| This study is mainly includes two parts:the first part is the establishment and application of efficient Tetrahymena thermophila RNA circularization system,and the second part is the evaluation and mechanism of the anti-tumor effect of the novel MDM2 inhibitor,which aims to study the molecular treatment pattern of cancer from different perspective of circRNA and small molecule inhibitor.Part I Establishment and application of efficient Tetrahymena thermophilabased RNA circularization systemObjective:Circular RNAs(circRNAs),a class of RNA molecules with covalently closed loop structures generated by back-splicing,have shown broad applicability in the diagnosis and treatment of diseases.However,due to their low expression abundance and the lack of effective,accurate,and stable overexpression tools,the biological functions of many endogenous circRNAs have not been extensively studied,which limits the wide application of circRNAs.Typically,exogenous circRNA is prepared in three ways:Chemical ligation,T4 DNA or RNA ligase-mediated circularization and permuted intronexon(PIE)strategy based on group Ⅰ intron ribozyme splicing activity.In addition,the classic strategy for constructing endogenous circRNA overexpression vectors is to retain both the circRNA-producing exons and their flanking intronic complementary sequences(ICSs).However,each of these methods has its disadvantages,such as the introduction of exogenous nucleotides and large numbers of linear transcripts due to the low efficiency.T.thermophila Group Ⅰ intron has proven to be a powerful ribozyme for Reactions such as internal cleavage of RNA and DNA,RNA polymerization,and gene delivery.In this study we aimed to develop a novel system for engineering circRNA synthesis in mammalian cells and in vitro based on the catalytic activity of thermophila group Ⅰ intron ribozyme.Methods:The indicated RNA circularization sequence was inserted downstream of the optimized T.thermophila ribozyme and a complementary antisense region was added to the pCDH backbone upstream of the ribozyme to establish a novel engineered T.thermophila ribozyme-based RNA circularization system.CircDnmt1 overexpression plasmid was constructed according to the above system,and its putative splice junction,expression level and splicing efficiency were analyzed.Then,the overexpression plasmids of CDR1as,circFOXO3 and circHIPK3 were constructed based on ribozyme or flanking ICSs,and the cellular biological function of CDR1as and circFOXO3 overexpressed by our system was further analyzed.Finally,we engineered a circRNA protein expression reporter gene system containing split green fluorescent protein(GFP)and an optimal CVB3 IRES sequence to initiate cap-independent translation.The circular sequence encoding GFP was generated from backsplicing,and its splicing junction,splicing efficiency,and GFP fluorescence were verified.Results:The T.thermophila ribozyme-based RNA circularization method was successfully established.Using this strategy,exogenous circRNAs can be synthesized.It can also perfectly mimic the sequence of naturally occurring endogenous circRNAs while retaining their original physiological functions.The qRT-PCR experiments in vitro and in mammalian cells showed that the circularization efficiency of circDnmt1 synthesized by this method can reach about 80%and can reach a higher expression level in various cells such as HEK293T,HeLa and MCF7.Moreover,the circular constructs of circDnmt1,CDR1as,circFOXO3,and circHIPK3 had higher overexpression levels than those based on flanking ICS-mediated circularization method,and the overexpressed CDRlas of our system inhibited the suppressive effect of miR-7 in colon cancer cells.The overexpressed circFOXO3 of our constructs retained its proven biological functions in regulating cell proliferation,migration and apoptosis.Moreover,the splicing efficiency of the circular sequence encoding GFP formed by our construct was higher than that of the control group,and the resulting open reading frame of GFP can be translated to generate a functional GFP protein.The green fluorescence was observed under the microscope.Conclusions:This study successfully established an RNA circularization strategy based on the efficient splicing activity of Tetrahymena thermophila ribozyme,which can efficiently generate circular RNA without introducing any additional exogenous sequences.Our research provides a efficient,accurate and simple strategy for the synthesis of circRNA in mammalian cells and in vitro,and the RNA circularization system can also express proteins by add internal ribosome entry site(IRES),which makes circRNA an effective and safe RNA therapeutic platform and alternative mRNA stable expression protein provide strong technical support.Part Ⅱ Evaluation and mechanism of anti-tumor effects of novel MDM2 inhibitorsObjective:The p53 protein encoded by the tumor suppressor gene TP53,is a transcription factor that regulates cell differentiation,DNA repair,cell cycle,and cell apoptosis.The inactivation of p5 3 protein is closely related to abnormal cell proliferation and tumor development.Generally speaking,the interaction between P53 protein and other proteins and the mutation of TP53 gene can lead to the loss of p53 function.Murine Double Minute 2(MDM2)is one of the key negative regulators of p53.It has been suggested that the p53 reactivation by inhibiting MDM2-p53 interaction represents a promising therapeutic option in cancers.MDM2 inhibitors can increase the protein level of wild-type p53 in tumor cells and enhance its tumor suppressor function by blocking the degradation of wild-type p53 by MDM2.Although some of agents had been developed based on the tactic of blocking the protein-protein interaction between p53 and MDM2,with some of them now being tested in clinical trials both in hematologic malignancies and solid tumors treatment,nevertheless,significant side effects,including hematotoxicity and gastrointestinal side effects,have limited rapid approval.Through the proteolysis targeting chimera(PROTAC)strategy,some MDM2-targeted heterobifunctional PROTAC have been designed and show potent MDM2 inhibition activities in recent years.Methods:In our previous works,we reported potently selective MDM2 inhibitors of PEGylation spirooxindole derivatives.In this work,based on our previous works,we developed a dimer spiroindolinone pyrrolidinecarboxamide XR-4 as potently MDM2p53 inhibitor.Here,to develop more effective MDM2 inhibitor with lower off-target toxicities,we synthesized a dimer spiroindolinone pyrrolidinecarboxamide XR-4 with potent MDM2-p53 inhibition activity.Western blot and qRT-PCR were performed to detect the influence of XR-4 on MDM2 and p53 protein levels and p53 downstream target gene levels in different cancers.Cancer cell proliferation and clonogenic activity were also investigated by CCK8 assay and colony formation assay.Results:As an effective MDM2 inhibitor,XR-4 could induce wild-type p53 accumulation in cancer cells by competitively binding to the binding sites of p53 and MDM2,upregulate the expression levels of p53 target genes P21 and PUMA,and then inhibit cancer cell proliferation and induce cell apoptosis.XR-4 could be regarded as a homo-PROTAC molecule consisting of 2 identical E3 ligase ligands targeting MDM2 so that it could break down MDM2 protein without adding additional targets and reducing possible side effects.XR-4 showed comparable cancer cell proliferation inhibition activity to RG7388 and has lower off-target toxicity in broad-spectrum p53 wild-type cancer cells including liver,prostate,breast,bladder,and colorectal cancers.Conclusions:In summary,XR-4 is a potent dimer spiroindolinone pyrrolidinecarboxamide derivative with effective p53 activation activity and cancer inhibition ability. |
