microRNAs在恶性黑色素瘤B16F0和B16F10细胞中差异表达的研究及miR-763对B16F10细胞增殖的影响
发布时间：2018-08-26 19:52
【摘要】：背景恶性黑色素瘤(malignant melanoma,MM)是一种皮肤肿瘤,其是由来源于神经脊的黑色素细胞异常增生而产生的,一旦产生并进入快速生长期,则具有易侵袭、易转移、预后差、死亡率高等特点。microRNA(mi RNA)是一类长度为21-25nt的内源性非编码单链RNA,它们通过与靶基因mRNA的3'UTRs(非编码区)或编码区的特异性结合,从而调控靶基因的表达。本课题旨在探究mi RNA在恶性黑色素瘤B16F0和B16F10细胞中的差异表达情况及过表达miR-763对B16F10细胞的影响[1]。目的1.探讨恶性黑色素瘤B16F0和B16F10细胞miRNA的差异表达情况;2.探讨miR-763对恶性黑色素瘤B16F10细胞的影响及潜在分子机制;方法使用恶性黑色素瘤B16F0和B16F10细胞作为研究对象,构建动物模型。1.观测在体内和体外恶性黑色素瘤B16F0和B16F10细胞迁移、侵袭和增殖方面的差异;2.检测恶性黑色素瘤B16F0和B16F10细胞mi RNA的表达情况,并利用生物信息学分析其差异性;3.利用Real-time PCR技术检测小鼠黑色素瘤细胞miR-763、miR-431-5p、miR-205-5p、let-7C-2-3p、mi R-26a-1-3p、miR-28b、miR-29b-3p和miR-24-2-5p的表达水平,验证miRNA芯片结果的准确性。4.在恶性黑色素瘤B16F10细胞过表达miR-763,观测其对B16F10细胞增殖的影响;5.检测生物信息学预测的miR-763靶基因的表达变化,利用激酶磷酸化特异性抗体检测各个信号通路中的关键激酶的磷酸化水平变化,确定miR-763通过靶基因影响哪些信号通路进而对B16F10细胞产生影响。结果1.恶性黑色素瘤B16F10细胞在体外的迁移、侵袭和增殖能力高于B16F0细胞。2.通过生物信息学分析发现恶性黑色素瘤B16F0和B16F10细胞在miRNA表达水平存在明显的差异性。3.Real-time PCR检测发现小鼠黑色素瘤细胞miR-763、miR-431-5p、miR-205-5p、let-7C-2-3p表达水平下调,miR-26a-1-3p、miR-28b、miR-29b-3p和miR-24-2-5p表达水平上调,结果与芯片结果相一致,miRNA芯片结果准确可信。4.过表达miR-763可以抑制恶性黑色素瘤B16F10细胞的体外增殖。5.miR-763下调潜在靶基因Grb2的表达,进而影响下游分子Cyclin D1的变化。结论1.生物信息学分析发现两株细胞之间在miRNA表达水平存在显著差异,这可能是恶性黑色素瘤B16F10与B16F0细胞迁移、侵袭和增殖能力差异的重要原因。2.miR-763可能通过靶向调节Grb2的表达,抑制恶性黑色素瘤B16F10细胞的增殖。
[Abstract]:Background malignant melanoma (malignant melanoma,MM) is a kind of skin tumor, which is produced by the abnormal proliferation of melanocytes from the spinal cord. Once it comes into rapid growth, it is prone to invasion, metastasis and poor prognosis. MicroRNAs are a class of endogenous non-coding single-stranded RNA, with length of 21-25nt which regulate the expression of target genes by binding specifically to the 3'UTRs (non-coding region) or coding region of the target gene mRNA. The purpose of this study was to investigate the differential expression of mi RNA in B16F0 and B16F10 cells of malignant melanoma and the effect of overexpression of miR-763 on B16F10 cells [1]. Objective 1. To investigate the differential expression of miRNA between B16F0 and B16F10 cells in malignant melanoma. To investigate the effect and potential molecular mechanism of miR-763 on B16F10 cells of malignant melanoma, the animal model was constructed by using B16F0 and B16F10 cells of malignant melanoma as research objects. To observe the difference in migration, invasion and proliferation of B16F0 and B16F10 cells in vivo and in vitro. To detect the expression of mi RNA in B16F0 and B16F10 cells of malignant melanoma and analyze its difference by bioinformatics. The expression levels of miR-763,miR-431-5p,miR-205-5p,let-7C-2-3p,mi R-26a-1-3pmmiR-28bmmiR-29b-3p and miR-24-2-5p in mouse melanoma cells were detected by Real-time PCR technique to verify the accuracy of miRNA microarray results. 4. Overexpression of miR-763, in B16F10 cells of malignant melanoma and its effect on the proliferation of B16F10 cells were observed. The expression of miR-763 target gene was detected by bioinformatics, and the phosphorylation level of key kinase in each signal pathway was detected by kinase phosphorylation specific antibody. Determine which signaling pathways miR-763 affect B16F10 cells through target genes. Result 1. The ability of migration, invasion and proliferation of malignant melanoma B16F10 cells was higher than that of B16F0 cells in vitro. By bioinformatics analysis, we found that there was significant difference between B16F0 and B16F10 cells in miRNA expression level. 3. Real-time PCR detection showed that miR-763,miR-431-5p,miR-205-5p,let-7C-2-3p expression level of murine melanoma cells down-regulated miR-26a-1-3pmmiR-28btimiR-29b-3p and miR-24-2-5p expression. The results were consistent with the results of microarray. Overexpression of miR-763 could inhibit the proliferation of malignant melanoma B16F10 cells in vitro. 5. 5. MiR-763 down-regulated the expression of potential target gene Grb2, and then affected the change of downstream molecule Cyclin D1. Conclusion 1. Bioinformatics analysis showed that there was a significant difference in miRNA expression between the two cells, which may be the important reason for the difference in migration, invasion and proliferation between B16F10 and B16F0 cells. 2. MiR-763 may regulate the expression of Grb2 by targeting. Inhibit the proliferation of malignant melanoma B16F10 cells.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5
本文编号：2205986
[Abstract]:Background malignant melanoma (malignant melanoma,MM) is a kind of skin tumor, which is produced by the abnormal proliferation of melanocytes from the spinal cord. Once it comes into rapid growth, it is prone to invasion, metastasis and poor prognosis. MicroRNAs are a class of endogenous non-coding single-stranded RNA, with length of 21-25nt which regulate the expression of target genes by binding specifically to the 3'UTRs (non-coding region) or coding region of the target gene mRNA. The purpose of this study was to investigate the differential expression of mi RNA in B16F0 and B16F10 cells of malignant melanoma and the effect of overexpression of miR-763 on B16F10 cells [1]. Objective 1. To investigate the differential expression of miRNA between B16F0 and B16F10 cells in malignant melanoma. To investigate the effect and potential molecular mechanism of miR-763 on B16F10 cells of malignant melanoma, the animal model was constructed by using B16F0 and B16F10 cells of malignant melanoma as research objects. To observe the difference in migration, invasion and proliferation of B16F0 and B16F10 cells in vivo and in vitro. To detect the expression of mi RNA in B16F0 and B16F10 cells of malignant melanoma and analyze its difference by bioinformatics. The expression levels of miR-763,miR-431-5p,miR-205-5p,let-7C-2-3p,mi R-26a-1-3pmmiR-28bmmiR-29b-3p and miR-24-2-5p in mouse melanoma cells were detected by Real-time PCR technique to verify the accuracy of miRNA microarray results. 4. Overexpression of miR-763, in B16F10 cells of malignant melanoma and its effect on the proliferation of B16F10 cells were observed. The expression of miR-763 target gene was detected by bioinformatics, and the phosphorylation level of key kinase in each signal pathway was detected by kinase phosphorylation specific antibody. Determine which signaling pathways miR-763 affect B16F10 cells through target genes. Result 1. The ability of migration, invasion and proliferation of malignant melanoma B16F10 cells was higher than that of B16F0 cells in vitro. By bioinformatics analysis, we found that there was significant difference between B16F0 and B16F10 cells in miRNA expression level. 3. Real-time PCR detection showed that miR-763,miR-431-5p,miR-205-5p,let-7C-2-3p expression level of murine melanoma cells down-regulated miR-26a-1-3pmmiR-28btimiR-29b-3p and miR-24-2-5p expression. The results were consistent with the results of microarray. Overexpression of miR-763 could inhibit the proliferation of malignant melanoma B16F10 cells in vitro. 5. 5. MiR-763 down-regulated the expression of potential target gene Grb2, and then affected the change of downstream molecule Cyclin D1. Conclusion 1. Bioinformatics analysis showed that there was a significant difference in miRNA expression between the two cells, which may be the important reason for the difference in migration, invasion and proliferation between B16F10 and B16F0 cells. 2. MiR-763 may regulate the expression of Grb2 by targeting. Inhibit the proliferation of malignant melanoma B16F10 cells.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.5
【参考文献】
相关期刊论文 前9条
1 王子月;刘萌;张春阳;;MicroRNA的超灵敏检测研究进展[J];高等学校化学学报;2017年01期
2 任雪维;张吉才;;miR-29在肿瘤中的研究进展[J];中国肿瘤临床;2016年17期
3 陈烈钳;邱明宁;谭国斌;柳建军;;MicroRNA let-7家族与肿瘤的研究进展[J];现代肿瘤医学;2015年04期
4 易韬;;Mir-30的研究进展[J];四川解剖学杂志;2013年04期
5 易小芳;邢荣春;郑家芬;;微小RNA-200家族与肿瘤关系的研究进展[J];中国全科医学;2013年14期
6 王华华;黄俊骏;梁卫红;;趣谈microRNA的命名[J];生物学教学;2013年03期
7 马丽霞;韦新焕;张晶;;miR-15家族在肝脏疾病中的作用[J];临床肝胆病杂志;2013年02期
8 ;中国黑色素瘤诊治指南(2011版)[J];临床肿瘤学杂志;2012年02期
9 赵军,李辰,狄静芳,冯峥,赵松滨;小鼠眼内移植三种B16黑色素瘤细胞系的比较研究[J];中国病理生理杂志;2003年07期
，本文编号：2205986
本文链接：https://www.wllwen.com/yixuelunwen/pifb/2205986.html
最近更新
教材专著
