宫颈癌淋巴结转移相关miRNA筛选及机制研究

发布时间：2018-06-27 11:52

  本文选题：宫颈癌 + 淋巴结转移　； 参考：《复旦大学》2014年博士论文

【摘要】：目的：淋巴结转移是宫颈癌主要转移途径,也是影响宫颈癌患者预后的重要因素。假设miRNA能调节宫颈癌淋巴结转移,研究不同淋巴结转移能力的宫颈癌组织中的miRNA表达差异,筛选出淋巴结转移相关的miRNA,并且研究其调节淋巴结转移的机制。材料和方法：在宫颈癌新鲜标本中,用miRNA芯片筛选出不同淋巴结转移能力的宫颈癌的miRNA的差异表达,Real-time PCR验证miRNA芯片筛选的结果。在SiHa细胞株中过表达或低表达候选miRNA含量,通过体外Transwell实验检测细胞的迁移和侵袭能力。用慢病毒载体构建稳定转染候选miRNA的稳转株,并建立裸鼠宫颈癌细胞转移模型。通过生物信息软件、靶基因mRNA real-time PCR和Western Blot蛋白检测三种方法综合推断miR-652的靶基因。结果：niRNA芯片筛查显示,对比无盆腔淋巴结转移的宫颈癌组织,有盆腔淋巴结转移的宫颈癌组织中2个miRNA高表达(miR-142-3p,miR-652) (P0.05),3个 miRNA低表达(miR-196b, miR-320b和miR-424) (P0.05)。Real-time PCR证实筛选出的miRNA表达变化趋势与芯片结果相似,但只有miR-196b及miR-652的差异表达具有统计学差异(P0.05)。Transwell实验显示,低表达miR-196b和过表达miR-652可以增进细胞迁移和侵袭能力,过表达miR-196b和低表达miR-652可以抑制细胞迁移和侵袭能力。未能发现改变niR-142-3p、miR-320b和miR-424表达量会影响细胞迁移或侵袭功能。综合上述结论,miR-196b和miR-652可能与宫颈癌淋巴结转移有关。从创新性考虑,暂选miR-652进行后续研究。在体内实验中,未有足够证据说明miR-652能增进肿瘤细胞的转移能力,但PET-CT显示miR-652可能有促进肝脏转移病变的趋势。综合Targetscan、miRDB、miRSearch、miRTarBase 和 miRwalk共5个miRNA数据库联合预测,并查阅靶基因资料文献,获得6个miR-652的候选靶基因,分别为BCL11A、ISL1、LLGL1、NPTN、NR4A3、YWHAH。在对以上基因mRNA表达水平的PCR检测中,发现miR-652过表达组ISL1、NR4A3和YWHAH基因的mRNA表达量下降(P0.05), miR-652低表达组ISL1、NR4A3和YWHAH基因的mRNA表达量上升(P0.05),变化趋势符合miRNA-mRNA结合改变。其余3个靶基因mRNA表达量变化未发现统计学差异(P0.05)。miR-652过表达组YWHAH蛋白表达量减少,miR-652低表达组YWHAH蛋白表达量上升。未能发现miR-652的低表达和过表达影响ISL1和NR4A3蛋白表达量的改变。结论：miR-196b有抑制宫颈癌淋巴结转移的功能,miR-652有促进宫颈癌淋巴结转移的功能。YWHAH可能是miR-652的靶基因。
[Abstract]:Objective: lymph node metastasis is the main metastasis pathway of cervical cancer and an important factor affecting the prognosis of cervical cancer patients. It is assumed that miRNA can regulate lymph node metastasis of cervical cancer. The difference of miRNA expression in cervical cancer tissues with different lymph node metastasis ability is studied. The miRNAs associated with lymph node metastasis are screened out and the mechanism of their regulation of lymph node metastasis is studied. Materials and methods: miRNA microarray was used to screen the differential expression of miRNA in cervical cancer with different lymph node metastasis ability. Real-time PCR was used to verify the results of miRNA microarray screening. Candidate miRNAs were overexpressed or low expressed in SIHA cell lines. The migration and invasion of SIHA cells were detected by Transwell assay in vitro. The stable transfer strain of candidate miRNA was constructed by using lentivirus vector and the metastasis model of cervical cancer cells in nude mice was established. The target gene of miR-652 was inferred by three methods: bioinformatics software, real-time PCR of target gene and Western Blot protein detection. Results compared with cervical cancer tissues without pelvic lymph node metastasis, the microarray screening showed that, In cervical carcinoma with pelvic lymph node metastasis, two miRNA overexpression (miR-142-3pnmiR-652), three miRNA low expression (miR-196b, miR-320b and miR-424) (P0.05). Real-time PCR confirmed that the change trend of miRNA expression was similar to that of microarray. However, only the difference of miR-196b and miR-652 expression was statistically significant (P0.05) .Transwell experiments showed that the low expression of miR-196b and over-expression of miR-652 could enhance the ability of cell migration and invasion, and over-expression of miR-196b and low-expression of miR-652 could inhibit the ability of cell migration and invasion. No change in the expression of niR-142-3pmmiR-320b and miR-424 was found to affect cell migration or invasion. These results suggest that miR-196b and miR-652 may be associated with lymph node metastasis of cervical cancer. From the innovative point of view, miR-652 was selected for follow-up study. In vivo, there was not enough evidence that miR-652 could enhance the metastasis ability of tumor cells, but PET-CT showed that miR-652 might promote liver metastasis. Combined prediction of 5 miRNA databases from TargetscanmiRDBmiRsearch chong miRTarBase and miRwalk, six candidate target genes of miR-652 were obtained, which were BCL11A, ISL1, LLGL1, NPNLGL1, NPNLGL1, NR4A3, YWHAH, respectively, and obtained six candidate target genes of miR-652 (BCL11A1, ISL1, LLGL1, NPNLGL1, NPNR4A3, YWHAH). In the PCR analysis of the mRNA expression level of the above genes, it was found that the mRNA expression of ISL1, NR4A3 and YWHAH gene decreased in the overexpression group of miR-652 (P0.05), and the mRNA expression of ISL1, NR4A3 and YWHAH gene increased in the low expression group of miR-652 (P0.05), and the change trend was consistent with the change of miRNA-mRNA binding. There was no significant difference in the mRNA expression of the other three target genes (P0.05). The expression of YWHAH protein decreased in the over-expression group of miR-652 and increased in the low expression group of miR-652. The low expression and overexpression of miR-652 had no effect on the expression of ISL1 and NR4A3. Conclusion: miR-196b can inhibit lymph node metastasis of cervical cancer. MiR-652 has the function of promoting lymph node metastasis of cervical cancer. YWHAH may be the target gene of miR-652.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.33

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