【作者】 刘斌；

【导师】 张灵；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2022， 硕士

【摘要】 背景:准确的RNA转录对维持细胞的生理功能至关重要,其中RNA聚合酶II(RNA polymerase II,RNAPII)对m RNA的合成至关重要。RNA调节因子的异常会诱导恶性肿瘤细胞的状态发生改变,表明其治疗靶点仍有待探索。目前有众多与m RNA转录起点及调控的相关研究,且其与癌症有广泛的影响。然而转录后调控却研究较少,尤其是在RNAPII的延长及终止之间的过渡。最近有研究首次发现两个m RNA抗终止子。作为富含丝氨酸/精氨酸的C末端重复结构域(C-terminal repeat domain,CTD)相关因子之一的SCAF8,在癌症中普遍表达,被首次确认为m RNA的抗终止蛋白。在早期poly A位点中,SCAF4和SCAF8通过维持m RNA的延长和防止m RNA的转录片段分裂,对m RNA转录成熟起着至关重要的作用。研究表明,SCAF4或SCAF8的异常与许多疾病过程有关。已有研究发现,RNA在癌症中受到系统调控,并有助于肿瘤的发生和进展。值得注意的是,在白血病中,一个广泛存在的内含子多聚腺苷酸化,被证明与一组肿瘤抑制因子失活有关。因此,m RNA抗终止子的功能障碍可能在癌症基因组调控中发挥重要作用,但目前仍未有相关研究。本文通过生物信息学分析发现,SCAF8在多种类型的癌症中表达缺失,其中在肾透明细胞癌(KIRC)中,SCAF8低表达与严重的临床病理特征和不良预后相关。结果表明,SCAF8的表达对KIRC的临床预后至关重要,由于转录终止而影响的肿瘤细胞基因组改变仍有待进一步探索。目的:在本研究中以KIRC患者为对象进行生信分析,通过分析SCAF8的表达及与SCAF8缺失相关的基因和信号通路,旨在探讨SCAF8在影响肾透明细胞癌预后中的作用。方法:(1)从TCGA数据库进行患者数据采集,利用UALCAN分析m RNA表达和基因甲基化状态。THPA数据库检测SCAF8蛋白水平。c Bio Portal(cbioportal.org)用于分析SCAF8基因组改变以及SCAF8 m RNA表达与甲基化之间的相关性。(2)采用Wilcoxon秩和检验、Logistic回归分析检验KIRC患者SCAF8表达与临床病理特征的相关性分析。(3)采用ROC(Receiver operating characteristic,ROC)、Kaplan-Meier(KM)生存曲线比较KIRC患者不同组间的总生存期(OS)、疾病特异性生存期(DSS)和无进展间隔期(PFI)。采用Cox回归分析以确定SCAF8表达作为KIRC预后的独立危险因素。采用UALCAN、c Bio Portal、Metascape(metascape.org)对KIRC中与SCAF8表达相关的通路进行富集分析和关键因子筛选。(4)采用R语言统计分析软件(3.6.3版本)及相关软件包进行数据分析和可视化。结果:(1)SCAF8在KIRC患者中基因和蛋白水平均低表达(P<0.001)且与KIRC患者的临床病理学特征相关。(2)在KIRC组织中SCAF8启动子甲基化水平明显增加,且在高临床分期患者的肿瘤组织中,甲基化水平更高,结果具有统计学差异(P<0.001)。SCAF8启动子甲基化水平与SCAF8 m RNA表达水平呈负相关。(3)SCAF8的表达与临床分期(T分期和N分期)、病理分期、性别和血钙水平显著相关,结果具有统计学差异(P<0.05)。(4)SCAF8对KIRC的诊断及不同临床亚型预后具有预测价值,SCAF8低表达患者其总生存期、疾病特异性生存期和无进展间隔期显著缩短(P<0.05),预示着患者预后不良。(5)SCAF8与KIRC个体发育中的关键因子VHL、SETD2及PBRM1呈高度相关,SCAF8作为RNAPII的抗终止子,其缺失可能与这些基因在转录水平上的缺失有关。FABP6和MFSD2A与SCAF8表达呈负相关,提示KIRC患者预后不良。结果均具有统计学差异(P<0.001)。结论:(1)SCAF8在KIRC患者中低表达,且与KIRC患者的临床特征相关。(2)SCAF8对KIRC的诊断及不同临床亚型预后具有预测价值。(3)SCAF8与KIRC个体发育中的关键因子高度相关,其中FABP6和MFSD2A与SCAF8表达呈负相关,FABP6和MFSD2A高表达提示KIRC患者预后不良。更多还原

【Abstract】 Background:Accurate RNA transcription is crucial for maintaining cell physiological function,and RNA polymerase II(RNAPII)is essential for m RNA synthesis.Dysregulation of RNA processing factors in cancer drives malignant cell status,which potentiates their attractive role as therapeutic targets to be explored.At present,there are many studies related to m RNA transcriptional initiation and its regulation,and their relevant impacts on cancer have been widely indicated.However,as the counterpart,post-initiation events of transcription has just been less discussed,especially in the transition between elongating and terminating PNAPII.Recently,two m RNA anti-terminators have been first identified.We observed that SCAF8,one of the serine/arginine rich-related and Cterminal repeat domain(CTD)-associated factors,draws widely alterations among cancers.SCAF4 and SCAF8 are required for correct m RNA transcript maturation through maintaining m RNA elongating and preventing m RNA transcript cleavage in early poly A sites.Studies suggested that aberrant expressions of SCAF4 or SCAF8 are associated with many diseases processes.RNA processing is found systematically regulated in cancer,and contributes to tumorigenesis and progression.Notably,a widespread intronic polyadenylation has been demonstrated responsible for inactivation of a cluster of tumor suppressors in leukemia.Hence,disfunction of m RNA anti-terminator may play an important role in cancer genome regulation.While as we known,no related researches have been reported.Here,we analyzed SCAF8 expression and found a profound reduction in kidney renal clear cell carcinoma(KIRC).Our study also indicated that SCAF8 loss altered a group of genes that might be related to poor KIRC prognosis.In conclusion,we recognized SCAF8 as a potential KIRC biomarker for prognosis,and indicated possible mechanisms influenced by SCAF8 loss in worse clinical outcome.Objective:In this study,we aimed to investigate the prognostic role of SCAF8 in KIRC by analyzing the expression of SCAF8,the genes and signaling pathways associated with loss of SCAF8,with KIRC patients as the object of bioinformatics analysis.Method:(1)Correlated clinicopathological information of KIRC patients and Pan-cancer were obtained from The Cancer Genome Atlas(TCGA)database(portal.gdc.cancer.gov).UALCAN(ualcan.path.uab.edu)was used to analyze m RNA expression and gene methylation status.c Bio Portal(cbioportal.org)was used to analyze SCAF8 genomic alterations and the correlation between SCAF8 m RNA expression and methylation status.(2)Wilcoxon rank sum test and Logistic regression analysis were used to compare and determine the correlation between SCAF8 expression and different clinicopathological characteristics in KIRC patients.(3)Receiver operating characteristic(ROC)and the Kaplan–Meier(K-M)plotter were used to compare overall survival(OS),disease specific survival(DSS)and progression free interval(PFI)between different subgroups in KIRC patients.Univariate and multivariate Cox regression analysis was used to determine SCAF8 expression as an independent risk factor in KIRC prognosis.(4)UALCAN and c Bio Portal were used to screen for genes correlated with SCAF8 expression alteration.Correlated genes of SCAF8 were performed for pathway enrichment analysis by Metascape(metascape.org).(5)R software(Version 3.6.3)and related packages were used for data analyzing and visualizing.P < 0.05 was considered significant threshold.Result:(1)SCAF8 is downregulated in KIRC and associated to clinical characteristics in KIRC patients.(2)Promoter methylation of SCAF8 significantly increased in KIRC tissues,and methylation levels of SCAF8 promoter showed negative correlation with SCAF8 m RNA expression.(3)SCAF8 is associated to clinical characteristics in KIRC patients,which displayed significant lower expression in T stage T3 and T4,N stage N1,M stage M1,clinical stages III-IV,and male sex patients.(4)SCAF8 shows a predictive value for KIRC diagnosis and prognosis,and lower SCAF8 expression was associated with poor overall survival(OS),disease specific survival(DSS)and progression free interval(PFI)in KIRC patients.(5)SCAF8 was highly related with crucial factors in KIRC ontogeny,and FABP6 and MFSD2A were negatively correlated with SCAF8 expression,indicating poor prognosis of KIRC patients.Conclusion:(1)Low SCAF8 expression indicates a poor KIRC prognosis.(2)SCAF8 shows a predictive value for KIRC diagnosis and prognosis.(3)SCAF8 was highly related with crucial factors in KIRC ontogeny,and FABP6 and MFSD2 A were negatively correlated with SCAF8 expression,indicating poor prognosis of KIRC patients.It provides a theoretical basis for further exploring the possible mechanisms of poor prognosis caused by SCAF8 loss.更多还原