【作者】 赵剑；

【导师】 张睿；

【作者基本信息】 中国医科大学 ， 外科学， 2023， 博士

【摘要】 目的:结肠癌是全球第三大常见恶性肿瘤,也是全球第四大癌症相关死亡原因。然而,结肠癌发展的详细机制目前尚不完全清楚,因此,探究结肠癌的发病机制对其早期诊断和治疗仍是必要的。lncRNA NFX1型锌指结构反义转录本1(NFX1-type zinc finger-containing protein 1 antisense RNA 1,ZFAS1)是定位于20q13.13号染色体的lncRNA,在结肠癌中表达上调,且其高表达与结肠癌患者TNM分期、淋巴浸润和较差的预后有关,可作为结肠癌的预后生物标志物和潜在治疗靶点,其参与调节结肠癌发展的具体机制有待进一步分析。综上所述,本研究旨在探究lncRNA ZFAS1对结肠癌进展的影响。此外,由于lncRNA常作为竞争性内源RNA(competing endogenous RNA,ce RNA)调节特定微小RNA(micro RNA,miRNAs)及他们的下游靶基因表达,本研究还筛选了lncRNA ZFAS1相关的miR-1271-5p/METTL1/ZEB1 ce RNA轴,并进一步验证了lncRNA ZFAS1通过miR-1271-5p/METTL1/ZEB1轴影响结肠癌进展及机制,为其作为结肠癌诊断及治疗的潜在靶点提供理论基础。研究方法:本研究第一部分:通过GSE126092数据集及生物信息学在线数据库筛选结肠癌中高表达的lncRNA,并通过q RT-PCR检测lncRNA ZFAS1在结肠癌组织和细胞中表达情况。将lncRNA ZFAS1敲减慢病毒转染结肠癌细胞,通过q RT-PCR验证转染效率。通过CCK-8、Ed U实验和平板克隆形成实验检测敲减lncRNA ZFAS1对结肠癌细胞增殖的影响。通过流式细胞术检测敲减lncRNA ZFAS1对结肠癌细胞凋亡的影响。通过Transwell迁移实验和侵袭实验检测敲减lncRNA ZFAS1对结肠癌细胞迁移和侵袭的影响。通过Western blot和免疫荧光检测敲减lncRNA ZFAS1对结肠癌细胞EMT相关蛋白表达的影响。将敲减lncRNA ZFAS1的结肠癌细胞皮下注射裸鼠,观察移植瘤肿瘤大小、肿瘤体积及瘤重,通过免疫组化检测移植瘤增殖标志物Ki-67表达,通过Western blot和免疫荧光检测移植瘤EMT相关蛋白表达。通过生物信息学预测了与lncRNA ZFAS1靶向结合的miR-1271-5p,并通过双荧光素酶报告基因实验和AGO2-RIP进行验证。通过q RT-PCR检测敲减lncRNA ZFAS1对结肠癌细胞miR-1271-5p表达的影响。通过q RT-PCR检测miR-1271-5p在结肠癌组织及细胞中表达情况。通过皮尔森相关分析分析结肠癌组织中lncRNA ZFAS1和miR-1271-5p表达相关性。将miR-1271-5p模拟物或抑制剂转染结肠癌细胞,通过q RT-PCR验证转染效率,并依次采用CCK-8、Transwell和Western blot检测转染miR-1271-5p模拟物或抑制剂对结肠癌细胞增殖、迁移和侵袭及EMT相关蛋白表达的影响。将miR-1271-5p抑制剂转染敲减lncRNA ZFAS1的结肠癌细胞并通过q RT-PCR验证转染效率。通过CCK-8、Ed U实验和平板克隆形成实验检测转染miR-1271-5p抑制剂对敲减lncRNA ZFAS1的结肠癌细胞增殖的影响。通过流式细胞术检测转染miR-1271-5p抑制剂对敲减lncRNA ZFAS1的结肠癌细胞凋亡的影响。通过Transwell迁移实验和侵袭实验检测转染miR-1271-5p抑制剂对敲减lncRNA ZFAS1的结肠癌细胞迁移和侵袭的影响。通过Western blot和免疫荧光检测转染miR-1271-5p抑制剂对敲减lncRNA ZFAS1的结肠癌细胞EMT相关蛋白表达的影响。瘤内注射antagomiR-1271-5p抑制敲减lncRNA ZFAS1的结肠癌细胞荷瘤小鼠miR-1271-5p表达,观察移植瘤肿瘤大小、肿瘤体积及瘤重,通过免疫组化检测移植瘤增殖标志物Ki-67表达,通过Western blot和免疫荧光检测移植瘤EMT相关蛋白表达。本研究第二部分:通过生物信息学预测与miR-1271-5p靶向结合的METTL1和ZEB1 m RNA,并通过双荧光素酶报告基因实验和AGO2-RIP进行验证。通过q RT-PCR和Western blot检测转染miR-1271-5p模拟物和抑制剂对结肠癌细胞METTL1和ZEB1表达的影响。用m7G抗体进行RIP实验检测过表达METTL1对ZEB1 m RNA m7G修饰的影响。通过q RT-PCR和Western blot检测过表达METTL1对结肠癌细胞ZEB1表达的影响。通过q RT-PCR和Western blot检测转染miR-1271-5p抑制剂对敲减lncRNA ZFAS1的结肠癌细胞METTL1和ZEB1表达的影响。通过q RT-PCR和Western blot检测过表达METTL1对敲减lncRNA ZFAS1的结肠癌细胞ZEB1表达的影响。通过CCK-8、Ed U实验和平板克隆形成实验检测过表达ZEB1对敲减lncRNA ZFAS1的结肠癌细胞增殖的影响。通过流式细胞术检测过表达ZEB1对敲减lncRNA ZFAS1的结肠癌细胞凋亡的影响。通过Transwell迁移实验和侵袭实验检测过表达ZEB1对敲减lncRNA ZFAS1的结肠癌细胞迁移和侵袭的影响。通过Western blot和免疫荧光检测过表达ZEB1对敲减lncRNA ZFAS1的结肠癌细胞EMT相关蛋白表达的影响。通过建立裸鼠移植瘤模型检测过表达ZEB1对敲减lncRNA ZFAS1的结肠癌细胞肿瘤生长的影响,观察移植瘤肿瘤大小、肿瘤体积及瘤重,通过免疫组化检测移植瘤增殖标志物Ki-67表达,通过Western blot和免疫荧光检测移植瘤EMT相关蛋白表达。结果:本研究第一部分:lncRNA ZFAS1在结肠癌组织和细胞中高表达。敲减lncRNA ZFAS1降低结肠癌细胞lncRNA ZFAS1表达,抑制细胞增殖、迁移、侵袭并促进细胞凋亡,下调细胞N-cadherin、Vimentin、Snail和Slug蛋白表达。敲减lncRNA ZFAS1降低结肠癌细胞移植瘤体积和质量,下调移植瘤Ki-67表达,降低移植瘤N-cadherin、Vimentin、Snail和Slug蛋白表达。lncRNA ZFAS1与miR-1271-5p具有潜在的结合位点,且lncRNA ZFAS1与miR-1271-5p结合。转染miR-1271-5p模拟物增加结肠癌细胞miR-1271-5p表达,抑制细胞增殖、迁移和侵袭,下调细胞N-cadherin、Vimentin、Snail和Slug蛋白表达;相反,转染miR-1271-5p抑制剂降低结肠癌细胞miR-1271-5p表达,促进细胞增殖、迁移和侵袭,上调细胞N-cadherin、Vimentin、Snail和Slug蛋白表达。敲减lncRNA ZFAS1增加结肠癌细胞miR-1271-5p表达。miR-1271-5p在结肠癌组织和细胞中低表达,且结肠癌组织中lncRNA ZFAS1与miR-1271-5p表达呈负相关。转染miR-1271-5p抑制剂降低敲减lncRNA ZFAS1的结肠癌miR-1271-5p表达,促进细胞增殖、迁移、侵袭并抑制细胞凋亡,上调细胞N-cadherin、Vimentin、Snail和Slug蛋白表达。转染antagomiR-1271-5p增加敲减lncRNA ZFAS1的结肠癌细胞移植瘤体积和质量,上调移植瘤Ki-67表达,增加移植瘤N-cadherin、Vimentin、Snail和Slug蛋白表达。本研究第二部分:METTL1和ZEB1 m RNA的3’-UTR存在miR-1271-5p的潜在结合位点,且miR-1271-5p与METTL1和ZEB1 m RNA靶向结合。转染miR-1271-5p模拟物降低结肠癌细胞METTL1和ZEB1表达,转染miR-1271-5p抑制剂增加结肠癌细胞METTL1和ZEB1表达。过表达METTL1增加ZEB1 m RNA m7G修饰,增加结肠癌细胞ZEB1蛋白表达但不影响细胞ZEB1 m RNA表达。转染miR-1271-5p抑制剂增加敲减lncRNA ZFAS1的结肠癌细胞METTL1和ZEB1表达。过表达ZEB1降低敲减lncRNA ZFAS1的结肠癌miR-1271-5p表达,促进细胞增殖、迁移、侵袭并抑制细胞凋亡,上调细胞N-cadherin、Vimentin、Snail和Slug蛋白表达。过表达ZEB1增加敲减lncRNA ZFAS1的结肠癌细胞移植瘤体积和质量,上调移植瘤Ki-67表达,增加移植瘤N-cadherin、Vimentin、Snail和Slug蛋白表达。结论:lncRNA ZFAS1在结肠癌中高表达,其通过海绵化miR-1271-5p上调其靶基因METTL1和ZEB1表达促进结肠癌细胞体外增殖、迁移、侵袭和EMT及体内肿瘤生长并抑制细胞凋亡,METTL1介导的m7G修饰促进ZEB1翻译并上调其表达。更多还原

【Abstract】 Objective: Colon cancer is the third most common malignancy in the world and the fourth leading cause of cancer-related deaths worldwide.However,the detailed mechanism of colon cancer development is currently not fully understood.Therefore,exploring the pathogenesis of colon cancer is still necessary for its early diagnosis and treatment.NFX1-type zinc finger containing protein 1 antisense RNA 1(ZFAS1)is a lncRNA located on chromosome 20q13.13.The lncRNA ZFAS1 is upregulated in colon cancer,and its high expression is associated with TNM staging,lymphatic infiltration,and poor prognosis in colon cancer patients,which can be used as a prognostic biomarker and potential therapeutic target for colon cancer.The the specific mechanism of its participation in regulating the development of colon cancer needs to be further analyzed.In summary,this study aims to investigate the impact of lncRNA ZFAS1 on the progression of colon cancer.In addition,lncRNA often acts as a competitive endogenous RNA(ce RNA)to regulate the expression of specific micro RNAs(miRNAs)and their downstream target genes.In this study,we also screened the miR-1271-5p/METTL1/ZEB1 ce RNA axis that related to lncRNA ZFAS1,and further validated the impact and mechanism of lncRNA ZFAS1 on colon cancer progression through the miR-1271-5p/METTL1/ZEB1 axis.This study will provide a theoretical basis for lncRNA ZFAS1 as a potential target for the diagnosis and treatment of colon cancer.Methods: Section 1: The highly expressed lncRNAs in colon cancer were screened by GSE126092 dataset and bioinformatics online database.The expression of lncRNA ZFAS1 in colon cancer tissues and cells was detected by q RT-PCR.The knockdown lncRNA ZFAS1 lentivirus were transfected into the colon cancer cells,and the transfection efficiency was verified by q RT-PCR.CCK-8,Ed U and plate clonal formation assay were carried out to analysis the effect of knockdown lncRNA ZFAS1 on the proliferation of colon cancer cells.Flow cytometry was used to detect the effect of knockdown lncRNA ZFAS1 on the apoptosis of colon cancer cells.Transwell migration and invasion assay were used to verify the effects of knockdown lncRNA ZFAS1 on migration and invasion of colon cancer cells.Western blot and immunofluorescence were performed to analysis the effect of knockdown lncRNA ZFAS1 on EMT-related protein expression in colon cancer cells The knockdown lncRNA ZFAS1 colon cancer cells were injected subcutaneously into the nude mice to observe the size,volume and weight of the transplanted tumors.The expression of proliferative marker Ki-67 was detected by immunohistochemistry,and the expression of EMT-related proteins in the transplanted tumors was detected by western blot and immunofluorescence.miR-1271-5p targeting binding to lncRNA ZFAS1 was predicted by bioinformatics and validated by double luciferase reporter gene experiments and AGO2-RIP.Then q RT-PCR was performed to detect the effect of knockdown lncRNA ZFAS1 on the expression of miR-1271-5p in colon cancer cells,as well as the expression of which in colon cancer tissues.At last,the correlation between lncRNA ZFAS1 and miR-1271-5p expression in colon cancer tissues was analyzed by Pearson correlation analysis.Colon cancer cells were transfected with miR-1271-5p mimetic or inhibitor,and the transfection efficiency was verified by q RT-PCR.The effects of miR-1271-5p mimetic or inhibitor on the proliferation,migration and invasion of colon cancer cells and the expression of EMT-related proteins were detected by CCK-8,Transwell and Western blot.The miR-1271-5p inhibitor was transfected with colon cancer cells that knocked down lncRNA ZFAS1 and verified the transfection efficiency by q RT-PCR CCK-8,Ed U and plate clonal formation assays were performed to detect the effect of transfected miR-1271-5p inhibitors on the proliferation of colon cancer cells with knockdown lncRNA ZFAS1.Flow cytometry was used to detect the effect of transfected miR-1271-5p inhibitors on the apoptosis of colon cancer cells with knockdown lncRNA ZFAS1.Transwell migration assay and invasion assay were used to detect the effect of transfection of miR-1271-5p inhibitors on the migration and invasion of colon cancer cells with knockout lncRNA ZFAS1.Western blot and immunofluorescence were used to detect the effect of miR-1271-5p inhibitors on the expression of EMT-related proteins in colon cancer cells with lncRNA ZFAS1 knockdown.After the antagomiR-1271-5p was injected into colon cancer cells to inhibit the expression of miR-1271-5p in tumorbearing mice with knockout lncRNA ZFAS1,the size,volume and weight of the transplanted tumor was observed,and the expression of Ki-67,a proliferative marker in the transplanted tumor,was detected by immunohistochemistry.Emt-related proteins were detected by western blot and immunofluorescence.Section 2: METTL1 and ZEB1 m RNAs that bind to miR-1271-5p were predicted by bioinformatics,and verified by dual luciferase reporter and AGO2-RIP assays.The effects of miR-1271-5p mimicry and inhibitors on the expression of METTL1 and ZEB1 in colon cancer cells were detected by q RT-PCR and western blot.RIP experiments with m7 G antibodies detected the effect of overexpression METTL1 on ZEB1 m RNA m7 G modification.The effect of overexpression of METTL1 on the expression of ZEB1 in colon cancer cells was detected by q RT-PCR and western blot.q RT-PCR and western blot assays were also used to detect the effect of miR-1271-5p inhibitor transfected on the expression of METTL1 and ZEB1 in colon cancer cells with lncRNA ZFAS1 knockdown,as well as the effect of METTL1 overexpression on ZEB1 expression of lncRNA ZFAS1 knockout colon cancer cells.The effect of overexpression of ZEB1 on the proliferation of colon cancer cells with knockdown lncRNA ZFAS1 was detected by CCK-8,Ed U and plate clonal formation assays.And flow cytometry was performed to analysis the effect of overexpression of ZEB1 on apoptosis of lncRNA ZFAS1 knockout colon cancer cells.The effect of overexpression of ZEB1 on the migration and invasion of lncRNA ZFAS1 knockdown colon cancer cells was examined by Transwell migration and invasion assays.Western blot and immunofluorescence were used to detect the effect of overexpression of ZEB1 on the expression of EMT-related proteins in colon cancer cells with lncRNA ZFAS1 knockdown.The effect of overexpression of ZEB1 on the tumor growth of colon cancer cells with knockdown lncRNA ZFAS1 was detected by establishing a nude mouse transplanted tumor model.The size,volume and weight of the transplanted tumor were monitored.The expression of Ki-67,a proliferative marker in the transplanted tumor,was detected by immunohistochemistry.Emt-related proteins were detected by western blot and immunofluorescence.Results: Section 1: LncRNA ZFAS1 is highly expressed in both colon cancer tissues and cells.Knocking down of lncRNA ZFAS1 reduced the expression of lncRNA ZFAS1 in colon cancer cells,inhibited cell proliferation,migration,invasion and promoted apoptosis,and downregulated the expression of N-cadherin,Vimentin,Snail and Slug proteins in cells.The knockdown of lncRNA ZFAS1 also decreased the tumor volume and mass of colon cancer cells,down-regulated the expression of Ki-67 and decreased the expressions of N-cadherin,Vimentin,Snail and Slug proteins.lncRNA ZFAS1 binds with miR-1271-5p,and has potential binding sites.Knockdown of lncRNA ZFAS1 increased the expression of miR-1271-5p in colon cancer cells.miR-1271-5p was low expressed in colon cancer tissues and cells,and which is negatively correlated with the lncRNA ZFAS1 in colon cancer tissues.Transfection of miR-1271-5p mimetic increased the expression of miR-1271-5p in colon cancer cells,inhibited cell proliferation,migration and invasion,and down-regulated the expression of N-cadherin,Vimentin,Snail and Slug.On the contrary,transfection of miR-1271-5p inhibitor decreased the expression of miR-1271-5p in colon cancer cells,promoted cell proliferation,migration and invasion,and up-regulated the expression of N-cadherin,Vimentin,Snail and Slug.Transfection with miR-1271-5p inhibitors decreased the expression of miR-1271-5p in colon cancer knockdown lncRNA ZFAS1,promoted cell proliferation,migration,invasion,inhibited cell apoptosis,and up-regulated the expression of N-cadherin,Vimentin,Snail and Slug proteins.Transfection of antagomiR-1271-5p increased the tumor volume and mass of colon cancer cell transplanted tumors with knockdown lncRNA ZFAS1,up-regulated the expression of Ki-67,and increased the expression of N-cadherin,Vimentin,Snail and Slug proteins in transplanted tumors.Section 2: The miR-1271-5p was targeted binding to METTL1 and ZEB1 m RNAs,and the 3’-UTR of METTL1 and ZEB1 m RNAs contained potential binding sites of miR-1271-5p.Transfection with miR-1271-5p mimicers decreased the expression of METTL1 and ZEB1 m RNAs in colon cancer cells,while transfection with miR-1271-5p inhibitors increased METTL1 and ZEB1 m RNAs expression.Overexpression of METTL1 increased ZEB1 m RNA and m7 G modification,and increased the expression of ZEB1 protein in colon cancer cells,but showed non effects on the ZEB1 m RNA expression.Transfection with miR-1271-5p inhibitor increased the expression of METTL1 and ZEB1 in colon cancer cells with knockdown lncRNA ZFAS1.Overexpression of ZEB1 decreased the expression of miR-1271-5p in colon cancer with knockdown lncRNA ZFAS1,promoted cell proliferation,migration and invasion,inhibited cell apoptosis,and upregulated the expression of N-cadherin,Vimentin,Snail and Slug proteins.Overexpression of ZEB1 also increased the tumor volume and weight of colon cancer cell transplant tumors with knockdown lncRNA ZFAS1,upregulated the expression of Ki-67,and increased the expressions of N-cadherin,Vimentin,Snail and Slug proteins.Conclusion: LncRNA ZFAS1 is highly expressed in colon cancer,and it promotes the proliferation,migration,invasion and EMT of colon cancer cells in vitro and increases tumor growth in vivo,and inhibits cell apoptosis by up-regulating the expression of target genes of METTL1 and ZEB1 by sponging miR-1271-5p.Mettl1-mediated m7 G modification promotes the translation and up-regulates the expression of ZEB1.更多还原