雙氫青蒿素調(diào)節(jié)核糖體蛋白L23抑制胃癌MKN45細(xì)胞增殖的機(jī)制研究
發(fā)布時(shí)間：2019-04-03 12:02
【摘要】：目的:研究雙氫青蒿素(dihydroartemisinin,DHA)對胃癌MKN45細(xì)胞增殖、凋亡的影響,并探索其可能的機(jī)制,明確DHA在胃癌治療中的可行性及其作用的發(fā)揮與核糖體蛋白L23(Ribosome protein L23,RPL23)活性狀態(tài)是否存在關(guān)系。方法:(1)根據(jù)Pubmed人RPL23基因序列,設(shè)計(jì)3條引物,通過脂質(zhì)體轉(zhuǎn)染化學(xué)合成的RPL23-si RNA干擾片段,使用實(shí)時(shí)熒光定量PCT(Real-time PCR,RT-PCR)、蛋白印記實(shí)驗(yàn)(Western blot,WB)進(jìn)行干擾效率鑒定;(2)使用不同濃度梯度的DHA作用MKN45細(xì)胞,MTT實(shí)驗(yàn)檢測細(xì)胞增殖情況,確定DHA的半數(shù)抑制濃度(Half maximal inhibitory concentration,IC50);(3)使用DHA及si RNA轉(zhuǎn)染共處理MKN45細(xì)胞,將實(shí)驗(yàn)分為4組:對照組(NC組)、RPL23-si RNA組、DHA組、DHA+RPL23-si RNA組,用甲基噻唑基四唑(Methylcyclopentadienyl Manganese Tricarbonyl,MTT)實(shí)驗(yàn)測定細(xì)胞增殖、流式細(xì)胞術(shù)(Flow cytometry,FCM)測定細(xì)胞凋亡及細(xì)胞周期,激光共聚焦掃描顯微鏡(Confocal laser scanning microscopy,CLSM)觀察細(xì)胞中RPL23、鼠雙微體2(Murine double minute 2,MDM2)蛋白定位,WB實(shí)驗(yàn)檢測RPL23、MDM2、B細(xì)胞易位基因2(B cell translation gene 2,BTG2)、細(xì)胞周期蛋白D1(Cyclin D1)表達(dá);(4)實(shí)驗(yàn)數(shù)據(jù)采取均數(shù)±標(biāo)準(zhǔn)差(?),SPSS 19.0進(jìn)行統(tǒng)計(jì)分析。結(jié)果:(1)構(gòu)建并篩選出最佳RPL23-si RNA干擾片段:RT-PCR鑒定結(jié)果顯示,正常細(xì)胞組、對照組、si RNA1組、si RNA2組、si RNA3組5組中RPL23m RNA表達(dá)水平具有具有顯著統(tǒng)計(jì)學(xué)差異(P0.01),多重比較發(fā)現(xiàn),si RNA1組干擾效率明顯高于RPL23-si RNA2組(P0.01)及RPL23-si RNA3組(P0.01),WB鑒定與上述結(jié)果一致,可以認(rèn)為,si RNA1是最佳干擾序列,可用于后續(xù)實(shí)驗(yàn)。(2)DHA IC50值的確定:MTT實(shí)驗(yàn)檢測了不同濃度梯度的DHA對于MKN45增殖的影響,MKN45的抑制率與藥物濃度之間呈現(xiàn)出劑量依賴性,隨著藥物濃度的增加,MKN45細(xì)胞的抑制率隨之增加,當(dāng)DHA濃度為160um/L時(shí),細(xì)胞增殖抑制率為50%左右,說明,160um/L可作為DHA的IC50值。(3)MTT實(shí)驗(yàn)及FCM實(shí)驗(yàn)顯示,DHA可明顯抑制胃癌MKN45增殖,誘導(dǎo)細(xì)胞凋亡,NC組、RPL23-si RNA組、DHA組、DHA+RPL23-si RNA組細(xì)胞的平均凋亡率之間存在統(tǒng)計(jì)學(xué)差異(P0.01)。細(xì)胞周期實(shí)驗(yàn)顯示,4組細(xì)胞數(shù)在G1期、S期均表現(xiàn)出統(tǒng)計(jì)學(xué)差異(P0.01),RPL23抑制表達(dá)后,MKN45細(xì)胞的G1期細(xì)胞數(shù)目減少,S期細(xì)胞數(shù)目增多,說明RPL23-si RNA促進(jìn)了細(xì)胞周期G1/S期轉(zhuǎn)化,而經(jīng)過DHA再次處理后,則出現(xiàn)了不同程度的G1/S期轉(zhuǎn)化抑制。用激光共聚焦顯微鏡觀察到,正常的MKN45細(xì)胞中,RPL23主要表達(dá)在細(xì)胞漿,MDM2則主要表達(dá)于細(xì)胞核漿,使用RPL23-si RNA轉(zhuǎn)染后,細(xì)胞漿中的RPL23明顯降低,而MDM2則出現(xiàn)了細(xì)胞核中的濃聚,而DHA處理后,兩組細(xì)胞中的RPL23再次出現(xiàn)了胞漿中的不同程度濃聚,而MDM2則在細(xì)胞核漿中出現(xiàn)了熒光減弱。WB實(shí)驗(yàn)觀察到4組細(xì)胞中RPL23(P0.01)、MDM2(P0.01)、Cyclin D1(P0.01)、BTG2(P0.01)均存在統(tǒng)計(jì)學(xué)差異。結(jié)論:DHA可通過RPL23-MDM2信號通路上調(diào)RPL23、BTG2表達(dá)、抑制MDM2、Cyclin D1表達(dá)發(fā)揮抗胃癌MKN45細(xì)胞增殖、誘導(dǎo)細(xì)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 鹵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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
本文編號：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 鹵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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
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