【作者】 王永强；

【导师】 洪新雨；

【作者基本信息】 吉林大学 ， 外科学， 2022， 硕士

【摘要】 目的:胶质母细胞瘤(Glioblastoma multiforme,GBM)是一种源自神经上皮的常见的原发性颅内恶性肿瘤,因其复发率高、患者死亡率高而臭名昭著。近年来,虽然在手术切除、放疗和化疗等方面有了一定进步,但由于其强大的侵袭和转移能力而使得这些进展微不足道,GBM患者的预后依然很差。因此,针对GBM的靶向治疗逐渐成为热门。长链非编码RNA(Long non-codingRNA,LncRNA)在多种癌症的发生和发展中发挥着关键作用。小核仁RNA宿主基因1(Small nucleolarRNA host gene 1,SNHG1)在人多种肿瘤中起致癌基因的作用。但SNHG1在GBM中的作用机制鲜为人知。为此,我们研究了SNHG1作为竞争性内源性RNA(competing endogenousRNA,ceRNA)对GBM中上皮间质转化(Epithelial-mesenchymal transition,EMT)过程的调控作用,为靶向治疗人类GBM提供了新思路。研究方法:在本研究中,对HA、H4、HS683、A172和U251细胞系进行细胞培养。采用Real-time PCR方法分别检测SNHG1和RRAS2在HA和胶质瘤细胞系中的内源性表达。构建敲降SNHG1和RRAS2的小干扰RNA载体,以及mi R-128-3p的过表达载体。采用双荧光素酶报告基因实验验证结合位点。采用划痕实验检测细胞的迁移能力。采用Transwell实验检测细胞迁移及侵袭能力。采用Western Blot检测敲降SNHG1和RRAS2后EMT相关蛋白的表达变化。统计学分析采用软件Graph Pad Prism 8.0分析数据和绘图。结果:1.TCGA数据库分析发现,与5个正常脑组织相比,SNHG1在156胶质瘤组织中表达明显升高(***p<0.001),通过q RT-PCR检测SNHG1在胶质瘤细胞中过表达(**p<0.01)。2.划痕实验与Transwell实验发现SNHG1增强GBM细胞的迁移和侵袭能力。Western Blot实验检测敲降SNHG1后GBM细胞中EMT相关蛋白表达降低。3.通过TCGA和KEGG分析构建LncRNA-miRNA-mRNA的ceRNA网络发现可能存在SNHG1/mi R-128-3p/RRAS2轴通路。4.TCGA数据库分析相关性分析发现mi R-128-3p的表达量与SNHG1呈显著负相关。敲降SNHG1后增强了mi R-128-3p的表达,证实SNHG1作为mi R-128-3P的海绵,mi R-128-3p的表达在GBM细胞中被下调。5.荧光素酶报告基因实验验证mi R-128-3p可通过与SNHG1 mRNA的3’UTR区域直接结合而降低SNHG1的表达水平,从而抑制GBM细胞的迁移和侵袭。6.TCGA数据库分析相关性分析发现RRAS2的表达量与mi R-128-3p呈显著负相关(P=0.02)。过表达mi R-128-3p后抑制了RRAS2的表达,证实mi R-128-3p可靶向调控RRAS2。7.Transwell实验发现敲降RRAS2后抑制了GBM细胞的侵袭和转移能力。Western Blot实验检测敲降RRAS2后GBM细胞中EMT相关蛋白表达降低。8.通过q RT-PCR检测发现敲降RRAS2也能够降低SNHG1的表达水平。结论:1.SNHG1作为促癌基因可促进GBM细胞恶性生物学行为。同时,SNHG1促进了GBM中的EMT过程。2.Mi R-128-3p可抑制GBM恶性生物学行为,SNHG1结合下调mi R-128-3p的表达发挥调控作用。3.RRAS2作为促癌基因可促进GBM细胞恶性生物学行为,mi R-128-3p可作用于RRAS2发挥生物学效应。同时,RRAS2促进了GBM细胞中的EMT过程。SNHG1通过抑制mi R-128-3p调节RRAS2的表达水平。4.综上,SNHG1通过SNHG1/mi R-128-3p/RRAS2轴激活EMT过程,进而促进GBM的恶性进展。更多还原

【Abstract】 Purpose:Glioblastoma multiforme(GBM)is a common primary intracranial malignancy of neuroepithelial origin,notorious for its high recurrence rate and high patient mortality.In recent years,although there have been advances in surgical resection,radiotherapy and chemotherapy,these advances have been rendered insignificant due to its powerful invasive and metastatic capabilities,and the prognosis of GBM patients remains poor.As a result,targeted therapies for GBM have become increasingly popular.Long non-codingRNA(LncRNA)plays a key role in the development and progression of many cancers,and small nucleolarRNA host gene 1(SNHG1)acts as an oncogene in many human tumors.However,the mechanism of action of SNHG1 in GBM is poorly understood.To this end,we investigated the role of SNHG1 as a competitive endogenousRNA(ceRNA)in regulating the Epithelial-mesenchymal transition(EMT)process in GBM mother cells,providing new ideas for the targeted treatment of human GBM.Methods:In this study,cell culture was performed on HA,H4,HS683,A172 and U251 cell lines.Real-time PCR was used to detect the endogenous expression of SNHG1 and RRAS2 in HA and glioma cell lines,respectively.Small interferingRNA vectors for knocking down SNHG1 and RRAS2 and overexpression vector for miR-128-3p were constructed.The binding sites were verified using a dual luciferase reporter gene assay.The migration ability of the cells was examined using a scratch assay.Transwell assay was used to detect cell migration and invasion ability.Western Blot was used to detect changes in the expression of EMT-related proteins after knockdown of SNHG1 and RRAS2.Statistical analysis was performed using the software Graph Pad Prism 8.0 to analyze data and plots.Results:1.TCGA database analysis revealed that SNHG1 expression was significantly higher in 156 glioma tissues compared to 5 normal brain tissues(***P < 0.001),and SNHG1 overexpression was detected in glioma cells by q RT-PCR(**P < 0.01).2.SNHG1 was found to enhance the migration and invasion ability of GBM cells by scratch assay and Transwell assay,and the expression of EMT-related protein was decreased in GBM cells after knocking down SNHG1 by Western Blot assay.3.The construction of LncRNA-miRNA-mRNA ceRNA network by TCGA and KEGG analysis revealed the possible existence of SNHG1/miR-128-3p/RRAS2 axis pathway.4.TCGA database analysis correlation analysis revealed that the expression of miR-128-3p was significantly and negatively correlated with SNHG1.Knockdown of SNHG1 enhanced miR-128-3p expression,confirming that SNHG1 acts as a sponge for miR-128-3P and miR-128-3p expression was downregulated in GBM cells.5.The luciferase reporter gene assay verified that miR-128-3p could reduce the expression level of SNHG1 by directly binding to the 3’UTR region of SNHG1 mRNA,thus inhibiting the migration and invasion of GBM cells.6.TCGA database analysis correlation analysis revealed that the expression of RRAS2 was significantly negatively correlated with miR-128-3p(P=0.02).Overexpression of miR-128-3p suppressed the expression of RRAS2,confirming that miR-128-3p could target and regulate RRAS2.7.Transwell assay revealed that knockdown of RRAS2 inhibited the invasion and metastatic ability of GBM cells.western blot assay detected that knockdown of RRAS2 decreased the expression of EMT-related proteins in GBM cells.8.QRT-PCR assay revealed that knockdown of RRAS2 could also reduce the expression level of SNHG1.Conclusions:1.SNHG1 as a pro-oncogene promotes malignant biological behavior of GBM cells.Meanwhile,SNHG1 promotes the EMT process in GBM.2.MiR-128-3p can inhibit the malignant biological behavior of GBM,and SNHG1 combined with down-regulation of miR-128-3p expression plays a regulatory role.3.MiR-128-3p acts as a pro-oncogene to promote the malignant biological behavior of GBM cells.miR-128-3p acts on RRAS2 to exert biological effects.Meanwhile,RRAS2 promotes the EMT process in GBM cells.SNHG1 regulates the expression level of RRAS2 by suppressing miR-128-3p.4.In summary,SNHG1 activates the EMT process through the SNHG1/miR-128-3p/RRAS2 axis,which in turn promotes the expression level of RRAS2 is regulated by SNHG1.更多还原