【作者】 张振；

【导师】 郭建强；

【作者基本信息】 山东大学 ， 内科学（消化系病）（专业学位）， 2023， 博士

【摘要】 前言我国的结直肠癌发病率和死亡率在逐年上升。多数散发性结直肠癌由癌前病变缓慢发展形成。早期筛查结合有效治疗可以降低发病率和死亡率。当前,手术切除、化疗、放疗、免疫疗法等治疗方法都具有副作用,如术后感染、放化疗毒性、免疫不良反应、复发率高等。因此,结直肠癌的早期诊断及有效治疗显得尤为重要。生物标志物侵入性小,预测疾病更为准确,有助于实现肿瘤的精准靶向治疗。lncRNA在癌症研究中展现了诊断治疗预后标志物的巨大潜力,其具有促进和抑制癌症发展的双重作用。lncRNA通过海绵化miRNA而抑制miRNA对靶标基因的作用,改变靶标基因的表达水平,在肿瘤的发生发展中发挥重要作用。本课题研究了 lncRNA PTOV1-AS2在结直肠癌发生发展中的作用,探讨PTOV1-AS2结合的miRNA及靶标基因之间的相互关系,并揭示了其内在调控机制,为结直肠癌诊断、治疗及预后提供新的靶点。第一部分 结直肠癌中PTOV1-AS2表达与临床相关性目的1.检测PTOV1-AS2在结直肠癌组织和细胞中的表达2.探讨PTOV1-AS2表达与患者临床病理及总生存期的相关性方法1.通过qRT-PCR技术检测PTOV1-AS2在结直肠癌组织和细胞系中的表达,并分析其与正常组织及细胞的表达差异。2.统计结直肠癌患者临床病理参数信息,并分析其与PTOV1-AS2表达水平的相关性。3.基于ENCORI分析PTOV1-AS2不同表达水平的结直肠癌患者的生存曲线。结果1.PTOV1-AS2在结直肠癌组织中的表达显著高于正常结直肠组织(P<0.01);PTOV1-AS2在不同人结直肠癌细胞系中的表达均显著高于正常人肠上皮细胞(P均<0.01)。2.PTOV1-AS2表达与结直肠癌患者的肿瘤大小、脉管侵犯、淋巴结转移、TNM分期显著相关(P均<0.05)。3.PTOV1-AS2高表达结直肠癌患者生存期更短(HR=1.56,P=0.028)。结论1.PTOV1-AS2在结直肠癌组织和细胞中高表达。2.PTOV1-AS2表达与结直肠癌患者的临床病理、生存期相关。第二部分 PTOV1-AS2表达对结直肠癌细胞的生物学功能影响目的1.探究PTOV1-AS2对结直肠癌细胞生物学功能的影响方法1.建立过表达PTOV1-AS2及沉默PTOV1-AS2结直肠癌细胞模型。2.通过qRT-PCR检测各模型组细胞中PTOV1-AS2的表达效率。3.通过CCK8检测、Transwell实验、流式细胞术检测,验证过表达及沉默表达PTOV1-AS2对结直肠癌细胞系增殖、迁移、侵袭及凋亡的影响。结果1.过表达组细胞模型PTOV1-AS2相对表达量均显著升高(P均<0.05);沉默组细胞模型PTOV1-AS2相对表达量均显著降低(P均<0.05)。2.CCK8检测显示,与对照组的OD 450 nm值比较,48、72和96小时时间点PTOV1-AS2过表达组均显著升高(P均<0.05),而PTOV1-AS2沉默组均显著降低(P均<0.05)。3.与对照组的细胞迁移和侵袭数目比较,PTOV1-AS2过表达组均显著升高(P均<0.05),而PTOV1-AS2沉默组均显著降低(P均<0.05)。4.与对照组的细胞总凋亡率比较,PTOV1-AS2过表达组均明显降低(P均<0.05),而PTOV1-AS2沉默组均明显升高(P均<0.05)。结论1.PTOV1-AS2过表达时促进结直肠癌细胞的增殖、迁移和侵袭,抑制细胞的凋亡。2.PTOV1-AS2沉默表达时抑制结直肠癌细胞的增殖、迁移和侵袭,促进细胞的凋亡。第三部分 结直肠癌中PTOV1-AS2通过靶向miR-145-5p调控FSCN1目的1.预测验证PTOV1-AS2靶向结合miR-145-5p及其靶向基因FSCN12.检测miR-145-5p及其靶向基因FSCN1在结直肠癌组织中的表达3.明确 PTOV1-AS2 对 miR-145-5p/FSCN1 的调控作用方法1.通过生物信息学分析预测PTOV1-AS2结合miR-145-5p,以及miR-145-5p靶向基因FSCN1,且通过双荧光素酶实验验证结合作用。2.采用qRT-PCR技术检测miR-145-5p及其靶向基因FSCN1在结直肠癌组织中的表达水平。3.基于过表达PTOV1-AS2结直肠癌细胞系模型,同时转染miR-145-5p mimic,通过Western blot 技术检测 PTOV1-AS2/miR-145-5p 对 FSCN1 表达的影响。结果1.PTOV1-AS2 靶向结合 miR-145-5p,miR-145-5p 靶向基因 FSCN1 3,-UTR 区域。2.miR-145-5p在结直肠癌组织中低表达,FSCN1在结直肠癌组织中高表达(P均<0.05)。3.过表达PTOV1-AS2引起的FSCN1蛋白表达升高,可以被miR-145-5p部分降低(P均<0.05)。结论1.PTOV1-AS2 通过靶向 miR-145-5p 上调 FSCN1 表达。第四部分 PTOV1-AS2/miR-145-5p/FSCN1对结直肠癌的生物学功能影响目的1.检测PTOV1-AS2/miR-145-5p/FSCN1轴对结直肠癌细胞的生物学功能影响2.探究PTOV1-AS2/miR-145-5p/FSCN1轴对结直肠癌细胞裸鼠成瘤能力的影响方法1.基于沉默表达PTOV1-AS2结直肠癌细胞系模型,同时转染miR-145-5p inhibitor或者过表达FSCN1载体。2.通过CCK8检测、Transwell实验、流式细胞术检测,衡量PTOV1-AS2/miR-145-5p/FSCN1轴对结直肠癌细胞增殖、迁移、侵袭及凋亡的影响。3.构建不同组结直肠癌细胞裸鼠皮下成瘤模型。4.记录瘤鼠肿瘤体积和重量。5.通过免疫组化实验检测Ki67在各组瘤体组织中的表达。6.基于Western blot方法检测FSCN1在各组瘤体组织中的表达。结果1.沉默PTOV1-AS2表达导致的结直肠癌细胞增殖、迁移、侵袭能力降低,凋亡水平的升高,可以被沉默miR-145-5p或者过表达FSCN1部分逆转(P均<0.05)。2.沉默PTOV1-AS2表达导致结直肠癌细胞致瘤体积、重量均显著降低(P<0.05);并可以被沉默miR-145-5p或者过表达FSCN1部分逆转(P均<0.05)。3.沉默PTOV1-AS2导致结直肠癌细胞成瘤瘤体中ki67和FSCN1表达显著降低(P<0.05);并可以被沉默miR-145-5p或者过表达FSCN1部分逆转(P均<0.05)。结论1.沉默miR-145-5p或者过表达FSCN1可以部分逆转沉默PTOV1-AS2表达对结直肠癌细胞功能的影响。2.沉默PTOV1-AS2表达抑制结直肠癌细胞致瘤能力。抑制miR-145-5p或者FSCN1过表达时部分逆转了沉默PTOV1-AS2的抑瘤作用。更多还原

【Abstract】 IntroductionThe incidence and mortality rate of colorectal cancer in China are increasing year by year.Most sporadic colorectal cancers are formed by the slow development of precancerous lesions.Early screening combined with effective treatment can reduce morbidity and mortality.Currently,surgical resection,chemotherapy,radiotherapy,immunotherapy and other treatments have side effects,such as postoperative infection,radiotherapy toxicity,immune adverse reactions,and high recurrence rates.Therefore,early diagnosis and effective treatment of colorectal cancer are particularly important.Biomarkers are less invasive and more accurate in predicting disease,which help to achieve precise targeting of tumors.LncRNAs have shown great potential in cancer research as prognostic markers for diagnosis and treatment,with dual roles in promoting and inhibiting cancer development.lncRNAs play an important role in tumor development by sponging miRNAs and thereby inhibiting the effects of miRNAs on target genes and altering the expression levels of target genes.In this study,we investigated the role of lncRNA PTOV1-AS2 in the development of colorectal cancer,explored the interrelationship between PTOV1-AS2-binding miRNAs and target genes,and revealed its intrinsic regulatory mechanism to provide new targets for the diagnosis,treatment and prognosis of colorectal cancer.Part Ⅰ:PTOV1-AS2 expression and clinical relevance in colorectal cancerObjectives1.Detection of PTOV1-AS2 expression in colorectal cancer tissues and cells2.To investigate the association of PTOV1-AS2 expression with patient clinicopathology and overall survival.Methods1.To detect the expression of PTOV1-AS2 in colorectal cancer tissues and cell lines by qRTPCR and to analyze the differences in expression with normal tissues and cells.2.To count the information of clinicopathological parameters of colorectal cancer patients and analyze their correlation with PTOV1-AS2 expression levels.3.To analyze the survival curves of colorectal cancer patients with different expression levels of PTOV1-AS2 based on ENCORI.Results1.The expression of PTOV1-AS2 was significantly higher in colorectal cancer tissues than in normal colorectal tissues(P<0.01).The expression of PTOV1-AS2 was significantly higher in different human colorectal cancer cell lines than in normal human intestinal epithelial cells(all P<0.01).2.PTOV1-AS2 expression was significantly correlated with tumor size,vascular invasion,lymph node metastasis and TNM analysis in colorectal cancer patients(all P<0.05).3.Patients with colorectal cancer with high PTOV1-AS2 expression had shorter survival(HR=1.56,P=0.028).Conclusion1.PTOV1-AS2 is highly expressed in colorectal cancer tissues and cells.2.PTOV1-AS2 expression was correlated with the clinicopathology and survival of colorectal cancer patients.Part Ⅱ:Effect of PTOV1-AS2 expression on the biological function of colorectal cancer cellsObjectives1.Investigating the effect of PTOV1-AS2 on the function of colorectal cancer cellsMethods1.To establish PTOV1-AS2 overexpression and PTOV1-AS2 silencing colorectal cancer cell lines HCT116 and SW620 cell models.2.To detect the expression efficiency of PTOV1-AS2 in each model group of cells by qRT-PCR.3.The effects of overexpression and silent expression of PTOV1-AS2 on proliferation,migration,invasion and apoptosis of colorectal cancer cell lines were measured by CCK8 assay,Transwell assay and flow cytometry assay.Results1.The relative expression of PTOV1-AS2 was significantly higher in the overexpression group(all P<0.05);the relative expression of PTOV1-AS2 was significantly lower in the silencing group(all P<0.05).2.The OD 450 nm values of the control group were significantly higher in the PTOV1-AS2 overexpression group at 48,72 and 96 hours(P<0.05),while the PTOV1-AS2 silencing group was significantly lower(P<0.05).3.The number of cell migration and invasion was significantly higher in the PTOV1-AS2 overexpression group and lower in the PTOV1-AS2 silencing group compared with the control group(both P<0.05).4.The total apoptotic rate was significantly lower in all PTOV1-AS2 overexpression groups and significantly higher in all PTOV1-AS2 silencing groups when compared with the control group(all P<0.05).Conclusion1.PTOV1-AS2 overexpression promoted the proliferation,migration and invasion of colorectal cancer cells and inhibited apoptosis.2.PTOV1-AS2 silent expression inhibited the proliferation,migration and invasion of colorectal cancer cells,and promoted apoptosis.Part Ⅲ:Regulation of FSCN1 by PTOV1-AS2 in colorectal cancer through targeting miR-145-5pObjectives1.Predictive validation of PTOV1-AS2 target binding to miR-145-5p and its target gene FSCN12.Detection of miR-145-5p and its target gene FSCN1 expression in colorectal cancer tissues3.To Clarity the regulatory role of PTOV1-AS2 on miR-145-5p/FSCN1Methods1.The binding miRNA miR-145-5p and its target gene FSCN 1 were predicted by bioinformatics analysis of PTOV1-AS2,and the binding effect was verified by dual luciferase assay.2.qRT-PCR was used to detect the expression levels of miR-145-5p and its target gene FSCN1 in colorectal cancer tissues.3.Based on the overexpression of PTOV1-AS2 colorectal cancer cell line model,miR-145-5p mimic was transfected simultaneously,and the effect of PTOV1-AS2/miR-145-5p on FSCN1 expression was detected by Western blot technique.Results1.PTOV1-AS2 targets binding to miR-145-5p and miR-145-5p targets the 3’-UTR region of gene FSCN 1.2.miR-145-5p was lowly expressed in colorectal cancer tissues and FSCN1 was highly expressed in colorectal cancer tissues(both P<0.05).3.Elevated FSCN1 protein expression caused by overexpression of PTOV1-AS2 could be partially reduced by miR-145-5p(both P<0.05).Conclusion1.PTOV1-AS2 upregulates FSCN1 expression by targeting miR-145-5pPart Ⅳ:Impact of PTOV1-AS2/miR-145-5p/FSCN1 on the biological function of colorectal cancerObjectives1.To detect the effect of PTOV1-AS2/miR-145-5p/FSCN1 axis on the biological function of colorectal cancer cells2.To investigate the effect of PTOV1-AS2/miR-145-5p/FSCN1 axis on the tumorigenic ability of colorectal cancer cells in nude miceMethods1.Based on silent expression of PTOV1-AS2 colorectal cancer cell line model with simultaneous transfection of miR-145-5p inhibitor or overexpression of FSCN1 vector.2.To measure the effect of PTOV1-AS2/miR-145-5p/FSCN1 axis on proliferation,migration,invasion and apoptosis of colorectal cancer cell lines by CCK8 assay,Transwell assay and flow cytometry assay.3.To construct different groups of colorectal cancer cells in a subcutaneous tumorigenic model in nude mice.4.To record the volume and weight of tumor in rats.5.The expression of Ki67 in each group of tumour tissues was detected by immunohistochemistry.6.Detection of FSCN1 expression in each group of tumor tissues based on Western blot method.Results1.Silencing of PTOV1-AS2 expression resulted in reduced proliferation,migration,and invasion ability and increased apoptosis of colorectal cancer cells,which could be partially reversed by silencing miR-145-5p or overexpression of FSCN1(both P<0.05).2.Silencing PTOV1-AS2 expression resulted in a significant decrease in tumorigenic volume and weight of colorectal cancer cells(P<0.05);and could be partially reversed by silencing miR-145-5p or overexpression of FSCN1(both P<0.05).3.Silencing PTOV1-AS2 resulted in a significant decrease in ki67 and FSCN1 expression in tumorigenic tumors of colorectal cancer cells(P<0.05);and could be partially reversed by silencing miR-145-5p or overexpression of FSCN1(both P<0.05).Conclusion1.Silencing miR-145-5p or overexpressing FSCN1 partially reversed the effect of silencing PTOV1-AS2 expression on the function of colorectal cancer cells.2.Silencing of PTOV1-AS2 expression inhibited the tumorigenic ability of colorectal cancer cells.Inhibition of miR-145-5p or FSCN1 overexpression partially reversed the tumor suppressive effect of silencing PTOV1-AS2.更多还原