【作者】 刘超；

【导师】 郑克彬；

【作者基本信息】 河北大学 ， 临床医学硕士（专业学位）， 2023， 硕士

【摘要】 研究背景:神经胶质瘤是中枢神经系统中常见的恶性肿瘤,尽管在胶质瘤治疗中,手术治疗联合放疗或化疗取得进展,但是患者复发率较高,预后较差,其原因除了胶质瘤增殖快、侵袭广、肿瘤内和肿瘤间遗传异质性、治疗耐药外,还源于对分子发病机制认识不深,缺乏及时诊断的手段。因此,阐明胶质瘤发生发展的分子机制,以及进一步探索可靠的诊断和治疗方法是至关重要的。Circ RNAs是一类新的具有闭环结构的内源性非编RNA,主要是由pre-m RNAs通过反向剪接产生的。circ RNAs最初被认为是RNA拼接的副产品。近年来,随着更多具有重要功能的circ RNAs的发现,人们对circ RNAs的性质和功能产生了极大的兴趣。此外,circ RNA表现出组织和发育阶段特异性的表达,并且它们的表达通常不依赖于相关的线性异构体。目前已有多项研究表明,circ RNA在大脑中的表达最丰富,在脑功能和脑疾病中发挥重要作用。虽然大多数circ RNAs的确切功能尚不清楚,但越来越多的证据表明,circ RNAs可能在多个水平上调节基因的表达,如调节其亲代基因的转录,影响其线性同源基因的剪接,作为mi RNA海绵,调节RNA结合蛋白的功能,以及编码肽。据报道,circ RNAs参与各种生理和病理过程,特别是在癌症中。许多研究表明,circ RNAs在包括胶质瘤在内的肿瘤中异常表达,在肿瘤的发生和发展中起着至关重要的作用。有研究发现hsa＿circ＿0006988在胰腺癌、胃癌、以及非小细胞肺癌中具有调节功能,但其对于胶质瘤的发病机制仍未完全明确。课题组前期对河北大学附属医院神经外科术后5对胶质母细胞瘤组织及5对正常脑组织circ RNA测序发现,hsa＿circ＿0006988在GBM中的基础表达水平很高,且与正常脑组织相比较,两者的表达水平具有差异性,其在GBM中差异性高表达,因此选取hsa＿circ＿0006988作为实验目标circ RNA分子。方法:课题组前期研究发现,hsa＿circ＿0006988在GBM中差异性高表达,因此选取hsa＿circ＿0006988作为实验目标circ RNA分子。通过实时荧光定量聚合酶链反应(quantitative Real-time polymerase chain reaction,q RT-PCR)检测hsa＿circ＿0006988在正常脑胶质细胞HA、胶质母细胞株A172和U343中的表达的相对表达水平。通过使用si-NC、si-hsa＿circ＿0006988转染A172和U343胶质母细胞,敲低胶质母细胞内hsa＿circ＿0006988相对表达量,q RT-PCR验证敲低效果,CCK-8实验、流式细胞实验、Transwell实验检测不同处理的胶质母细胞的增殖、凋亡、侵袭和迁移能力变化。通过蛋白质印迹法(Western Blot,WB)检测β-catenin、activeβ-catenin蛋白的相对表达量。结果:与正常胶质细胞HA相比,hsa＿circ＿0006988在胶质母细胞A172和U343中表达量明显增高,且以A172细胞系表达更高(P<0.01)。在A172细胞系中转染si-hsa＿circ＿0006988后,其表达水平明显降低(P<0.01)。CCK-8实验结果显示,沉默hsa＿circ＿0006988的表达后,A172细胞实验组在24 h和48 h的增殖活性明显低于对照组(P<0.05),且实验组在72 h的增殖活性显著低于对照组(P<0.01),结果显示,沉默hsa＿circ＿0006988的表达后,A172细胞的增殖能力较对照组明显降低(P<0.01)。流式细胞实验结果显示,沉默hsa＿circ＿0006988的表达后,A172细胞的凋亡能力较对照组明显增高(P<0.01)。Transwell实验结果显示,沉默hsa＿circ＿0006988的表达后,A172细胞的侵袭和迁移能力较对照组明显降低(P<0.01)。Western blot实验结果显示,沉默hsa＿circ＿0006988的表达后,A172细胞系中的β-catenin、activeβ-catenin蛋白表达显著降低(P<0.05)。结论:hsa＿circ＿0006988在胶质母A172细胞中高表达,沉默hsa＿circ＿0006988表达可显著抑制胶质母细胞的增殖和侵袭,并促进其凋亡,其机制可能是hsa＿circ＿0006988通过β-catenin信号通路影响胶质母细胞的增殖凋亡、侵袭和迁移能力,这为胶质母细胞瘤的治疗提供了新的治疗靶点。更多还原

【Abstract】 Background: Glioma is a common malignant tumour in the central nervous system.Despite advances in glioma treatment with surgical treatment combined with radiotherapy or chemotherapy,patients have a high recurrence rate and a poor prognosis due to a lack of understanding of the molecular pathogenesis and a lack of means for timely diagnosis,in addition to the rapid proliferation,widespread invasion,intra-and inter-tumour genetic heterogeneity and treatment resistance of gliomas.Therefore,it is crucial to elucidate the molecular mechanisms of glioma development and to further explore reliable diagnostic and therapeutic approaches.Circ RNAs are a new class of endogenous non-codified RNAs with a closed-loop structure,mainly produced by reverse splicing of pre-m RNAs.circ RNAs were initially thought to be by-products of RNA splicing.In recent years,with the discovery of more functionally important circ RNAs,there has been a great deal of interest in the nature and function of circ RNAs.Furthermore,circ RNAs exhibit tissue-and developmental stage-specific expression,and their expression is often independent of the associated linear isoforms.Several studies have now shown that circ RNAs are most abundantly expressed in the brain and play an important role in brain function and brain disease.Although the exact function of most circ RNAs is unknown,there is growing evidence that circ RNAs may regulate gene expression at multiple levels,such as regulating the transcription of their parental genes,influencing the splicing of their linear homologues,acting as mi RNA sponges,regulating the function of RNA-binding proteins,and encoding peptides.Circ RNAs have been reported to be involved in a variety of physiological and pathological processes,particularly in cancer.Many studies have shown that circ RNAs are aberrantly expressed in tumours,including gliomas,and play a critical role in tumourigenesis and progression.Some studies have found that hsa＿circ＿0006988 has regulatory functions in pancreatic cancer,gastric cancer,and non-small cell lung cancer,but its pathogenesis for glioma is still not fully understood.The group’s preliminary sequencing of circ RNA in five pairs of glioma tissues and five pairs of normal brain tissues after neurosurgery at the Affiliated Hospital of Hebei University revealed that hsa＿circ＿0006988 had high basal expression levels in GBM and its differential high expression levels in GBM when compared with normal brain tissues.Therefore,hsa＿circ＿0006988 was selected as the target circ RNA molecule for the experiment.Methods: The relative expression of hsa＿circ＿0006988 in glioma cells was knocked down by transfection of A172 and U343 glioma cells with si-NC and si-hsa＿circ＿0006988.q RT-PCR was used to verify the knockdown effect.CCK-8 assay,flow cytometry assay and Transwell assay were performed to detect changes in the proliferation,apoptosis,invasion and migration ability of the glioma cells treated with different treatments.The relative expression of β-catenin and active β-catenin proteins was measured by protein blotting(Western Blot,WB).Results: Compared with normal glial cell HA,hsa＿circ＿0006988 expression was significantly higher in glioma A172 and U343 cells,and was higher in the A172 cell line(P <0.01).After transfection of si-hsa＿circ＿0006988 in the A172 cell line,its expression level was significantly reduced(P < 0.01).The results of CCK-8 assay showed that after silencing the expression of hsa＿circ＿0006988,the proliferative activity of the experimental group of A172 cells at 24 h and 48 h was significantly lower than that of the control group(P<0.05),and the proliferation of the experimental group at 72 h was significantly lower than that of the control group(P<0.05).The proliferation activity at 72 h was significantly lower in the experimental group than in the control group(P<0.01).The results showed that the proliferation ability of A172 cells was significantly reduced after silencing the expression of hsa＿circ＿0006988compared with the control group(P<0.01).The results of flow cytometry assay showed that the apoptotic ability of A172 cells was significantly increased after silencing the expression of hsa＿circ＿0006988 compared with the control group(P<0.01).Transwell assay results showed that silencing the expression of hsa＿circ＿0006988 significantly decreased the invasion and migration ability of A172 cells compared with the control group(P<0.01).Western Blot assay showed that the expression of β-catenin,active β-catenin protein in A172 cell line was significantly reduced after silencing the expression of hsa＿circ＿0006988(P<0.05).Conclusion: hsa＿circ＿0006988 is highly expressed in glioma A172 cells.Silencing hsa＿circ＿0006988 expression significantly inhibited the proliferation and invasion of glioma cells and promoted their apoptosis.The mechanism may be that hsa＿circ＿0006988 affects the proliferation,apoptosis,invasion and migration of glioma cells through the β-catenin signaling pathway,which provides a new therapeutic target for the treatment of glioma.更多还原