Let-7b和miR-199a对B16F10细胞生长与增殖的影响
发布时间：2019-03-05 14:32
【摘要】： 目的：在前期研究基础上,进一步探讨let-7b和miR-199a对黑色素瘤增殖生长的影响。 方法：首先构建靶向let-7b和miR-199a的过表达质粒及inhibitor干扰单链,运用基因转染技术将其导入黑色素瘤高转移细胞B16F10中,使let-7b基因和miR-199a基因过表达或表达抑制,以实时荧光定量PCR(Real-time PCR)验证对应基因的差异表达,应用蛋白免疫印迹方法(Western Blot)检测相关蛋白(cyclinD1、Met)的差异表达,再以MTT绘制细胞生长曲线,并用流式细胞术检测细胞凋亡率,最后统计分析let-7b和miR-199a对B16F10细胞增殖生长的影响。 结果： 1.质粒及inhibitor转染后,各组转染细胞均可见荧光细胞,提示转染成功。 2. Real-time PCR检测发现,let-7b质粒组中let-7b基因相对表达量(3.8776±0.1372)明显高于空白组和空质粒组(1.1400±0.2769)(P0.05),let-7b inhibitor组中let-7b基因相对表达量(0.2057±0.0263)明显低于空白组和inhibitor对照组(0.9760±0.2300)(P0.05)；miR-199a质粒组中miR-199a基因相对表达量(2.8660±0.2821)明显高于空白组和空质粒组(0.9809±0.1703) (P0.05), miR-199a inhibitor组中miR-199a基因相对表达量(0.2656±0.0253)明显低于空白组和inhibitor对照组(0.7512±0.0690)(P0.05)；而空质粒组,inhibitor对照组和空白组比较无显著差异性(P0.05)。 3. Western-blot检测发现,let-7b质粒组中cyclinDl蛋白相对含量(2.023±0.315)与let-7b inhibitor组中cyclinD1蛋白相对含量(1.857±0.377)均明显高于空白组(0.997±0.041)(P0.05)；而miR-199a质粒组中cyclinD1蛋白相对含量(1.763±0.172),空质粒组(1.490±0.292), miR-199a inhibitor组(1.590±0.286)及inhibitor对照组(1.443±0.099)和空白组比较无显著差异性(P0.05)；miR-199a质粒组中Met蛋白相对含量(5.19±0.309)与miR-199a inhibitor组中Met蛋白相对含量(4.87±0.044)均明显高于空白组(2.2±0.198)(P0.05)；而let-7b质粒组中Met蛋白相对含量(3.24±0.340),空质粒组(2.85±0.047),let-7b inhibitor组(2.49±0.068)及inhibitor对照组(2.73±0.033)和空白组比较均无显著差异性(P0.05)。 4.MTT绘制细胞生长曲线发现,与空白组相比,let-7b质粒组及miR-199a质粒组生长呈下降趋势,且到转染后第三天,此趋势最明显,具有统计学意义(P0.05)；而空质粒组及let-7b inhibitor组,miR-199a inhibitor和inhibitor对照组细胞生长趋势与空白组比较无显著差异性(P0.05)。 5.流式细胞术检测细胞凋亡率发现,let-7b质粒组细胞凋亡率(11.8±1.19)%与miR-199a质粒组细胞凋亡率(11.3±1.59)%均明显高于空白组(5.77±1.74)%(P0.05)；而空质粒组细胞凋亡率(6.75±1.59)%, let-7b inhibitor组(4.39±1.52)%,miR-199a inhibitor组(4.97±1.47)%及inhibitor对照组(6.68±1.71)%与空白组比较无显著差异性(P0.05)。 结论：let-7b和miR-199a负调控B16F10细胞的增殖生长。
[Abstract]:Aim: to explore the effects of let-7b and miR-199a on the proliferation and growth of melanoma. Methods: the overexpression plasmids targeting let-7b and miR-199a and inhibitor interference single strand were constructed first, and then transfected into melanoma high metastatic cells B16F10 by gene transfection. The overexpression or inhibition of let-7b gene and miR-199a gene were induced by the transfection of let-7b gene and miR-199a gene, and the expression of let-7b gene and miR-199a gene were inhibited by RT-PCR. Real-time fluorescent quantitative PCR (Real-time PCR) was used to detect the differential expression of the corresponding genes, Western blot (Western Blot) was used to detect the differential expression of related proteins (cyclinD1,Met), and MTT was used to draw the cell growth curve. The apoptosis rate of B16F10 cells was measured by flow cytometry. Finally, the effects of let-7b and miR-199a on the proliferation and growth of B16F10 cells were analyzed statistically. Results: 1. After transfection with plasmid and inhibitor, fluorescent cells could be seen in all the transfected cells, suggesting that the transfection was successful. 2. Real-time PCR analysis showed that the relative expression of let-7b gene in let-7b plasmid group (3.8776 卤0.1372) was significantly higher than that in blank group and blank particle group (1.1400 卤0.2769) (P0.05). The relative expression of let-7b gene in let-7b inhibitor group (0.2057 卤0.0263) was significantly lower than that in blank group and inhibitor control group (0.9760 卤0.2300) (P0.05). The relative expression of miR-199a gene in miR-199a plasmid group (2.8660 卤0.2821) was significantly higher than that in blank group and blank group (0.9809 卤0.1703) (P0.05). The relative expression of miR-199a gene in miR-199a inhibitor group (0.2656 卤0.0253) was significantly lower than that in blank group and inhibitor control group (0.7512 卤0.0690) (P0.05). There was no significant difference among empty plasmid group, inhibitor control group and blank group (P0.05). 3. The relative content of cyclinDl protein in let-7b plasmid group (2.023 卤0.315) and cyclinD1 protein in let-7b inhibitor group (1.857 卤0.377) was significantly higher than that in blank group (0.997 卤0.041) (P0.05). The relative content of cyclinDl protein in let-7b plasmid group (2.023 卤0.315) was significantly higher than that in let-7b inhibitor group (1.857 卤0.377) by Western-blot. The relative content of cyclinD1 protein in miR-199a plasmid group (1.763 卤0.172), empty granule group (1.490 卤0.292), miR-199a inhibitor group (1.590 卤0.286), inhibitor control group (1.443 卤0.099) and blank group had no significant difference (P0.05). The relative content of Met protein in miR-199a plasmid group (5.19 卤0.309) and Met protein in miR-199a inhibitor group (4.87 卤0.044) was significantly higher than that in blank group (2.22 卤0.198) (P0.05). The relative content of Met protein in let-7b plasmid group was (3.24 卤0.340) and that in empty plasmid group was (2.85 卤0.047). There was no significant difference between let-7b inhibitor group (2.49 卤0.068), inhibitor control group (2.73 卤0.033) and blank group (P0.05). 4.MTT plotted cell growth curve, compared with the blank group, the growth of let-7b plasmid group and miR-199a plasmid group showed a downward trend, and the third day after transfection, this trend was the most obvious, with statistical significance (P0.05). However, there was no significant difference in cell growth trend between empty plasmid group, let-7b inhibitor group, miR-199a inhibitor and inhibitor control group and blank group (P0.05). 5. The apoptosis rate was (11.8 卤1.19)% in let-7b plasmid group and (11.3 卤1.59)% in miR-199a plasmid group, which was significantly higher than that in blank group (5.77 卤1.74)% (P0.05). However, there was no significant difference in the apoptosis rate of the blank plasmid group (6.75 卤1.59)%, the let-7b inhibitor group (4.39 卤1.52)%, the miR-199a inhibitor group (4.97 卤1.47)% and the inhibitor control group (6.68 卤1.71)% compared with the blank group (P0.05). Conclusion: let-7b and miR-199a negatively regulate the proliferation and growth of B16F10 cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5
本文编号：2435008
[Abstract]:Aim: to explore the effects of let-7b and miR-199a on the proliferation and growth of melanoma. Methods: the overexpression plasmids targeting let-7b and miR-199a and inhibitor interference single strand were constructed first, and then transfected into melanoma high metastatic cells B16F10 by gene transfection. The overexpression or inhibition of let-7b gene and miR-199a gene were induced by the transfection of let-7b gene and miR-199a gene, and the expression of let-7b gene and miR-199a gene were inhibited by RT-PCR. Real-time fluorescent quantitative PCR (Real-time PCR) was used to detect the differential expression of the corresponding genes, Western blot (Western Blot) was used to detect the differential expression of related proteins (cyclinD1,Met), and MTT was used to draw the cell growth curve. The apoptosis rate of B16F10 cells was measured by flow cytometry. Finally, the effects of let-7b and miR-199a on the proliferation and growth of B16F10 cells were analyzed statistically. Results: 1. After transfection with plasmid and inhibitor, fluorescent cells could be seen in all the transfected cells, suggesting that the transfection was successful. 2. Real-time PCR analysis showed that the relative expression of let-7b gene in let-7b plasmid group (3.8776 卤0.1372) was significantly higher than that in blank group and blank particle group (1.1400 卤0.2769) (P0.05). The relative expression of let-7b gene in let-7b inhibitor group (0.2057 卤0.0263) was significantly lower than that in blank group and inhibitor control group (0.9760 卤0.2300) (P0.05). The relative expression of miR-199a gene in miR-199a plasmid group (2.8660 卤0.2821) was significantly higher than that in blank group and blank group (0.9809 卤0.1703) (P0.05). The relative expression of miR-199a gene in miR-199a inhibitor group (0.2656 卤0.0253) was significantly lower than that in blank group and inhibitor control group (0.7512 卤0.0690) (P0.05). There was no significant difference among empty plasmid group, inhibitor control group and blank group (P0.05). 3. The relative content of cyclinDl protein in let-7b plasmid group (2.023 卤0.315) and cyclinD1 protein in let-7b inhibitor group (1.857 卤0.377) was significantly higher than that in blank group (0.997 卤0.041) (P0.05). The relative content of cyclinDl protein in let-7b plasmid group (2.023 卤0.315) was significantly higher than that in let-7b inhibitor group (1.857 卤0.377) by Western-blot. The relative content of cyclinD1 protein in miR-199a plasmid group (1.763 卤0.172), empty granule group (1.490 卤0.292), miR-199a inhibitor group (1.590 卤0.286), inhibitor control group (1.443 卤0.099) and blank group had no significant difference (P0.05). The relative content of Met protein in miR-199a plasmid group (5.19 卤0.309) and Met protein in miR-199a inhibitor group (4.87 卤0.044) was significantly higher than that in blank group (2.22 卤0.198) (P0.05). The relative content of Met protein in let-7b plasmid group was (3.24 卤0.340) and that in empty plasmid group was (2.85 卤0.047). There was no significant difference between let-7b inhibitor group (2.49 卤0.068), inhibitor control group (2.73 卤0.033) and blank group (P0.05). 4.MTT plotted cell growth curve, compared with the blank group, the growth of let-7b plasmid group and miR-199a plasmid group showed a downward trend, and the third day after transfection, this trend was the most obvious, with statistical significance (P0.05). However, there was no significant difference in cell growth trend between empty plasmid group, let-7b inhibitor group, miR-199a inhibitor and inhibitor control group and blank group (P0.05). 5. The apoptosis rate was (11.8 卤1.19)% in let-7b plasmid group and (11.3 卤1.59)% in miR-199a plasmid group, which was significantly higher than that in blank group (5.77 卤1.74)% (P0.05). However, there was no significant difference in the apoptosis rate of the blank plasmid group (6.75 卤1.59)%, the let-7b inhibitor group (4.39 卤1.52)%, the miR-199a inhibitor group (4.97 卤1.47)% and the inhibitor control group (6.68 卤1.71)% compared with the blank group (P0.05). Conclusion: let-7b and miR-199a negatively regulate the proliferation and growth of B16F10 cells.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5
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相关期刊论文 前2条
1 肖国宏;罗成群;唐国茂;周建大;;人内皮抑素基因转染原代成人黑色素瘤细胞[J];中南大学学报(医学版);2005年06期
2 徐毅;周建大;罗成群;贺全勇;聂新民;赵颜忠;PASHUPATI Babu Pokharel;王少华;徐丹;;中国汉族人黑色素瘤家系临床特点分析[J];激光生物学报;2007年06期
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