【作者】 高杰；

【导师】 胡孔旺；

【作者基本信息】 安徽医科大学 ， 普通外科学（专业学位）， 2024， 博士

【摘要】 研究背景在结直肠癌(CRC)中,RAC1信号通路在肿瘤细胞的增殖、迁移和侵袭中发挥关键作用。RAC1是一种小GTP结合蛋白,通过调节WNT、MAPK/ERK和PI3K/AKT等信号通路,影响肿瘤的发生和发展。研究表明,RAC1的过度激活与肿瘤的耐药性和免疫逃避密切相关,使其成为潜在的治疗靶点。环状RNA(circ RNA)是一类具有稳定环状结构的非编码RNA,广泛存在于多种细胞中。circ RNA可以作为微小RNA(miRNA)的海绵,调节基因表达。在CRC中,特定的circ RNA通过结合miRNA,影响RAC1的功能,从而参与肿瘤的进展。鉴于RAC1在CRC中的多重作用,以及circ RNA在调节RAC1功能中的作用,深入研究circ RNA与RAC1的相互作用机制,有望为CRC的治疗提供新的策略和靶点。研究目的本研究旨在探讨环状RNA circ＿0058123在结直肠癌(CRC)中的功能及其分子机制,重点研究其通过吸附miR-939-5p调控Rac1信号通路的作用。研究结合生物信息学预测与分子实验,验证了circ＿0058123—miR-939-5p—Rac1这一信号轴在CRC细胞的增殖、迁移、侵袭和血管生成中的关键调控作用。同时,通过体外细胞实验和体内动物实验进一步验证了circ＿0058123作为CRC潜在治疗靶点的价值。研究方法(1)信号通路分析:利用Circbank和Circular RNA Interactome数据库预测circ＿0058123的潜在靶向miRNA,通过RNA pull-down实验筛选出与circ＿0058123结合力最强的miRNA。使用Target Scan Human7.2预测该miRNA的下游靶基因,并根据Rac1结合位点推测可能的上游miRNA。进一步,通过双荧光素酶报告基因实验和RIP实验确认miR-939-5p与circ＿0058123以及miR-939-5p和RAC1的相互作用。(2)circ＿0058123/miR-939-5p/RAC1轴的分子机制及功能研究:培养SW480和HCT-116两组细胞,使用qRT-PCR和Western blot检测上述两组细胞,以及结直肠癌标本CRC细胞和配对正常细胞中circ＿0058123、miR-939-5p、RAC1的表达水平,通过si RNA技术敲低circ＿0058123,以qRT-PCR检测其表达、EDU实验检测其对细胞增殖影响、并通过迁移及侵袭实验、血管生成实验、细胞凋亡实验、以及Western blot检测BAX和Bcl2蛋白表达情况来多方面评估敲低circ＿0058123对CRC细胞功能影响;通过敲低miR-939-5p,qRT-PCR验证敲低效率,重复上述实验评估细胞功能变化情况;通过转染miR-939-5p mimic,以Western blot检测Rac1蛋白表达情况,并分别转染miR-939-5p mimic和pc-RAC1,评价各组SW480、HCT-116细胞的血管生成、细胞凋亡、凋亡蛋白表达、迁移及侵袭等情况。(3)动物实验:将12只雄性裸鼠(18-20 g)随机分为sh-NC组和sh-circ＿0058123组,每组注射2×10^6稳定转染的SW480细胞。每7天测量肿瘤体积,五周后处死裸鼠并称重肿瘤组织。对肿瘤组织进行免疫组化染色,使用Ki67标记增殖细胞,利用共聚焦显微镜观察并分析结果。qRT-PCR和Western blot检测肿瘤细胞RAC1表达情况。研究结果(1)信号通路验证:研究首先检测CRC细胞和正常细胞的circ＿0058123表达情况(t=-9.511,p<0.001),并检测FHC、SW480、HCT-116细胞的circ＿0058123表达情况(F=25.187,p<0.05),证实circ＿0058123在结直肠癌细胞中表达明显升高,随后通过预测circ＿0058123的潜在靶向miRNA,发现了与其相关的376个已知miRNA。在这些miRNA中,筛选出相对评分较高的一组miRNA,选定hsa-miR-939-5p、hsa-miR-613、hsa-miR-605-5p、hsa-miR-590-5p和hsa-miR-574-3p作为circ＿0058123的潜在下游调控miRNA,Target Scan Human7.2工具进一步预测,通过分析RAC1的3’UTR,确认了这些miRNA可能与其结合。为了验证这一假设,进行了RNA pull-down实验,结果显示miR-939-5p与circ＿0058123的结合最强。因此,miR-939-5p被选择作为进一步研究circ＿0058123靶向miRNA的对象。通过RIP实验揭示了circ-0058123和miR-939-5p在结直肠癌细胞中与AGO2的特异性相互作用;双荧光素酶报告基因实验成功验证了miR-939-5p与RAC1的结合关系。(2)circ＿0058123的CRC细胞功能分析:qRT-PCR检测CRC细胞和正常细胞circ＿0058123可得出其在CRC,尤其是III期CRC细胞中表达明显增加,Ed U测定结果表明circ＿0058123的敲低显著减少了CRC细胞的增殖(SW480:t=10.22,p<0.001;HCT-116:t=15.93,p<0.001)。Transwell实验进一步显示,与si-NC组相比,si-circ＿0058123显著抑制了CRC细胞的迁移和侵袭(迁移试验SW480:t=7.08,p=0.002;HCT-116:t=15.44,p=0.001;侵袭试验SW480:t=13.36,p<0.001;HCT-116:t=15.15,p<0.001)。此外,circ＿0058123的下调明显抑制了CRC细胞的血管生成(SW480细胞:t=8.37,p=0.001;HCT-116细胞:t=12.99,p<0.001),而si-circ＿0058123转染显著提高了CRC细胞的凋亡率(t=-23.94、p<0.0001;t=-30.86,p<0.001)。circ＿0058123沉默提高了Bax蛋白的表达水平,同时降低了Bcl-2蛋白的表达水平(SW480细胞:Bax蛋白t=-13.44,p<0.001,Bcl-2蛋白t=-13.44,p<0.001;HCT-116细胞:t=-14.94,p<0.001,Bcl-2蛋白t=10.38,p<0.001)。(3)miR-939-5p的CRC细胞功能分析:qRT-PCR检测CRC细胞和正常细胞miR-939-5p可得出其在CRC细胞中表达降低,Ed U测定发现,circ＿0058123敲低显著抑制了CRC细胞的增殖,而转染miR-939-5p抑制剂后增殖增加明显。Transwell检测结果显示circ＿0058123敲低抑制了CRC细胞的迁移和侵袭,而miR-939-5p可以逆转这一抑制作用(迁移试验,SW480细胞:F=116.6,p<0.001;HCT-116细胞:F=105.72,p<0.001;侵袭试验,SW480细胞:F=102.55,p<0.001;HCT-116细胞:F=101.40,p<0.001)。管形成实验结果表明,circ＿0058123沉默对CRC细胞血管生成的抑制作用可以通过下调miR-939-5p来逆转(SW480细胞:t=7.34,p<0.01;HCT-116细胞:t=13.20,p<0.001)。类似地,circ＿0058123敲低引起的促凋亡作用被miR-939-5p抑制剂部分抵消。总体而言,circ＿0058123通过miR-939-5p影响了CRC的增殖、迁移、侵袭、血管生成和凋亡等功能。(4)RAC1的CRC细胞功能分析:qRT-PCR和Western blot检测正常和CRC组织中RAC1表达情况可知其在CRC细胞中表达明显上调,上面的结果我们可知miR-939-5p会抑制CRC细胞增殖及侵袭,而RAC1的过表达逆转了miR-939-5p模拟物对CRC细胞进程的抑制效果。通过pc-RAC1转染后,RAC1蛋白表达水平显著增加(SW480细胞:t=-17.94,p<0.001;HCT-116细胞:t=-24.76,p<0.001)。功能实验验证了miR-939-5p对CRC细胞的增殖、迁移和侵袭的抑制作用,而RAC1过表达则明显恢复了增殖、迁移和侵袭特性。血管生成抑制和促进凋亡的效果在RAC1过表达条件下被显著减弱,验证了miR-939-5p通过RAC1通路调控CRC细胞生物学功能的机制。(5)体内实验:测量肿瘤体积和重量,使用qRT-PCR、western blot、IHC等方法检测裸鼠肿瘤组织中circ＿0058123、miR-939-5p、RAC1和Ki67的表达。结果显示sh-circ＿0058123组相较于sh-NC组,肿瘤体积和重量明显下降,circ＿0058123、RAC1和Ki67的表达水平降低,而miR-939-5p的表达明显升高。研究结论(1)通过生物信息学网站的预测,我们证实了circ＿0058123与miR-939-5p以及Rac1之间存在一个信号通路。通过RNA pull-down实验、双荧光素酶报告实验和RIP实验的验证,我们确认了该通路在结直肠癌(CRC)细胞中是一个完整的信号传递通路。(2)分子及功能层面显示,circ＿0058123、RAC1在CRC细胞中高表达、miR-939-5p表现为低表达,circ＿0058123的高表达在CRC细胞中促进了增殖、迁移、侵袭和血管生成,且其敲低显著抑制这些过程并诱导细胞凋亡。miR-939-5p作为该通路中的关键调节因子,通过靶向RAC1来介导circ＿0058123对CRC细胞的生物学功能调控。(3)在体内试验中,circ＿0058123的上调显著降低了miR-939-5p的表达,导致RAC1表达的增加,从而促进肿瘤的生长和进展。(4)本研究首次系统地证明了circ＿0058123通过吸附miR-939-5p调控RAC1表达,并在CRC细胞中调节增殖、迁移、侵袭和血管生成等关键生物学过程。更多还原

【Abstract】 Research BackgroundIn colorectal cancer(CRC),the RAC1 signaling pathway plays a crucial role in tumor cell proliferation,migration,and invasion.RAC1,a small GTP-binding protein,influences tumor development and progression by regulating signaling pathways such as WNT,MAPK/ERK,and PI3K/AKT.Studies have shown that the overactivation of RAC1 is closely associated with tumor resistance and immune evasion,making it a potential therapeutic target.Circular RNAs(circ RNAs)are a class of non-coding RNAs with a stable circular structure that are widely found in various cells.Circ RNAs can act as sponges for micro RNAs(miRNAs),regulating gene expression.In CRC,specific circ RNAs can bind to miRNAs,affecting RAC1 function and thereby participating in tumor progression.Given RAC1’s multifaceted role in CRC and the involvement of circ RNAs in modulating RAC1 function,exploring the interaction mechanisms between circ RNAs and RAC1 may provide novel strategies and targets for CRC treatment.Research ObjectiveThis study aims to investigate the function and molecular mechanisms of circular RNA circ＿0058123 in CRC,focusing on its role in regulating the RAC1 signaling pathway by sponging miR-939-5p.The study combines bioinformatics prediction with molecular experiments to verify the regulatory axis of circ＿0058123 — miR-939-5p — RAC1 in CRC cell proliferation,migration,invasion,and angiogenesis.Furthermore,in vitro cell experiments and in vivo animal models will be employed to validate circ＿0058123 as a potential therapeutic target for CRC.Research Methods(1)Signaling Pathway Analysis: The potential target miRNAs of circ＿0058123 were predicted using the Circbank and Circular RNA Interactome databases.RNA pull-down experiments were used to select the miRNA with the strongest binding affinity to circ＿0058123.Target Scan Human7.2 was used to predict the downstream target genes of this miRNA and to infer possible upstream miRNAs based on the Rac1 binding site.Further,the interaction between miR-939-5p and circ＿0058123 as well as between miR-939-5p and RAC1 was confirmed through dual-luciferase reporter gene assays and RIP experiments.(2)Molecular Mechanism and Functional Study of the circ＿0058123/miR-939-5p/RAC1 Axis: SW480 and HCT-116 cell lines were cultured,and the expression levels of circ＿0058123,miR-939-5p,and RAC1 in these cells,as well as in CRC cell and matched normal cells,were detected using qRT-PCR and Western blot.Knockdown of circ＿0058123 was performed using si RNA technology,and its effects on cell proliferation(EDU assay),migration and invasion(transwell assay),angiogenesis,apoptosis(Western blot for BAX and Bcl2 protein expression),and other cellular functions were assessed.Knockdown efficiency of miR-939-5p was verified by qRT-PCR,and the same experiments were repeated to evaluate changes in cellular functions.Overexpression of miR-939-5p was achieved by transfection of miR-939-5p mimic,and the expression of Rac1 protein was detected by Western blot.The effects of miR-939-5p mimic and pc-RAC1 transfection on angiogenesis,apoptosis,apoptotic protein expression,migration,and invasion in SW480 and HCT-116 cells were evaluated.(3)Animal Experiments: Twelve male nude mice(18-20 g)were randomly divided into sh-NC and sh-circ＿0058123 groups,with each group injected with 2×10^6 stably transfected SW480 cells.Tumor volume was measured every 7 days,and after five weeks,the nude mice were sacrificed,and tumor tissue was weighed.Immunohistochemical staining was performed on tumor tissue,with Ki67 marking proliferating cells,and results were observed and analyzed using confocal microscopy.qRT-PCR and Western blot were used to detect the expression of RAC1 in tumor cells.Research Results(1)Signaling Pathway Validation: The study initially detected the expression of circ＿0058123 in CRC cells and normal cells(t =-9.511,p < 0.001),and the expression of circ＿0058123 in FHC,SW480,and HCT-116 cells(F = 25.187,p < 0.05),confirming that circ＿0058123 is significantly upregulated in colorectal cancer cells.Subsequently,by predicting the potential target miRNAs of circ＿0058123,376 known miRNAs related to it were identified.Among these miRNAs,a group with relatively high scores was selected,and hsa-miR-939-5p,hsa-miR-613,hsa-miR-605-5p,hsa-miR-590-5p,and hsa-miR-574-3p were chosen as potential downstream regulatory miRNAs of circ＿0058123.Target Scan Human7.2 tool further predicted that these miRNAs might bind to RAC1 by analyzing its 3’UTR.To verify this hypothesis,RNA pull-down experiments were conducted,showing that miR-939-5p has the strongest binding to circ＿0058123.Therefore,miR-939-5p was selected for further study as a target miRNA of circ＿0058123.RIP experiments revealed the specific interaction between circ-0058123 and miR-939-5p with AGO2 in colorectal cancer cells;dual-luciferase reporter gene assays successfully validated the binding relationship between miR-939-5p and RAC1.(2)Functional Analysis of circ＿0058123 in CRC Cells: qRT-PCR detection of CRC cells and normal cells for circ＿0058123 indicated a significant increase in its expression in CRC,especially in stage IV CRC cells.Ed U assay results showed that the knockdown of circ＿0058123 significantly reduced the proliferation of CRC cells(SW480: t = 10.22,p < 0.001;HCT-116: t = 15.93,p < 0.001).Transwell assays further demonstrated that si-circ＿0058123 significantly inhibited the migration and invasion of CRC cells compared to the si-NC group(migration assay SW480: t = 7.08,p = 0.002;HCT-116: t = 15.44,p = 0.001;invasion assay SW480: t = 13.36,p < 0.001;HCT-116:t = 15.15,p < 0.001).Additionally,the downregulation of circ＿0058123 significantly inhibited angiogenesis in CRC cells(SW480 cells: t = 8.37,p = 0.001;HCT-116 cells: t= 12.99,p < 0.001),and transfection with si-circ＿0058123 significantly increased the apoptosis rate of CRC cells(t =-23.94,p < 0.0001;t =-30.86,p < 0.001).Silence of circ＿0058123 increased the expression level of Bax protein while decreasing the expression level of Bcl-2 protein(SW480 cells: Bax protein t =-13.44,p < 0.001,Bcl-2protein t =-13.44,p < 0.001;HCT-116 cells: t =-14.94,p < 0.001,Bcl-2 protein t =10.38,p < 0.001).(3)Functional Analysis of miR-939-5p in CRC Cells: qRT-PCR detection of CRC cells and normal cells for miR-939-5p indicated a decrease in its expression in CRC cells.Ed U assay found that the knockdown of circ＿0058123 significantly inhibited the proliferation of CRC cells,while transfection with miR-939-5p inhibitor significantly increased proliferation.Transwell detection results showed that the knockdown of circ＿0058123 inhibited the migration and invasion of CRC cells,and miR-939-5p could reverse this inhibitory effect(migration assay,SW480 cells: F = 116.6,p < 0.001;HCT-116 cells: F = 105.72,p < 0.001;invasion assay,SW480 cells: F = 102.55,p <0.001;HCT-116 cells: F = 101.40,p < 0.001).Tube formation assay results indicated that the inhibitory effect of circ＿0058123 silence on angiogenesis in CRC cells could be reversed by downregulating miR-939-5p(SW480 cells: t = 7.34,p < 0.01;HCT-116 cells: t = 13.20,p < 0.001).Similarly,the pro-apoptotic effect induced by the knockdown of circ＿0058123 was partially counteracted by the miR-939-5p inhibitor.Overall,circ＿0058123 affects the functions of CRC such as proliferation,migration,invasion,angiogenesis,and apoptosis through miR-939-5p.(4)Functional Analysis of RAC1 in CRC Cells: qRT-PCR and Western blot detection of RAC1 expression in normal and CRC tissues showed a significant upregulation in CRC cells.The aforementioned results indicated that miR-939-5p inhibits the proliferation and invasion of CRC cells,while the overexpression of RAC1 reverses the inhibitory effects of miR-939-5p mimics on CRC cell processes.Transfection with pc-RAC1 significantly increased the expression level of RAC1 protein(SW480 cells: t=-17.94,p < 0.001;HCT-116 cells: t =-24.76,p < 0.001).Functional experiments confirmed the inhibitory effects of miR-939-5p on the proliferation,migration,and invasion of CRC cells,while the overexpression of RAC1 significantly restored these characteristics.The inhibitory effects on angiogenesis and the promotion of apoptosis were significantly weakened under the condition of RAC1 overexpression,verifying the mechanism by which miR-939-5p regulates the biological functions of CRC cells through the RAC1 pathway.(5)In Vivo Experiments: Tumor volume and weight were measured,and the expression of circ＿0058123,miR-939-5p,RAC1,and Ki67 in nude mouse tumor tissue was detected using qRT-PCR,Western blot,and IHC methods.The results showed that compared to the sh-NC group,the sh-circ＿0058123 group had significantly reduced tumor volume and weight,with decreased expression levels of circ＿0058123,RAC1,and Ki67,and a significant increase in the expression of miR-939-5p.Research Conclusions(1)Through predictions from bioinformatics websites,we confirmed the existence of a signaling pathway between circ＿0058123,miR-939-5p,and Rac1.Validation through RNA pull-down experiments,dual-luciferase reporter assays,and RIP experiments confirmed that this pathway is an intact signal transduction pathway in colorectal cancer(CRC)cells.(2)At the molecular and functional levels,circ＿0058123 and RAC1 are highly expressed,and miR-939-5p is lowly expressed in CRC cells.High expression of circ＿0058123 promotes proliferation,migration,invasion,and angiogenesis in CRC cells,and its knockdown significantly inhibits these processes and induces apoptosis.miR-939-5p,as a key regulatory factor in this pathway,mediates the biological function regulation of CRC cells by circ＿0058123 by targeting RAC1.(3)In vivo experiments showed that upregulation of circ＿0058123 significantly reduced the expression of miR-939-5p,leading to increased expression of RAC1,thereby promoting tumor growth and progression.(4)This study systematically demonstrated for the first time that circ＿0058123 regulates the expression of RAC1 by adsorbing miR-939-5p and modulates key biological processes such as proliferation,migration,invasion,and angiogenesis in CRC cells.更多还原