| Part ⅠNonsense-mediated mRNA decay(NMD)is a critical post-transcriptional mechanism that guards cells against potential poisonous proteins.NMD mainly degrades mRNA that harbors premature terminal codon(PTC)resulting from alternative splicing(AS)or missense mutations,thus blocking the production of C-terminal truncated protein.NMD also plays a role in the regulation of 10-20%of normal transcripts.NMD is widely involved in pathological and physiological conditions of cells,such as amino acids starvation,oxidative stress,regulation of the immune,nervous system development,genetic diseases,cancer,etc.Recently,therapy that targets NMD has become a promising novel adjuvant treatment for many cancers and genetic diseases.However,not all patients can benefit from NMD inhibition.It is necessary to detect the activity of NMD and to assess whether they are appropriate for NMD adjuvant therapy.Currently,methods have been widely used to evaluate NMD activity,such as the NMD reporter gene system and mRNA sequencing.Conventional NMD reporters reflect the activity of NMD by calculating the ratio of the truncated molecules(PTC contained,NMD sensitive)to full-length molecules(NMD insensitive)at the mRNA or protein level.These NMD reporters could measure NMD activity directly,but there are limitations in measuring clinical samples.Another approach is short-read sequencing,which was represented by Illumina sequencing.Short-read sequencing could reflect NMD activity at the mRNA level.In recent years,the third generation long-read sequencing methods,Oxford Nanopores and Pacbio as the representative,have been developed rapidly.Theoretically,long-read sequencing could detect RNA or DNA directly without reads length limitations.Nanopore direct RNA sequencing provides more information about the full-length sequences,transcriptional isoforms of genes,base modifications,etc.The first part of our study adopted the Nanopore direct RNA sequencing with short-read sequencing to evaluate the activity of NMD and transcripts features.Firstly,we used the traditional NMD reporter to confirm the validity of NMD inhibitors.Then,we performed short-read sequencing using RNA from HEK293T cells treated with DMSO or SMG1i.Our results indicated that short-read sequencing could reflect NMD in several aspects,such as enriched signaling pathways,the biotype of transcripts,etc.However,we did not detect any novel transcripts,and there was no authoritative software to predict whether a specific transcript is a substrate for NMD.We further conducted Nanopore direct RNA sequencing using the same sample as short-read sequencing.We detected about 40%of the novel transcripts and more transcriptional isoforms per gene.We also applied the software FLAIR to analyze the productivity of transcripts and to predict whether they were NMD substrates.The results suggested that transcripts annotated with PTC were significantly upregulated after NMD inhibition.We further screened and chose five transcripts that upregulated markedly after NMD inhibition and the major transcripts of their corresponding gene as our NMD markers.Verification experiments were performed by qRT-PCR in three human cell lines.The results indicated that five markers were significantly increased in the mRNA level after NMD inhibition.We further applied our NMD markers in tumor samples and para-carcinoma samples of nine newly diagnosed prostate cancer patients.Although clinical samples showed wide heterogeneity,the activity of NMD in tumor samples still showed an upward trend in general.To sum up,our results revealed the superiority of long-read sequencing in NMD activity measurement and transcripts features evaluation.The method we established based on Nanopore sequencing results was expected to be applied to measure the NMD activity of the clinical sample and ultimately promote the application of the NMD-targeted adjuvant therapy for tumors or other diseases.Part ⅡTP53 is the most important and first discovered tumor suppressor gene.As a transcription factor,p53 protein could bind to specific DNA sequences and activates its transcription.Consequently,p53 is extensively involved in many aspects of cells,such as apoptotic,cell cycle progression,DNA damage repair,aging,metabolism,etc.The expression level of p53 is mainly under the regulation of MDM2.MDM2 ubiquitinates lysine in the C-terminal of p53 and mediates the degradation of p53,thus keeping p53 at a low level.In addition to MDM2,the E6 protein,which is integrated into host cells and expressed by the high-risk HPV virus,is also involved in p53 inactivation.Besides,missense mutations of p53 are another cause of the dysfunction of p53.Therefore,restoring the function of p53 is an attractive method for cancer therapy.Small molecule inhibitors that block the interaction of p53 and MDM2 have been developed,e.g.,Nutlin3 and its derivatives RG7388 and RG7112,and all of them exhibit excellent antitumor effects in the TP53 wild-type tumor.p53 has 12 isoforms and can be divided into three categories,p53α,p53β,and p53γ.p53αis the full-length p53 protein,carrying the major functions of p53.p53β and p53γ are generated from the alternative splicing of the intron nine.Since the introduction of a premature stop codon,p53β and p53γ are C-terminal truncated proteins with 341 and 345 amino acid residues,respectively.p53β and p53γ are less susceptible to MDM2 mediated degradation due to the lack of ubiquitination sites in the C-terminal.However,p53β and p53γ keep the intact DNA binding domain and the transactivation domain,so they remain the function of the tumor suppressor as full-length p53.Studies have shown that tumors that express p53β or p53γ have a better prognosis than those without p53β or p53γ expression,which implied the importance of these two isoforms.Inhibition of NMD can significantly increase the mRNA and protein expression levels of p53β or p53γ,which can assist p53α in further activating the transcription of p53 target genes.In the second part of this study,we combined the MDM2 inhibitors(XR-2)and NMD inhibitors(SMG1i)in TP53 wide-type tumor cell lines and HPV18 positive cervical cancer cell line HeLa,and all these cell lines achieved profound synergistic effects.The combination of these two agents could significantly reduce cell proliferation,promote cell apoptosis,obstacle cell cycle progression,and disturb DNA repair.We further elucidated the mechanism underlying the synergistic effect.In TP53 wide-type cells,XR-2 and SMG1i upregulated the protein level of p53α and p53β,respectively.p53β modulated the transactivate ability of p53αand increased the expression of p53 target genes,thus achieving the synergistic effects.In HeLa cells,MDM2 inhibitor and NMD inhibitor may act synergistically through the E6 protein or truncated E6 protein.Our study provides a rationale for the MDM2 inhibitors combination in the treatment of TP53 wide-type and HPV-positive tumors. |
