双氢青蒿素调节核糖体蛋白L23抑制胃癌MKN45细胞增殖的机制研究
发布时间：2019-04-03 12:02
【摘要】：目的:研究双氢青蒿素(dihydroartemisinin,DHA)对胃癌MKN45细胞增殖、凋亡的影响,并探索其可能的机制,明确DHA在胃癌治疗中的可行性及其作用的发挥与核糖体蛋白L23(Ribosome protein L23,RPL23)活性状态是否存在关系。方法:(1)根据Pubmed人RPL23基因序列,设计3条引物,通过脂质体转染化学合成的RPL23-si RNA干扰片段,使用实时荧光定量PCT(Real-time PCR,RT-PCR)、蛋白印记实验(Western blot,WB)进行干扰效率鉴定;(2)使用不同浓度梯度的DHA作用MKN45细胞,MTT实验检测细胞增殖情况,确定DHA的半数抑制浓度(Half maximal inhibitory concentration,IC50);(3)使用DHA及si RNA转染共处理MKN45细胞,将实验分为4组:对照组(NC组)、RPL23-si RNA组、DHA组、DHA+RPL23-si RNA组,用甲基噻唑基四唑(Methylcyclopentadienyl Manganese Tricarbonyl,MTT)实验测定细胞增殖、流式细胞术(Flow cytometry,FCM)测定细胞凋亡及细胞周期,激光共聚焦扫描显微镜(Confocal laser scanning microscopy,CLSM)观察细胞中RPL23、鼠双微体2(Murine double minute 2,MDM2)蛋白定位,WB实验检测RPL23、MDM2、B细胞易位基因2(B cell translation gene 2,BTG2)、细胞周期蛋白D1(Cyclin D1)表达;(4)实验数据采取均数±标准差(?),SPSS 19.0进行统计分析。结果:(1)构建并筛选出最佳RPL23-si RNA干扰片段:RT-PCR鉴定结果显示,正常细胞组、对照组、si RNA1组、si RNA2组、si RNA3组5组中RPL23m RNA表达水平具有具有显著统计学差异(P0.01),多重比较发现,si RNA1组干扰效率明显高于RPL23-si RNA2组(P0.01)及RPL23-si RNA3组(P0.01),WB鉴定与上述结果一致,可以认为,si RNA1是最佳干扰序列,可用于后续实验。(2)DHA IC50值的确定:MTT实验检测了不同浓度梯度的DHA对于MKN45增殖的影响,MKN45的抑制率与药物浓度之间呈现出剂量依赖性,随着药物浓度的增加,MKN45细胞的抑制率随之增加,当DHA浓度为160um/L时,细胞增殖抑制率为50%左右,说明,160um/L可作为DHA的IC50值。(3)MTT实验及FCM实验显示,DHA可明显抑制胃癌MKN45增殖,诱导细胞凋亡,NC组、RPL23-si RNA组、DHA组、DHA+RPL23-si RNA组细胞的平均凋亡率之间存在统计学差异(P0.01)。细胞周期实验显示,4组细胞数在G1期、S期均表现出统计学差异(P0.01),RPL23抑制表达后,MKN45细胞的G1期细胞数目减少,S期细胞数目增多,说明RPL23-si RNA促进了细胞周期G1/S期转化,而经过DHA再次处理后,则出现了不同程度的G1/S期转化抑制。用激光共聚焦显微镜观察到,正常的MKN45细胞中,RPL23主要表达在细胞浆,MDM2则主要表达于细胞核浆,使用RPL23-si RNA转染后,细胞浆中的RPL23明显降低,而MDM2则出现了细胞核中的浓聚,而DHA处理后,两组细胞中的RPL23再次出现了胞浆中的不同程度浓聚,而MDM2则在细胞核浆中出现了荧光减弱。WB实验观察到4组细胞中RPL23(P0.01)、MDM2(P0.01)、Cyclin D1(P0.01)、BTG2(P0.01)均存在统计学差异。结论:DHA可通过RPL23-MDM2信号通路上调RPL23、BTG2表达、抑制MDM2、Cyclin D1表达发挥抗胃癌MKN45细胞增殖、诱导细胞凋亡的作用。
[Abstract]:Objective: to study the effect of dihydroartemisinin (dihydroartemisinin,DHA) on proliferation and apoptosis of gastric cancer MKN45 cells and explore its possible mechanism, and to clarify the feasibility of DHA in the treatment of gastric cancer and its function and the expression of ribosomal protein L23 (Ribosome protein L23, Whether there is a relationship between the active state of RPL23 or not. Methods: (1) according to the sequence of human RPL23 gene of Pubmed, three primers were designed and transfected into chemically synthesized RPL23-si RNA interference fragments by liposome. Real-time fluorescence quantitative PCT (Real-time PCR,RT-PCR) and protein imprinting test (Western blot, were used. WB) for the identification of interference efficiency; (2) MKN45 cells were treated with DHA with different concentration gradient. The proliferation of MKN45 cells was detected by MTT assay, and the 50% inhibitory concentration of DHA (Half maximal inhibitory concentration,IC50) was determined. (3) MKN45 cells were transfected with DHA and si RNA, and were divided into 4 groups: control group (NC group), RPL23-si RNA group, DHA RPL23-si RNA group, and the proliferation of cells was measured by methylthiazolyl tetrazolium (Methylcyclopentadienyl Manganese Tricarbonyl,MTT assay. Flow cytometry (Flow cytometry,FCM) was used to detect apoptosis and cell cycle, laser confocal scanning microscope (Confocal laser scanning microscopy,CLSM) was used to observe the localization of RPL23, mouse double microsomes 2 (Murine double minute 2, MDM 2) protein, and WB assay was used to detect RPL23,MDM2,. B cell translocation gene 2 (B cell translation gene 2, BTG2) and cyclin D1 (Cyclin D1) expression; (4) the experimental data were statistically analyzed by mean 卤standard deviation (?), SPSS 19.0). Results: (1) the optimal RPL23-si RNA interference fragment was constructed and screened. The results of RT-PCR identification showed that the normal cell group, control group, si RNA1 group, si RNA2 group, and normal cell group, control group, si RNA1 group, si RNA2 group. The expression level of RPL23m RNA in si RNA3 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). Multiple comparisons showed that the interference efficiency in si RNA1 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). WB identification is consistent with the above results, it can be considered that si RNA1 is the best interference sequence and can be used in subsequent experiments. (2) determination of DHA IC50 value: MTT test detected the effect of DHA of different concentration gradient on MKN45 proliferation. There was a dose-dependent relationship between the inhibition rate of MKN45 and the drug concentration. With the increase of the drug concentration, the inhibition rate of MKN45 cells increased. When the concentration of DHA was 160um/L, the inhibition rate of cell proliferation was about 50%, which indicated that the inhibitory rate of cell proliferation was about 50%. 160um/L can be used as the IC _ (50) value of DHA. (3) MTT and FCM test showed that DHA could significantly inhibit MKN45 proliferation and induce apoptosis of gastric cancer, NC group, RPL23-si RNA group, DHA group, NC group, RPL23-si RNA group, DHA group. There was statistical difference between the average apoptosis rate of DHA RPL23-si RNA group (P0.01). Cell cycle test showed that the number of MKN45 cells in G1 phase and S phase showed statistical difference (P0.01). After the expression of RPL23 was inhibited, the number of G1 phase cells in MKN45 cells decreased, and the number of S phase cells increased. The results showed that RPL23-si RNA promoted G1 / S phase transformation in cell cycle, but after retreatment with DHA, there were different degrees of G1 / S phase transformation inhibition. It was observed by confocal laser microscopy that RPL23 was mainly expressed in cytoplasm and MDM2 was mainly expressed in nuclear cytoplasm in normal MKN45 cells. After transfected with RPL23-si RNA, the RPL23 in cytoplasm decreased significantly. On the other hand, MDM2 showed the accumulation of nucleus, and after DHA treatment, the RPL23 in the two groups again showed different degrees of accumulation in the cytoplasm, and the concentration of RPL23 in the cytoplasm of the two groups of cells was similar to that of the control group. RPL23 (P0.01), MDM2 (P0.01) and BTG2 (P0.01) were observed in the four groups of cells by WB test. The fluorescence of MDM2 decreased in the nuclear plasma of the cells, and there were significant differences between the four groups (P0.01). Conclusion: DHA can up-regulate the expression of RPL23,BTG2 through RPL23-MDM2 signaling pathway, inhibit the expression of MDM2,Cyclin D1 and play an anti-proliferation and apoptosis-inducing role in gastric cancer MKN45 cells.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2453194
[Abstract]:Objective: to study the effect of dihydroartemisinin (dihydroartemisinin,DHA) on proliferation and apoptosis of gastric cancer MKN45 cells and explore its possible mechanism, and to clarify the feasibility of DHA in the treatment of gastric cancer and its function and the expression of ribosomal protein L23 (Ribosome protein L23, Whether there is a relationship between the active state of RPL23 or not. Methods: (1) according to the sequence of human RPL23 gene of Pubmed, three primers were designed and transfected into chemically synthesized RPL23-si RNA interference fragments by liposome. Real-time fluorescence quantitative PCT (Real-time PCR,RT-PCR) and protein imprinting test (Western blot, were used. WB) for the identification of interference efficiency; (2) MKN45 cells were treated with DHA with different concentration gradient. The proliferation of MKN45 cells was detected by MTT assay, and the 50% inhibitory concentration of DHA (Half maximal inhibitory concentration,IC50) was determined. (3) MKN45 cells were transfected with DHA and si RNA, and were divided into 4 groups: control group (NC group), RPL23-si RNA group, DHA RPL23-si RNA group, and the proliferation of cells was measured by methylthiazolyl tetrazolium (Methylcyclopentadienyl Manganese Tricarbonyl,MTT assay. Flow cytometry (Flow cytometry,FCM) was used to detect apoptosis and cell cycle, laser confocal scanning microscope (Confocal laser scanning microscopy,CLSM) was used to observe the localization of RPL23, mouse double microsomes 2 (Murine double minute 2, MDM 2) protein, and WB assay was used to detect RPL23,MDM2,. B cell translocation gene 2 (B cell translation gene 2, BTG2) and cyclin D1 (Cyclin D1) expression; (4) the experimental data were statistically analyzed by mean 卤standard deviation (?), SPSS 19.0). Results: (1) the optimal RPL23-si RNA interference fragment was constructed and screened. The results of RT-PCR identification showed that the normal cell group, control group, si RNA1 group, si RNA2 group, and normal cell group, control group, si RNA1 group, si RNA2 group. The expression level of RPL23m RNA in si RNA3 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). Multiple comparisons showed that the interference efficiency in si RNA1 group was significantly higher than that in RPL23-si RNA2 group (P0.01) and RPL23-si RNA3 group (P0.01). WB identification is consistent with the above results, it can be considered that si RNA1 is the best interference sequence and can be used in subsequent experiments. (2) determination of DHA IC50 value: MTT test detected the effect of DHA of different concentration gradient on MKN45 proliferation. There was a dose-dependent relationship between the inhibition rate of MKN45 and the drug concentration. With the increase of the drug concentration, the inhibition rate of MKN45 cells increased. When the concentration of DHA was 160um/L, the inhibition rate of cell proliferation was about 50%, which indicated that the inhibitory rate of cell proliferation was about 50%. 160um/L can be used as the IC _ (50) value of DHA. (3) MTT and FCM test showed that DHA could significantly inhibit MKN45 proliferation and induce apoptosis of gastric cancer, NC group, RPL23-si RNA group, DHA group, NC group, RPL23-si RNA group, DHA group. There was statistical difference between the average apoptosis rate of DHA RPL23-si RNA group (P0.01). Cell cycle test showed that the number of MKN45 cells in G1 phase and S phase showed statistical difference (P0.01). After the expression of RPL23 was inhibited, the number of G1 phase cells in MKN45 cells decreased, and the number of S phase cells increased. The results showed that RPL23-si RNA promoted G1 / S phase transformation in cell cycle, but after retreatment with DHA, there were different degrees of G1 / S phase transformation inhibition. It was observed by confocal laser microscopy that RPL23 was mainly expressed in cytoplasm and MDM2 was mainly expressed in nuclear cytoplasm in normal MKN45 cells. After transfected with RPL23-si RNA, the RPL23 in cytoplasm decreased significantly. On the other hand, MDM2 showed the accumulation of nucleus, and after DHA treatment, the RPL23 in the two groups again showed different degrees of accumulation in the cytoplasm, and the concentration of RPL23 in the cytoplasm of the two groups of cells was similar to that of the control group. RPL23 (P0.01), MDM2 (P0.01) and BTG2 (P0.01) were observed in the four groups of cells by WB test. The fluorescence of MDM2 decreased in the nuclear plasma of the cells, and there were significant differences between the four groups (P0.01). Conclusion: DHA can up-regulate the expression of RPL23,BTG2 through RPL23-MDM2 signaling pathway, inhibit the expression of MDM2,Cyclin D1 and play an anti-proliferation and apoptosis-inducing role in gastric cancer MKN45 cells.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
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