MiR-29c在骨肉瘤中的表达及对骨肉瘤细胞系SOSP9607增殖、转移能力的影响

发布时间：2017-12-26 21:41

本文关键词：MiR-29c在骨肉瘤中的表达及对骨肉瘤细胞系SOSP9607增殖、转移能力的影响　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：骨肉瘤是一种少见的骨原发恶性肿瘤,早期肺转移、临床预后差是骨肉瘤的特点,也是影响患者生存率的重要因素。microRNA是一种具有转录后调控作用的小分子RNA,具有多种生理功能,参与多种恶性肿瘤增殖、转移的调控。研究发现,miR-29家族中的miR-29c在多种恶性肿瘤中表达下调,并验证其对多种恶性肿瘤的转移和增殖起到抑制作用,前期实验证实mi R-29c在骨肉瘤组织和细胞系中异常表达,提示其可能参与了骨肉瘤的增殖和转移。目的:验证miR-29c在骨肉瘤组织中的异常表达和不同侵袭能力骨肉瘤细胞系中的差异表达;探究miR-29c对骨肉瘤细胞系增殖、侵袭和迁移能力的影响,为骨肉瘤的分子靶向治疗提供理论基础。方法:1.采用实时定量PCR检测68例骨肉瘤、正常组织标本miR-29c的表达;用此法检测SOSP-9607骨肉瘤细胞系的两组侵袭性不同的细胞亚系F5M2和F4中miR-29c的表达。2.采用Lipofectamine2000脂质体法分别将miR-29c mimics和inhibitor瞬时转染入sosp-9607的两个细胞亚系,构建mir-29c高表达、低表达骨肉瘤细胞模型。3.对所构建的细胞模型进行肿瘤细胞功能实验,包括transwell侵袭和迁移实验、mtt法检测肿瘤细胞增殖曲线;流式细胞仪法检测肿瘤细胞周期和凋亡比例。结果:1.骨肉瘤组织mir-29c表达明显低于瘤旁正常组织;高侵袭性f5m2骨肉瘤细胞系mir-29c表达明显低于低侵袭性f4骨肉瘤细胞系。2.用lipofectamine2000脂质体分别将mir-29cmimics和inhibitor成功转入f5m2骨肉瘤细胞系和f4骨肉瘤细胞系。rt-pcr结果验证f5m2/29cmimics细胞中mir-29c表达增加;f4/29cinhibitor细胞通过rt-pcr间接验证其靶基因的表达,证明f4骨肉瘤细胞mir-29c下调成功。3.transwell细胞侵袭和迁移实验显示,mir-29c上调的骨肉瘤细胞f5m2侵袭和迁移能力较对照组均明显下降;mir-29c下调的骨肉瘤细胞f4侵袭和迁移能力较对照组明显提升。mtt细胞增殖实验显示,mir-29c上调的骨肉瘤细胞f5m2增殖能力较对照组减弱,细胞增殖曲线下移;mir-29c下调的骨肉瘤细胞f4增殖能力较对照组增强,细胞增殖曲线上移。4.流式细胞仪细胞周期和细胞凋亡实验显示,mir-29c上调的骨肉瘤细胞f5m2与对照组相比,各周期肿瘤细胞比例无明显差异,凋亡细胞比例也无明显差异;mir-29c下调的骨肉瘤细胞f4与对照组相比,各周期肿瘤细胞比例亦无明显差异,凋亡细胞比例无统计学差异。结论:1.mir-29c骨肉瘤组织中表达含量下调,且高侵袭性骨肉瘤细胞mir-29c表达含量低于低侵袭性骨肉瘤细胞系,提示mir-29c与肿瘤侵袭能力呈负相关,mir-29c可能参与了骨肉瘤细胞的增殖和转移能力的调控。2.通过构建mir-29c上、下调肿瘤细胞模型,验证了mir-29c对骨肉瘤细胞增殖、侵袭及迁移能力的抑制作用,为下一步研究mir-29c抑制骨肉瘤细胞增殖和转移的分子机制研究打下基础3.mir-29c对骨肉瘤细胞系sosp9607的细胞周期和凋亡无明显影响,mir-29c对肿瘤细胞周期和凋亡的作用可能具有组织特异性。
[Abstract]:Osteosarcoma is a rare primary malignant tumor of bone. Early pulmonary metastasis and poor clinical prognosis are the characteristics of osteosarcoma and also an important factor affecting the survival rate of the patients. MicroRNA, a small molecule RNA with post transcriptional regulation, has many physiological functions and participates in the regulation of multiple malignant tumor proliferation and metastasis. The study found that the expression of miR-29 family miR-29c in a variety of malignant tumors is down regulated, and verifies its effect on malignant tumor metastasis and proliferation play an inhibitory effect, previous experiments confirmed that the abnormal expression of MI and R-29c in osteosarcoma tissues and cell lines, suggesting that it may be involved in the proliferation and metastasis of osteosarcoma. Objective: to verify the abnormal expression of miR-29c in osteosarcoma and the differential expression of different invasiveness in osteosarcoma cell lines, and to explore the effect of miR-29c on the proliferation, invasion and migration of osteosarcoma cell lines, so as to provide a theoretical basis for molecular targeted therapy of osteosarcoma. Methods: 1.. Real time quantitative PCR was used to detect the expression of miR-29c in 68 cases of osteosarcoma and normal tissue. The expression of miR-29c in two groups of SOSP-9607 osteosarcoma cell lines and two subsets of invasive F5M2 and F4 were detected by this method. 2., Lipofectamine2000 liposome method was used to transfect miR-29c mimics and inhibitor into two subsets of sosp-9607 respectively, and the mir-29c expression model with high expression and low expression in osteosarcoma cells was constructed. 3., we conducted tumor cell function experiments on the constructed cell models, including Transwell invasion and migration experiments, MTT assay for tumor cell proliferation curve, and flow cytometry to detect tumor cell cycle and apoptosis ratio. Results: the expression of mir-29c in 1. osteosarcoma tissues was significantly lower than that in adjacent normal tissues. The expression of mir-29c in highly invasive f5m2 osteosarcoma cell lines was significantly lower than that in low invasive F4 osteosarcoma cell lines. 2. mir-29cmimics and inhibitor were successfully transferred into f5m2 osteosarcoma cell line and F4 osteosarcoma cell line with lipofectamine2000 liposomes. RT-PCR results showed that the expression of mir-29c increased in f5m2/29cmimics cells. F4/29cinhibitor cells indirectly verified the expression of target genes by RT-PCR, which showed that mir-29c of F4 osteosarcoma cells was down regulated successfully. 3.transwell cell invasion and migration experiments showed that mir-29c up-regulated f5m2 invasion and migration ability of osteosarcoma cells were significantly lower than that of the control group; mir-29c down regulated the invasion and migration ability of osteosarcoma cell F4, which was significantly higher than that of the control group. MTT cell proliferation assay showed that the proliferation ability of osteosarcoma cell f5m2 increased with the increase of mir-29c, and the cell proliferation curve decreased. Compared with the control group, the cell proliferation curve of mir-29c decreased, and the proliferation ability of osteosarcoma cells increased. 4. flow cytometry cell cycle and cell apoptosis in experimental mir-29c, upregulation of f5m2 osteosarcoma cells compared with the control group, no significant difference in the proportion of the tumor cells in each cycle, and no significant difference in the proportion of apoptotic cells; mir-29c downregulation of F4 osteosarcoma cells compared with the control group, no significant differences in the proportion of the tumor cells in each cycle. There was no significant difference in the proportion of apoptotic cells. Conclusion: the content of expression of 1.mir-29c in osteosarcoma, and invasion of osteosarcoma cell mir-29c expression was lower than that of low invasive osteosarcoma cell lines, suggesting that mir-29c was negatively related to invasion and tumor, the regulation of mir-29c may be involved in the proliferation and metastasis of osteosarcoma cells. 2. through the construction of mir-29c, down-regulation of tumor cell model, verified the inhibitory effect of mir-29c on the proliferation of osteosarcoma cell invasion and migration, the next step for the study of molecular mechanism of inhibitory effect of mir-29c on proliferation and metastasis of osteosarcoma cells lay the foundation of 3.mir-29c has no obvious effect on osteosarcoma cell line sosp9607 cell cycle and apoptosis and the effect of mir-29c on tumor cell cycle and apoptosis may have tissue specificity.
【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】：R738.1

【共引文献】