miR-483在食管癌发生中的功能研究

发布时间：2018-05-25 00:07

本文选题：食管癌 + microRNA　；参考：《第四军医大学》2015年硕士论文

【摘要】：【背景】食管癌是世界上第八大常见恶性肿瘤,第六大因癌症导致死亡的原因。目前全世界有45万多食管癌患者,并且其发病率仍在上升。食管癌诊断越早,预后越好。然而,由于缺乏有效的早期诊断手段,大部分患者首诊时已届进展期,其总的五年生存率仅为15%~25%,因此有必要探索有应用价值的食管癌分子标志物和靶分子,从而为食管癌的早期诊断和治疗提供线索。micro RNAs(mi RNAs)是一类在进化过程中高度保守的长度在18~25个核苷酸的单链非编码小RNA,主要通过抑制靶基因的翻译影响人体的各种生理和病理过程。前期我们通过mi RNA芯片检测了25例食管鳞癌及其癌旁组织的差异表达分子,率先发现并报道了一个食管癌发生相关的mi RNA分子群。其中,mi R-483在食管癌组织中高表达,但其对食管癌细胞恶性表型的影响及分子机制尚未阐明,本课题将对此展开研究。【目的】1.检测mi R-483-3p在食管鳞癌细胞系及正常食管鳞状上皮细胞系中的表达;2.构建mi R-483-3p过表达和低表达的食管癌细胞模型,通过体内外实验研究mi R-483-3p对食管癌生物学行为的影响;3.探讨mi R-483-3p影响食管癌发生的分子机制。【方法】1.q PCR检测mi R-483-3p在食管鳞癌细胞系及正常食管鳞状上皮细胞系中的表达;2.构建mi R-483-3p的慢病毒表达载体及空载体,转染食管癌细胞,建立过表达mi R-483-3p的细胞模型及相应对照细胞模型;3.合成mi R-483-3p inhibitor及inhibitor NC,转染食管癌细胞,建立低表达mi R-483-3p的细胞模型及相应对照细胞模型;4.MTT实验研究mi R-483-3p对食管癌细胞增殖的影响;5.Transwell实验观察mi R-483-3p对食管癌细胞迁移的影响;6.流式细胞仪检测mi R-483-3p对食管鳞癌细胞周期的影响;7.流式细胞仪检测mi R-483-3p对食管癌细胞凋亡的影响;8.MTT法体外药物敏感性实验检测mi R-483-3p对食管癌细胞药物敏感性的影响;9.裸鼠成瘤实验观察mi R-483-3p对食管癌细胞体内成瘤能力的影响;10.通过生物信息学及全基因组表达谱芯片联合预测的方法筛选mi R-483-3p的靶基因;11.通过q PCR、Western blot及双荧光素酶报告基因实验验证mi R-483-3p的靶基因。【结果】1.与正常食管鳞状上皮细胞系Het-1A相比,mi R-483-3p在食管鳞癌细胞系EC109、EC9706及TE-1中表达升高。2.成功建立了过表达和低表达mi R-483-3p的食管癌细胞模型。3.上调mi R-483-3p可促进食管癌细胞的增殖、迁移,促进细胞周期由G1期向G2期转化,减少ADR诱导的细胞凋亡,减弱细胞对化疗药物的敏感性,增强食管癌细胞的体内成瘤能力。4.下调mi R-483-3p可抑制食管癌细胞的增殖、迁移,导致细胞周期G1期阻滞,增加ADR诱导的细胞凋亡,增强细胞对化疗药物的敏感性。5.通过生物信息学和全基因组表达谱芯片联合预测,得到7个mi R-483-3p的靶基因:EI24、KLHL12、GRSF1、MKRN1、LEPROTL1、CREBL2、MAPKAPK2。6.上调食管癌细胞中mi R-483-3p的表达对以上7个靶基因的m RNA表达水平无影响。7.上调食管癌细胞中mi R-483-3p的表达可导致抑癌基因EI24的表达下降。【结论】1.与正常食管鳞状上皮细胞相比,mi R-483-3p在食管鳞癌细胞系中表达升高。2.上调mi R-483-3p表达可通过调节细胞周期促进食管癌细胞的增殖、迁移,增强细胞对化疗药物的耐药性;下调mi R-483-3p则起到相反的效果。3.mi R-483-3p可能通过调节EI24等靶基因的表达影响食管癌的发生发展。
[Abstract]:[background] esophageal cancer is the 8th most common malignant tumor in the world. The sixth major cause of death is cancer. There are more than 450 thousand esophageal cancer patients worldwide, and the incidence is still rising. The earlier the diagnosis of esophageal cancer is, the better the prognosis. However, because of the lack of effective early diagnosis, most of the patients have progressing in the first visit. The total five year survival rate is only 15%~25%, so it is necessary to explore the useful molecular markers and target molecules of esophageal cancer, thus providing clues to the early diagnosis and treatment of esophageal cancer,.Micro RNAs (MI RNAs) is a class of highly conserved single strand non coded small RNA, which is highly conservative in the course of evolution, with the length of 18~25 nuclear acid, mainly through the suppression of the target. The translation of the gene affects the physiological and pathological processes of the human body. In the earlier period, we detected the differential expression molecules of 25 cases of esophageal squamous cell carcinoma and its adjacent tissues by Mi RNA chip, and first discovered and reported a mi RNA group related to the occurrence of esophageal cancer. Among them, Mi R-483 was highly expressed in the esophageal cancer tissue, but it was bad for the cancer of the esophagus. The effects and molecular mechanisms of sexual phenotype have not been elucidated. [Objective] [Objective] 1. to detect the expression of MI R-483-3p in esophageal squamous cell carcinoma cell lines and normal esophageal squamous cell lines; 2. to construct mi R-483-3p overexpressed and low expressed esophageal cancer cell models, and to study mi R-483-3p for esophageal cancer in vitro and in vivo The influence of physical behavior; 3. to explore the molecular mechanism of MI R-483-3p affecting the occurrence of esophageal cancer. [Methods] 1.q PCR was used to detect the expression of MI R-483-3p in esophageal squamous cell carcinoma cell lines and normal esophageal squamous cell lines; 2. to construct the MI R-483-3p Lentivirus Expression Vector and unloaded body, transfection of esophageal cancer cells, and to establish a fine expression of MI R-483-3p. Cell model and corresponding control cell model; 3. mi R-483-3p inhibitor and inhibitor NC were synthesized to transfect esophageal cancer cells to establish a cell model with low expression of MI R-483-3p and a corresponding control cell model. 4.MTT experiment studied the effect of MI R-483-3p on the proliferation of esophageal cancer cells; 5.Transwell experiment observed the migration of the esophagus cancer cells by MI. The effect of MI R-483-3p on the cell cycle of esophageal squamous cell carcinoma was detected by 6. flow cytometry, and the effect of MI R-483-3p on the apoptosis of esophageal cancer cells was detected by 7. flow cytometry; the effect of MI R-483-3p on the drug sensitivity of esophageal cancer cells was detected by 8.MTT assay in vitro; and 9. in nude mice to observe the effect of MI R-483-3p on esophageal cancer cells. The effect of tumor forming ability; 10. screening target genes of MI R-483-3p by bioinformatics and whole genome expression chip combined prediction; 11. test the target gene of MI R-483-3p by Q PCR, Western blot and double luciferase reporter gene test. [results] 1., MI R-483-3p is compared with the normal esophageal squamous cell line Het-1A. The expression of esophageal squamous cell carcinoma cell lines EC109, EC9706 and TE-1 elevated.2. successfully established the overexpression and low expression of MI R-483-3p in the esophageal cancer cell model.3. to promote the proliferation and migration of the esophageal cancer cells and promote the transformation of the cell cycle from G1 to G2 phase, reducing the apoptosis of ADR induced cells and reducing the sensitivity of cell to chemotherapeutic drugs. Sex, enhancing the tumorigenicity of esophageal cancer cells in vivo.4. down regulation of MI R-483-3p can inhibit the proliferation, migration, cell cycle G1 block, increase ADR induced apoptosis and enhance cell sensitivity to chemotherapeutic drugs,.5. through bioinformatics and total gene group expression chip combined prediction to obtain 7 mi R-483-3p Target genes: EI24, KLHL12, GRSF1, MKRN1, LEPROTL1, CREBL2, MAPKAPK2.6. up-regulated the expression of MI R-483-3p in esophageal cancer cells. The expression of M RNA on the above 7 target genes has no effect on the expression of.7. up esophageal cancer cells. [Conclusion] 1. compared with normal esophageal squamous cells. I R-483-3p increased the expression of.2. in esophageal squamous cell carcinoma cell line and increased the expression of MI R-483-3p by regulating cell cycle to promote the proliferation, migration, and drug resistance of esophageal cancer cells. The downregulation of MI R-483-3p may play the opposite effect,.3.mi R-483-3p may affect the development of esophageal cancer by regulating the expression of EI24 and other target genes. The development of life.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

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