Let-7b和miR-199a對B16F10細(xì)胞生長與增殖的影響
發(fā)布時(shí)間：2019-03-05 14:32
【摘要】： 目的：在前期研究基礎(chǔ)上,進(jìn)一步探討let-7b和miR-199a對黑色素瘤增殖生長的影響。 方法：首先構(gòu)建靶向let-7b和miR-199a的過表達(dá)質(zhì)粒及inhibitor干擾單鏈,運(yùn)用基因轉(zhuǎn)染技術(shù)將其導(dǎo)入黑色素瘤高轉(zhuǎn)移細(xì)胞B16F10中,使let-7b基因和miR-199a基因過表達(dá)或表達(dá)抑制,以實(shí)時(shí)熒光定量PCR(Real-time PCR)驗(yàn)證對應(yīng)基因的差異表達(dá),應(yīng)用蛋白免疫印跡方法(Western Blot)檢測相關(guān)蛋白(cyclinD1、Met)的差異表達(dá),再以MTT繪制細(xì)胞生長曲線,并用流式細(xì)胞術(shù)檢測細(xì)胞凋亡率,最后統(tǒng)計(jì)分析let-7b和miR-199a對B16F10細(xì)胞增殖生長的影響。 結(jié)果： 1.質(zhì)粒及inhibitor轉(zhuǎn)染后,各組轉(zhuǎn)染細(xì)胞均可見熒光細(xì)胞,提示轉(zhuǎn)染成功。 2. Real-time PCR檢測發(fā)現(xiàn),let-7b質(zhì)粒組中l(wèi)et-7b基因相對表達(dá)量(3.8776±0.1372)明顯高于空白組和空質(zhì)粒組(1.1400±0.2769)(P0.05),let-7b inhibitor組中l(wèi)et-7b基因相對表達(dá)量(0.2057±0.0263)明顯低于空白組和inhibitor對照組(0.9760±0.2300)(P0.05)；miR-199a質(zhì)粒組中miR-199a基因相對表達(dá)量(2.8660±0.2821)明顯高于空白組和空質(zhì)粒組(0.9809±0.1703) (P0.05), miR-199a inhibitor組中miR-199a基因相對表達(dá)量(0.2656±0.0253)明顯低于空白組和inhibitor對照組(0.7512±0.0690)(P0.05)；而空質(zhì)粒組,inhibitor對照組和空白組比較無顯著差異性(P0.05)。 3. Western-blot檢測發(fā)現(xiàn),let-7b質(zhì)粒組中cyclinDl蛋白相對含量(2.023±0.315)與let-7b inhibitor組中cyclinD1蛋白相對含量(1.857±0.377)均明顯高于空白組(0.997±0.041)(P0.05)；而miR-199a質(zhì)粒組中cyclinD1蛋白相對含量(1.763±0.172),空質(zhì)粒組(1.490±0.292), miR-199a inhibitor組(1.590±0.286)及inhibitor對照組(1.443±0.099)和空白組比較無顯著差異性(P0.05)；miR-199a質(zhì)粒組中Met蛋白相對含量(5.19±0.309)與miR-199a inhibitor組中Met蛋白相對含量(4.87±0.044)均明顯高于空白組(2.2±0.198)(P0.05)；而let-7b質(zhì)粒組中Met蛋白相對含量(3.24±0.340),空質(zhì)粒組(2.85±0.047),let-7b inhibitor組(2.49±0.068)及inhibitor對照組(2.73±0.033)和空白組比較均無顯著差異性(P0.05)。 4.MTT繪制細(xì)胞生長曲線發(fā)現(xiàn),與空白組相比,let-7b質(zhì)粒組及miR-199a質(zhì)粒組生長呈下降趨勢,且到轉(zhuǎn)染后第三天,此趨勢最明顯,具有統(tǒng)計(jì)學(xué)意義(P0.05)；而空質(zhì)粒組及l(fā)et-7b inhibitor組,miR-199a inhibitor和inhibitor對照組細(xì)胞生長趨勢與空白組比較無顯著差異性(P0.05)。 5.流式細(xì)胞術(shù)檢測細(xì)胞凋亡率發(fā)現(xiàn),let-7b質(zhì)粒組細(xì)胞凋亡率(11.8±1.19)%與miR-199a質(zhì)粒組細(xì)胞凋亡率(11.3±1.59)%均明顯高于空白組(5.77±1.74)%(P0.05)；而空質(zhì)粒組細(xì)胞凋亡率(6.75±1.59)%, let-7b inhibitor組(4.39±1.52)%,miR-199a inhibitor組(4.97±1.47)%及inhibitor對照組(6.68±1.71)%與空白組比較無顯著差異性(P0.05)。 結(jié)論：let-7b和miR-199a負(fù)調(diào)控B16F10細(xì)胞的增殖生長。
[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 鹵0.1372) was significantly higher than that in blank group and blank particle group (1.1400 鹵0.2769) (P0.05). The relative expression of let-7b gene in let-7b inhibitor group (0.2057 鹵0.0263) was significantly lower than that in blank group and inhibitor control group (0.9760 鹵0.2300) (P0.05). The relative expression of miR-199a gene in miR-199a plasmid group (2.8660 鹵0.2821) was significantly higher than that in blank group and blank group (0.9809 鹵0.1703) (P0.05). The relative expression of miR-199a gene in miR-199a inhibitor group (0.2656 鹵0.0253) was significantly lower than that in blank group and inhibitor control group (0.7512 鹵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 鹵0.315) and cyclinD1 protein in let-7b inhibitor group (1.857 鹵0.377) was significantly higher than that in blank group (0.997 鹵0.041) (P0.05). The relative content of cyclinDl protein in let-7b plasmid group (2.023 鹵0.315) was significantly higher than that in let-7b inhibitor group (1.857 鹵0.377) by Western-blot. The relative content of cyclinD1 protein in miR-199a plasmid group (1.763 鹵0.172), empty granule group (1.490 鹵0.292), miR-199a inhibitor group (1.590 鹵0.286), inhibitor control group (1.443 鹵0.099) and blank group had no significant difference (P0.05). The relative content of Met protein in miR-199a plasmid group (5.19 鹵0.309) and Met protein in miR-199a inhibitor group (4.87 鹵0.044) was significantly higher than that in blank group (2.22 鹵0.198) (P0.05). The relative content of Met protein in let-7b plasmid group was (3.24 鹵0.340) and that in empty plasmid group was (2.85 鹵0.047). There was no significant difference between let-7b inhibitor group (2.49 鹵0.068), inhibitor control group (2.73 鹵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 鹵1.19)% in let-7b plasmid group and (11.3 鹵1.59)% in miR-199a plasmid group, which was significantly higher than that in blank group (5.77 鹵1.74)% (P0.05). However, there was no significant difference in the apoptosis rate of the blank plasmid group (6.75 鹵1.59)%, the let-7b inhibitor group (4.39 鹵1.52)%, the miR-199a inhibitor group (4.97 鹵1.47)% and the inhibitor control group (6.68 鹵1.71)% compared with the blank group (P0.05). Conclusion: let-7b and miR-199a negatively regulate the proliferation and growth of B16F10 cells.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R739.5
本文編號：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 鹵0.1372) was significantly higher than that in blank group and blank particle group (1.1400 鹵0.2769) (P0.05). The relative expression of let-7b gene in let-7b inhibitor group (0.2057 鹵0.0263) was significantly lower than that in blank group and inhibitor control group (0.9760 鹵0.2300) (P0.05). The relative expression of miR-199a gene in miR-199a plasmid group (2.8660 鹵0.2821) was significantly higher than that in blank group and blank group (0.9809 鹵0.1703) (P0.05). The relative expression of miR-199a gene in miR-199a inhibitor group (0.2656 鹵0.0253) was significantly lower than that in blank group and inhibitor control group (0.7512 鹵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 鹵0.315) and cyclinD1 protein in let-7b inhibitor group (1.857 鹵0.377) was significantly higher than that in blank group (0.997 鹵0.041) (P0.05). The relative content of cyclinDl protein in let-7b plasmid group (2.023 鹵0.315) was significantly higher than that in let-7b inhibitor group (1.857 鹵0.377) by Western-blot. The relative content of cyclinD1 protein in miR-199a plasmid group (1.763 鹵0.172), empty granule group (1.490 鹵0.292), miR-199a inhibitor group (1.590 鹵0.286), inhibitor control group (1.443 鹵0.099) and blank group had no significant difference (P0.05). The relative content of Met protein in miR-199a plasmid group (5.19 鹵0.309) and Met protein in miR-199a inhibitor group (4.87 鹵0.044) was significantly higher than that in blank group (2.22 鹵0.198) (P0.05). The relative content of Met protein in let-7b plasmid group was (3.24 鹵0.340) and that in empty plasmid group was (2.85 鹵0.047). There was no significant difference between let-7b inhibitor group (2.49 鹵0.068), inhibitor control group (2.73 鹵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 鹵1.19)% in let-7b plasmid group and (11.3 鹵1.59)% in miR-199a plasmid group, which was significantly higher than that in blank group (5.77 鹵1.74)% (P0.05). However, there was no significant difference in the apoptosis rate of the blank plasmid group (6.75 鹵1.59)%, the let-7b inhibitor group (4.39 鹵1.52)%, the miR-199a inhibitor group (4.97 鹵1.47)% and the inhibitor control group (6.68 鹵1.71)% compared with the blank group (P0.05). Conclusion: let-7b and miR-199a negatively regulate the proliferation and growth of B16F10 cells.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R739.5
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1 肖國宏;羅成群;唐國茂;周建大;;人內(nèi)皮抑素基因轉(zhuǎn)染原代成人黑色素瘤細(xì)胞[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年06期
2 徐毅;周建大;羅成群;賀全勇;聶新民;趙顏忠;PASHUPATI Babu Pokharel;王少華;徐丹;;中國漢族人黑色素瘤家系臨床特點(diǎn)分析[J];激光生物學(xué)報(bào);2007年06期
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