骨肉瘤组织内miR-29b作用于CDK6抑制肿瘤细胞增殖和迁移
发布时间：2018-07-25 20:12
【摘要】：[目的]文献研究表明,miR-29b具有抑制肿瘤细胞增殖和迁移的作用,而CDK6可以促进肿瘤细胞增殖和迁移。研究骨肉瘤细胞中,miR-29b是否通过作用于CDK6抑制骨肉瘤细胞增殖和迁移,为骨肉瘤的临床治疗进展提供理论依据,探寻骨肉瘤治疗新方向。[方法]临床中选取无合并其他肿瘤、非复发、未放化疗的骨肉瘤患者,留取手术切除的骨肉瘤组织和瘤旁正常组织。通过Western blotting和qRT-PCR的方法测定骨肉瘤组织内CDK6蛋白及其mRNA,同时测定细胞内miR-29b水平,寻找miR-29b与CDK6相关性。使用网络共享的miRNA靶点预测软件,预测miR-29b的靶点为CDK6 mRNA 3'-UTR。通过pre-miR-29b重组质粒转染细胞,测定转染后细胞内CDK6蛋白水平,然后诱导细胞内CDK6mRNA3'-UTR突变,消除miR-29b的预测结合位点,接着再使用pre-miR-29b重组质粒转染,测定转染后细胞内CDK6蛋白水平,验证miR-29b靶位点是否为CDK6 mRNA3'-UTR。最后,将 pre-miR-29b 重组质粒、pre-miR-control 重组质粒、CDK6质粒及control质粒四种物质按照对照的原则组合,经脂质体包裹后进行基因转染体外培养MG-63细胞系,通过CCK8检测分析转染细胞的增殖能力,Transwell试验检测转染细胞的迁移能力。[结果](1)骨肉瘤组织中表达上调的是CDK6,而不是CDK6mRNA水平;(2)骨肉瘤组织内miR-29b与CDK6蛋白水平负相关;(3)确定CDK6 mRNA3'-UTR是miR-29b的作用位点;(4)miR-29b作用于CDK6抑制骨肉瘤细胞增殖和迁移。[结论]在骨肉瘤细胞中,miR-29b通过作用于CDK6抑制肿瘤细胞增殖和迁移,为临床中骨肉瘤的治疗可能打开了新的思路。
[Abstract]:[objective] it has been shown that miR-29b can inhibit the proliferation and migration of tumor cells, while CDK6 can promote the proliferation and migration of tumor cells. To study whether miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6 in order to provide theoretical basis for the progress of clinical treatment of osteosarcoma and explore a new direction for the treatment of osteosarcoma. [methods] Osteosarcoma patients with no tumor, no recurrence, no radiotherapy and chemotherapy were selected, and the resected osteosarcoma tissues and adjacent normal tissues were retained. The CDK6 protein and its mRNAs in osteosarcoma tissues were determined by Western blotting and qRT-PCR, and the intracellular miR-29b levels were measured to find the correlation between miR-29b and CDK6. Using the miRNA target prediction software shared by the network, the target of miR-29b prediction is CDK6 mRNA 3U -UTR. After transfection with pre-miR-29b recombinant plasmid, the level of CDK6 protein in the transfected cells was measured, and then the CDK6mRNA3'-UTR mutation was induced to eliminate the predicted binding site of miR-29b. Then, the CDK6 protein level of the transfected cells was determined by using pre-miR-29b recombinant plasmid transfection. To verify whether the miR-29b target site is CDK6 mRNA3-UTR. Finally, the pre-miR-29b recombinant plasmid pre-miR-control recombinant plasmid CDK6 and control plasmid were combined according to the control principle, and then the MG-63 cell line was transfected with liposome. The proliferation ability of transfected cells was analyzed by CCK8 and the migration ability of transfected cells was detected by Transwell test. [results] (1) the expression of CDK6 was up-regulated in osteosarcoma, not the level of CDK6mRNA; (2) miR-29b was negatively correlated with CDK6 protein in osteosarcoma; (3) CDK6 mRNA3'-UTR was identified as the action site of miR-29b; (4) miR-29b inhibited proliferation and migration of osteosarcoma cells induced by CDK6. [conclusion] in osteosarcoma cells, miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6, which may open a new way for the treatment of osteosarcoma.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738.1
，
本文编号：2144950
[Abstract]:[objective] it has been shown that miR-29b can inhibit the proliferation and migration of tumor cells, while CDK6 can promote the proliferation and migration of tumor cells. To study whether miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6 in order to provide theoretical basis for the progress of clinical treatment of osteosarcoma and explore a new direction for the treatment of osteosarcoma. [methods] Osteosarcoma patients with no tumor, no recurrence, no radiotherapy and chemotherapy were selected, and the resected osteosarcoma tissues and adjacent normal tissues were retained. The CDK6 protein and its mRNAs in osteosarcoma tissues were determined by Western blotting and qRT-PCR, and the intracellular miR-29b levels were measured to find the correlation between miR-29b and CDK6. Using the miRNA target prediction software shared by the network, the target of miR-29b prediction is CDK6 mRNA 3U -UTR. After transfection with pre-miR-29b recombinant plasmid, the level of CDK6 protein in the transfected cells was measured, and then the CDK6mRNA3'-UTR mutation was induced to eliminate the predicted binding site of miR-29b. Then, the CDK6 protein level of the transfected cells was determined by using pre-miR-29b recombinant plasmid transfection. To verify whether the miR-29b target site is CDK6 mRNA3-UTR. Finally, the pre-miR-29b recombinant plasmid pre-miR-control recombinant plasmid CDK6 and control plasmid were combined according to the control principle, and then the MG-63 cell line was transfected with liposome. The proliferation ability of transfected cells was analyzed by CCK8 and the migration ability of transfected cells was detected by Transwell test. [results] (1) the expression of CDK6 was up-regulated in osteosarcoma, not the level of CDK6mRNA; (2) miR-29b was negatively correlated with CDK6 protein in osteosarcoma; (3) CDK6 mRNA3'-UTR was identified as the action site of miR-29b; (4) miR-29b inhibited proliferation and migration of osteosarcoma cells induced by CDK6. [conclusion] in osteosarcoma cells, miR-29b inhibits the proliferation and migration of osteosarcoma cells by acting on CDK6, which may open a new way for the treatment of osteosarcoma.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738.1
，
本文编号：2144950
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