LMP1基因特异性沉默对GT38细胞影响的初步研究
发布时间：2018-09-05 11:23
【摘要】： EB病毒(Epstein-Barr virus,EBV)属疱疹病毒γ亚科,是重要的DNA肿瘤病毒,与鼻咽癌(NPC)、Burkitt淋巴瘤(BL)、胃癌(GC)等多种肿瘤的发生有关。在EBV编码基因中,潜伏膜蛋白1(Latent membrane protein 1,LMP1)编码基因已被确认具有癌基因的功能。研究表明,LMP1具有介导细胞增殖、抑制细胞凋亡和细胞分化,以及促进肿瘤的侵袭和转移等多种生物学活性。 siRNA是一种短片段双链RNA分子,能以同源互补序列的mRNA为靶目标降解特定的mRNA,从而高效、特异地阻断体内特定基因的表达,诱使细胞表现出特定基因缺失表型。因此采用siRNA技术深入探讨LMP1的生物活性对于揭示EBV致癌机制以及开展EBV相关肿瘤的特异性治疗具有实际意义。 本研究选择EBV阳性胃上皮细胞GT38作为靶细胞,采用人工合成的siRNA特异性阻断LMP1的表达,检测LMP1沉默对GT38增殖和凋亡的影响,探讨LMP1在EBV相关肿瘤发生发展中的分子机制,为EBV相关肿瘤的生物治疗提供实验依据。 目的探讨化学合成小干涉RNA(small interfering RNA,siRNA)对EBV阳性胃上皮细胞(GT38)LMP1编码基因表达的抑制作用以及对细胞增殖和凋亡的影响。 方法①采用脂质体法分别以20、30、50、80和100nM终浓度荧光标记的阴性对照siRNA(FAM-siRNA)转染GT38细胞,转染12h内用倒置荧光显微镜检测转染效率。②化学合成靶向LMP1 mRNA 649、979和1348位点的3对siRNA,以EBVⅢ型潜伏的胃上皮细胞GT38为靶细胞,采用脂质体法分别将3组siRNA转染GT38细胞,同时设非特异性对照和细胞对照。采用RT-PCR和Western blotting检测靶基因LMP1的转录表达水平,筛选出抑制效果最为理想的siRNA链并检测对LMP1表达影响的时效关系。③选择抑制效果最好的siRNA链以最佳转染浓度转染靶细胞,采用Hoechst 33258、透射电镜和流式细胞术检测GT38细胞凋亡和周期的变化;Western blotting检测LMP1沉默对GT38细胞、细胞浆和细胞核内NFκB基因表达的影响;RT-PCR检测对Bcl-2、Bax、MMP9、ICAM-1转录表达的影响。 结果①FAM-siRNA转染GT38细胞后12h各浓度组在荧光显微镜下均可观察到细胞内有点状绿色荧光出现,表明转染成功。siRNA终浓度为50,80和100nM时转染效率较高,本实验选用50nM浓度siRNA转染GT38细胞。②RT-PCR检测结果表明,与转染非特异性siRNA的细胞相比,siRNA649、siRNA979和siRNA1348对靶细胞LMP1的转录表达均具有抑制作用,差异均有统计学意义(F=235.99,P＜0.05);其中以siRNA649对LMP1转录表达的抑制作用最为明显,转染后24,48和72h时LMP1mRNA的转录水平均明显低于细胞对照组(F=89.93,P＜0.05),而转染后48和72h时LMP1mRNA的转录表达水平明显低于转染后24h时LMP1mRNA的转录表达水平。Western blotting结果显示,与细胞对照比较,转染siRNA649后48h未检测到LMP1的表达,转染后72和96h时LMP1的表达明显减弱。③Hochest 33258染色可见siRNA649作用后的GT38细胞发生凋亡,透射电镜观察到GT38细胞线粒体空泡化,染色质固缩;而转染非特异性对照siRNA的GT38细胞未观察到上述改变。流式细胞分析显示,与非特异性对照组和细胞对照组比较,siRNA649作用24,72以及120h后的细胞其细胞周期无明显改变。④Western blotting结果显示与细胞对照比较,siRNA649转染后GT38细胞总蛋白和核蛋白中NFκB的表达水平降低,细胞浆蛋白中NFκB表达水平增高。⑤RT-PCR检测Bcl-2、Bax、MMP9和ICAM-1 mRNA的转录表达,电泳结果显示与细胞对照比较,转染后24,48和72h GT38细胞Bcl-2(F=39.83,P＜0.05)、MMP9(F=177.47,P＜0.05)、ICAM-1(F=467.69,P＜0.05)的转录表达明显下调,差别有统计学意义,对Bax的转录表达无明显影响(F=0.75,P＞0.05),Bcl-2/Bax比值降低,差别有统计学意义(F=5.45,P＜0.05)。 结论化学合成siRNA能有效抑制GT38细胞中LMP1编码基因的表达,其抑制作用具有时间依赖性,在一定浓度范围内呈明显的量效关系,且对靶基因的特定序列具有较强的选择偏向性。LMP1特异性沉默可抑制NFκB的表达和核转移,进而下调Bcl-2/Bax、MMP9、ICAM-1等下游基因的表达,最终导致靶细胞的凋亡。GT38细胞可用作研究LMP1生物活性及其在EBV相关肿瘤发生中所起作用的理想的靶细胞。
[Abstract]:Epstein-Barr virus (EBV) belongs to the herpesvirus gamma subfamily. It is an important DNA oncovirus. It is associated with nasopharyngeal carcinoma (NPC), Burkitt lymphoma (BL), gastric cancer (GC) and many other tumors. In the EBV coding gene, the latent membrane protein 1 (LMP1) coding gene has been confirmed to have the function of oncogene. It has many biological activities, such as mediating cell proliferation, inhibiting cell apoptosis and differentiation, and promoting tumor invasion and metastasis.
SiRNA is a short-fragment double-stranded RNA molecule, which can degrade specific mRNA with the target of homologous complementary sequences, thus effectively and specifically block the expression of specific genes in vivo and induce cells to show specific gene deletion phenotypes. Therefore, the biological activity of LMP1 is studied by siRNA technology to reveal the carcinogenic mechanism of EBV and to carry out EBV carcinogenesis. The specific treatment of V related tumors is of practical significance.
In this study, we selected EBV-positive gastric epithelial cells GT38 as target cells, and used synthetic siRNA to specifically block the expression of LMP1. We examined the effects of LMP1 silencing on the proliferation and apoptosis of GT38, and explored the molecular mechanism of LMP1 in the development of EBV-related tumors.
Objective To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of LMP1 gene in EBV-positive gastric epithelial cells (GT38) and its effect on cell proliferation and apoptosis.
Methods (1) GT38 cells were transfected with 20,30,50,80 and 100 nM fluorescent-labeled negative control siRNA (FAM-siRNA) by liposome method, and the transfection efficiency was detected by inverted fluorescence microscopy within 12 hours. 2) Three pairs of siRNA targeting at sites 649,979 and 1348 of LMP1 mRNA were synthesized by chemosynthesis, and GT38 cells were taken as target cells. Three groups of siRNA were transfected into GT38 cells by liposome method, and non-specific control and cell control were set up. The target gene LMP1 was detected by RT-PCR and Western blotting. The siRNA chains with the best inhibitory effect were screened and the time-dependent relationship of LMP1 expression was detected. 3. The siRNA chains with the best inhibitory effect were selected. Hoechst 33258 was used to detect the apoptosis and cell cycle of GT38 cells; Western blotting was used to detect the effect of LMP1 silencing on the expression of NF-kappa B gene in GT38 cells, cytoplasm and nucleus; RT-PCR was used to detect the effect of Bcl-2, Bax, MMP9, ICAM-1 transcription and expression.
Results: (1) The dot-like green fluorescence was observed in all concentration groups 12 hours after transfection of FAM-siRNA into GT38 cells, indicating that the transfection was successful. The transfection efficiency was high when the final concentration of siRNA was 50,80 and 100 nM. The 50 nM concentration of siRNA was selected to transfect GT38 cells in this experiment. The results of RT-PCR showed that the transfection was successful. Compared with the control cells, siRNA 649, siRNA 979 and siRNA 1348 all inhibited the transcriptional expression of LMP1, and the difference was statistically significant (F = 235.99, P < 0.05); among them, siRNA 649 inhibited the transcriptional expression of LMP1 most significantly, and the transcriptional level of LMP1 mRNA at 24, 48 and 72 hours after transfection was significantly lower than that of the control group (F = 89.93, P < 0.05). The expression of LMP1 mRNA at 48 and 72 hours after transfection was significantly lower than that at 24 hours after transfection. Western blotting showed that LMP1 expression was not detected at 48 hours after transfection, but decreased significantly at 72 and 96 hours after transfection. Mitochondrial vacuolation and chromatin condensation were observed in GT38 cells after apoptosis, but not in GT38 cells transfected with non-specific siRNA. Flow cytometry analysis showed that the cell cycle of GT38 cells treated with siRNA 649 at 24, 72 and 120 h was not observed in non-specific control group and cell control group. Western blotting showed that the expression of NF-kappa B in the total protein and nucleoprotein of GT38 cells decreased and the expression of NF-kappa B in cytoplasm protein increased after transfection with siRNA 649. _The transcriptional expression of Bcl-2, Bax, MMP9 and ICAM-1 mRNA was detected by RT-PCR, and the electrophoresis results showed that compared with the cell control, the expression of NF-kappa B in the transfected GT38 cells decreased and the expression of NF-kappa B in the cytoplasm protein increased. Bcl-2 (F = 39.83, P < 0.05), MMP-9 (F = 177.47, P < 0.05), ICAM-1 (F = 467.69, P < 0.05) were significantly down-regulated, and the difference was statistically significant. There was no significant effect on the transcriptional expression of Bax (F = 0.75, P > 0.05), and the ratio of Bcl-2 to Bax was decreased (F = 5.45, P < 0.05).
Conclusion Chemically synthesized siRNA can effectively inhibit the expression of LMP1-encoded gene in GT38 cells. The inhibitory effect is time-dependent and dose-dependent in a certain concentration range. Specific silencing of LMP1 can inhibit the expression and nuclear transfer of NF-kappa B and then down-regulate Bcl-2/Ba. The expression of downstream genes such as x, MMP9 and ICAM-1 eventually leads to the apoptosis of target cells. GT38 cells can be used to study the biological activity of LMP1 and its role in EBV-related tumorigenesis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
本文编号：2224125
[Abstract]:Epstein-Barr virus (EBV) belongs to the herpesvirus gamma subfamily. It is an important DNA oncovirus. It is associated with nasopharyngeal carcinoma (NPC), Burkitt lymphoma (BL), gastric cancer (GC) and many other tumors. In the EBV coding gene, the latent membrane protein 1 (LMP1) coding gene has been confirmed to have the function of oncogene. It has many biological activities, such as mediating cell proliferation, inhibiting cell apoptosis and differentiation, and promoting tumor invasion and metastasis.
SiRNA is a short-fragment double-stranded RNA molecule, which can degrade specific mRNA with the target of homologous complementary sequences, thus effectively and specifically block the expression of specific genes in vivo and induce cells to show specific gene deletion phenotypes. Therefore, the biological activity of LMP1 is studied by siRNA technology to reveal the carcinogenic mechanism of EBV and to carry out EBV carcinogenesis. The specific treatment of V related tumors is of practical significance.
In this study, we selected EBV-positive gastric epithelial cells GT38 as target cells, and used synthetic siRNA to specifically block the expression of LMP1. We examined the effects of LMP1 silencing on the proliferation and apoptosis of GT38, and explored the molecular mechanism of LMP1 in the development of EBV-related tumors.
Objective To investigate the inhibitory effect of small interfering RNA (siRNA) on the expression of LMP1 gene in EBV-positive gastric epithelial cells (GT38) and its effect on cell proliferation and apoptosis.
Methods (1) GT38 cells were transfected with 20,30,50,80 and 100 nM fluorescent-labeled negative control siRNA (FAM-siRNA) by liposome method, and the transfection efficiency was detected by inverted fluorescence microscopy within 12 hours. 2) Three pairs of siRNA targeting at sites 649,979 and 1348 of LMP1 mRNA were synthesized by chemosynthesis, and GT38 cells were taken as target cells. Three groups of siRNA were transfected into GT38 cells by liposome method, and non-specific control and cell control were set up. The target gene LMP1 was detected by RT-PCR and Western blotting. The siRNA chains with the best inhibitory effect were screened and the time-dependent relationship of LMP1 expression was detected. 3. The siRNA chains with the best inhibitory effect were selected. Hoechst 33258 was used to detect the apoptosis and cell cycle of GT38 cells; Western blotting was used to detect the effect of LMP1 silencing on the expression of NF-kappa B gene in GT38 cells, cytoplasm and nucleus; RT-PCR was used to detect the effect of Bcl-2, Bax, MMP9, ICAM-1 transcription and expression.
Results: (1) The dot-like green fluorescence was observed in all concentration groups 12 hours after transfection of FAM-siRNA into GT38 cells, indicating that the transfection was successful. The transfection efficiency was high when the final concentration of siRNA was 50,80 and 100 nM. The 50 nM concentration of siRNA was selected to transfect GT38 cells in this experiment. The results of RT-PCR showed that the transfection was successful. Compared with the control cells, siRNA 649, siRNA 979 and siRNA 1348 all inhibited the transcriptional expression of LMP1, and the difference was statistically significant (F = 235.99, P < 0.05); among them, siRNA 649 inhibited the transcriptional expression of LMP1 most significantly, and the transcriptional level of LMP1 mRNA at 24, 48 and 72 hours after transfection was significantly lower than that of the control group (F = 89.93, P < 0.05). The expression of LMP1 mRNA at 48 and 72 hours after transfection was significantly lower than that at 24 hours after transfection. Western blotting showed that LMP1 expression was not detected at 48 hours after transfection, but decreased significantly at 72 and 96 hours after transfection. Mitochondrial vacuolation and chromatin condensation were observed in GT38 cells after apoptosis, but not in GT38 cells transfected with non-specific siRNA. Flow cytometry analysis showed that the cell cycle of GT38 cells treated with siRNA 649 at 24, 72 and 120 h was not observed in non-specific control group and cell control group. Western blotting showed that the expression of NF-kappa B in the total protein and nucleoprotein of GT38 cells decreased and the expression of NF-kappa B in cytoplasm protein increased after transfection with siRNA 649. _The transcriptional expression of Bcl-2, Bax, MMP9 and ICAM-1 mRNA was detected by RT-PCR, and the electrophoresis results showed that compared with the cell control, the expression of NF-kappa B in the transfected GT38 cells decreased and the expression of NF-kappa B in the cytoplasm protein increased. Bcl-2 (F = 39.83, P < 0.05), MMP-9 (F = 177.47, P < 0.05), ICAM-1 (F = 467.69, P < 0.05) were significantly down-regulated, and the difference was statistically significant. There was no significant effect on the transcriptional expression of Bax (F = 0.75, P > 0.05), and the ratio of Bcl-2 to Bax was decreased (F = 5.45, P < 0.05).
Conclusion Chemically synthesized siRNA can effectively inhibit the expression of LMP1-encoded gene in GT38 cells. The inhibitory effect is time-dependent and dose-dependent in a certain concentration range. Specific silencing of LMP1 can inhibit the expression and nuclear transfer of NF-kappa B and then down-regulate Bcl-2/Ba. The expression of downstream genes such as x, MMP9 and ICAM-1 eventually leads to the apoptosis of target cells. GT38 cells can be used to study the biological activity of LMP1 and its role in EBV-related tumorigenesis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373
【参考文献】
相关期刊论文 前10条
1 刘克拉,宗永生,张昌卿,张锋,冯凯涛;鼻咽癌组织中EB病毒LMP1的表达与癌细胞增殖和凋亡的关系[J];癌症;1999年01期
2 胡利富;EBV编码的潜伏性膜蛋白[J];癌症;2002年05期
3 苟新敏,陈燕,陈小毅,John R Arrand;EBV-LMP1对鼻咽癌细胞系CNE1细胞转移相关因素的影响[J];癌症;2003年05期
4 黄河,黄培春;不同来源LMP1基因转染对CNE1细胞TGFβ1抗性的影响[J];癌症;2003年12期
5 唐发清,唐敏,夏林庆,顾焕华,王海,邓锡云,曹亚;EB病毒潜伏膜蛋白1调控细胞凋亡的cDNA阵列分析[J];病毒学报;2001年02期
6 燕晓雯,石群立;EB病毒与T细胞淋巴瘤[J];医学研究生学报;2001年05期
7 王学文;EB病毒感染相关的噬血细胞性淋巴组织细胞增生症的临床特征和治疗策略[J];医学研究生学报;2004年07期
8 倪海滨,黄介飞,毛振彪,肖明兵,施公胜;胃癌患者细胞间黏附分子-1表达意义的研究[J];交通医学;2003年02期
9 马先勇,曹亚,姚开泰;EB病毒BNLF-1基因的分子生物学研究进展[J];生物化学与生物物理进展;1997年05期
10 卢忠心,曹亚;LMP1与细胞凋亡[J];国际病理科学与临床杂志;2005年05期
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