleic acid drug molecules（therapeutic siRNA）.In addition,a block copolymer micelle（PHB-PDMAEMA）modified with Angiopep-2 was designed and constructed as a nucleic acid drug carrier.In PHB-PDMAEMA,the PHB fragment can bind to the peptide Angiopep-2 through a decarboxylation reaction,and the PDMAEMA fragment can adsorb therapeutic siRNA through electrostatic force,forming a polymer/gene complex（RNAi nanodrug）.ThisRNAi nanodrug can bind to low-density lipoprotein receptor related protein-1（LRP-1）membrane receptors and mediate targeting of the BBB and GBM,specifically reducing the expression level of the oncogenic gene circ＿TNFRSF19 in cells,thereby exerting an antitumour effect in the malignant progression of GBM through its competing endogenousRNA（ceRNA）regulatory mechanism.In summary,this study is the first attempt to use Angiopep-2-modified PHB-PDMAEMA as a nucleic acid drug carrier to mediate the targeted reversal of endogenous circ＿TNFRSF19 expression in GBM cells through therapeutic siRNA,thereby inhibiting malignant tumour biological behaviour.On the one hand,this study aims to achieve a stable interaction platform using polymer nanomicelles as nucleic acid drug carriers and to deeply explore the practical value of nanomedicine in clinical disease treatment.On the other hand,this study confirms the emerging role of circRNA-mediated epigenetic regulatory mechanisms in tumour treatment,further promotingRNAi therapy based on circRNAs.This study provides new ideas for the targeted treatment of GBM.Research Methods:（1）We conducted a full transcriptome sequencing study based on 5 sets of clinical postoperative GBM tissues and traumatic decompression normal cortical tissues and conducted advanced bioinformatics analysis of the gene sequencing results.A circRNA/lncRNA miRNA ceRNA regulatory network was constructed,key circRNA molecules were selected from the interaction network,and RT-qPCR technology was used to verify gene expression levels,ultimately obtaining the target circRNA.（2）Implement circRNA knockdown based onRNAi technology and RT-qPCR validation.Ed U,Transwell,and flow cytometry experiments were used to screen the target circRNAs for cellular function experiments.The stability,ROS response,p H response and cytotoxicity of theRNAi nanodrugs were determined by agarose gel electrophoresis,dynamic light scattering,transmission electron microscopy and CCK-8 technology.（3）BBB and 3D tumour ball in vitro models were constructed,and flow cytometry,Transwell experiments,and laser scanning confocal microscopy were used to detect the targeting properties of theRNAi nanomedicine（cell uptake,BBB penetration,and tumour tissue infiltration）.RT-qPCR,CCK-8,Transwell,and flow cytometry detection experiments further validated the gene silencing and antitumour effects ofRNAi nanodrugs.Bioinformatics analysis predicts and verifies the downstream target genes of circ＿TNFRSF19through RT-qPCR and Western blot.Research Results:（1）Compared with the non-tumour control group,a total of 5341 differentially expressed（DE）mRNAs,259 DEmiRNAs,3122 DElncRNAs,and 2135 DEcircRNAs were found in GBM samples.In addition,a circRNA/lncRNA miRNA ceRNA regulatory network was integrated and constructed,and based on this,five upregulated circRNAs were screened.Three molecules of interest were identified as preliminary candidate circRNAs for subsequent experimental studies through RT-qPCR validation.（2）The siRNA transient knockdown U343 cell model was successfully constructed.After the downregulation of the target circRNA（circ＿TNFRSF19）expression level,the malignant biological behaviour of GBM cells was significantly inhibited.In addition,the PHB-PDMAEMA nucleic acid drug delivery vector was successfully prepared,which can adsorb siRNA to form stable polymer/gene complexes.It can undergo degradation under high H2O2or weakly acidic conditions（simulating the tumour microenvironment in vitro）,rapidly releasing siRNA molecules.（3）Angiopep-2-modifiedRNAi nanodrugs can bind to LRP-1 membrane receptors and actively target BBB and GBM cells.Compared with the nontargeted modified group,their cell uptake,BBB penetration,and tumour tissue penetration abilities are the strongest.In addition,it has been proven to significantly reduce the expression level,proliferation,migration,and invasion ability of circ＿TNFRSF19 in GBM cells and promote tumour cell apoptosis.The main mechanism is that circ＿TNFRSF19 acts as a miRNA molecule sponge,inducing cell apoptosis by regulating the mi R-324-3p/Bcl-2 axis in GBM cells.Research Conclusions:（1）This study utilized full transcriptome sequencing technology to deeply explore the pathogenesis of GBM and constructed a circRNA/lncRNA miRNA mRNA interaction network diagram,further revealing the potential correlation between noncodingRNA（ncRNA）-mediated epigenetic regulation and GBM malignant transformation.These findings not only demonstrate the complexity of the GBM genome but also provide evidence for the search for GBM gene therapy-related targets.Targeting the correction of the dysregulation of endogenous circRNA expression during malignant tumour formation will be an ideal treatment strategy to overcome the bottleneck of GBM therapy,which is of great significance.（2）Knocking down the expression of circ＿TNFRSF19 significantly inhibited the proliferation,invasion,and migration of GBM cells while promoting their apoptosis.PHB-PDMAEMA polymer nanocarriers can stably adsorb siRNA to form polymer/gene complexes.It has the dual response ability of ROS and p H,which can undergo depolymerization under high H2O2or weakly acidic（in vitro simulated tumour microenvironment）conditions,rapidly releasing nucleic acid molecules and significantly improving the bioavailability of siRNA.This study provides an experimental basis for further exploration of the antitumour effect and related mechanisms ofRNAi nanomedicine in GBM targeted therapy.（3）Angiopep-2-modifiedRNAi nanomedicine has an active targeting function,which can not only cross the BBB but also actively target GBM cells and penetrate deep into tumour tissue.In addition,it also has good gene silencing and antitumour effects.Silencing circ＿TNFRSF19 significantly inhibits GBM cell proliferation,migration,and malignant progression phenotypes and promotes tumour cell apoptosis.The potential mechanism is that circ＿TNFRSF19 acts as a molecular sponge and significantly promotesRNAi nanodrug-induced cell apoptosis through the mi R-324-3p/Bcl-2 signalling pathway.In summary,this study is the first attempt to use Angiopep-2-modified PHB-PDMAEMA as a nucleic acid drug carrier,mediating therapeutic siRNA molecules to exert antitumour effects in GBM malignant progression by regulating the circ＿TNFRSF19/mi R-324-3p/Bcl-2signalling axis.This study not only provides new targets and a theoretical basis forRNAi therapy of GBM but also provides a good interactive platform for GBM targeted therapy,showcasing the broad prospects of nanomedicine in the future field of tumour therapy. |
PDF Full Text Request