shRNA沉默Annexin A2基因对人肺腺癌细胞A549生物学行为的影响
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摘要
背景与目的：肺癌是世界上发病率较高、治疗效果相对较差的恶性肿瘤,并已成为绝大多数国家癌症死亡的首要原因。恶性增殖和侵袭转移是肺癌恶性表型的最本质表现,是影响生存率和病死率的主要因素。膜联蛋白A2 (Annexin A2)是一类钙依赖性磷脂结合蛋白,具有多种重要的生物学功能,与多种疾病的发生有关。已有研究显示Annexin A2参与肿瘤细胞的DNA合成和侵袭转移,与大多数肿瘤的发生发展有关。我们在人支气管上皮癌变各阶段组织、肺鳞癌及癌旁组织的比较蛋白质组学、肺癌血清蛋白质组学的前期研究中发现Annexin A2在癌组织中高表达,且具有抗原性,但其机制不明。为进一步探讨Annexin A2与肺癌之间的关系,本课题应用RNA干扰(RNA interference, RNAi)技术研究Annexin A2基因沉默对人肺腺癌细胞A549生物学行为的影响,为进一步阐明Annexin A2在肺癌恶性进展中的分子机制、寻找预防及治疗肺癌侵袭转移的潜在靶标提供实验依据。
方法：(1)以人肺腺癌A549细胞株为研究对象,体外构建Annexin A2序列特异性双链短发卡RNA (short hairpin RNA, shRNA)质粒,脂质体介导转染A549细胞,同时设无关序列组、空载体组、空白组为对照组,于转染后24小时荧光显微镜观察转染效率和转染后48小时收集shRNA质粒干扰细胞。(2)采用半定量RT-PCR方法检测各组细胞Annexin A2 mRNA的表达。(3)用Western blot和免疫细胞化学方法检测各组细胞Annexin A2蛋白质表达。(4)通过MTT法测定各组细胞增殖能力。(5)用Transwell小室模型检测各组细胞体外侵袭能力。(6)利用ELISA方法检测各组培养细胞上清中基质金属蛋白酶-2(matrix metalloproteinase-2, MMP-2)和组织蛋白酶B(cathepsin B, CB)的浓度。
结果：(1)AnnexinA2 shRNA质粒成功转染人肺腺癌A549细胞,转染效率达60%。(2)转染48小时后,shRNA转染组Annexin A2的mRNA及蛋白质表达水平均明显下调(p<0.05)、(p<0.05),细胞增殖活性显著降低(p<0.05),细胞的体外侵袭能力明显减弱(p<0.05),其培养上清中MMP-2和CB的浓度显著减少(p<0.05)、(p<0.05)。
结论：(1)采用RNA干扰技术能有效降解Annexin A2 mRNA,抑制其蛋白的表达,显著降低人肺癌细胞增殖、体外侵袭能力。(2)Annexin A2基因表达的下调能引起下游靶基因MMP-2及CB的表达下降。(3)表明Annexin A2在肺癌的发生发展及侵袭转移中起着重要的作用,调控MMP-2及CB的表达可能是Annexin A2影响人肺腺癌A549细胞的生物学行为的途径之一。
Background and Objective:Lung cancer is a kind of malignant tumor which has higher level of incidence and worse therapeutic efficacy in the world, and has become the first cause of malignancy-related deaths in most countries. Malignant proliferation, invasion and metastasis are the essence for malignant phenotype of lung cancer, and the main factor impacting on survival rate and case fatality. Annexin A2 is a calcium-dependent phosphnolipid-binding protein that possesses many biology functions and is related to many diseases. A number of studies have discovered that Annexin A2 participates in tumor cells DNA synthesis, invasion, metastasis and relates to development of majority tumors. In previous work, we utilized serum and comparison proteome system to study the normal-metaplasia-dysplasia-carcinoma tissue of human bronchial epithelium, lung squamous cancer and normal tissue beside lung cancer, then discovered that the expression of Annexin A2 was obviously ascended in carcinoma tissue and possessed antigenicity. However the mechanism was unknown. In order to further investigate the relationship between Annexin A2 and lung cancer, our study is to apply RNA interference technique for researching the effect of silencing of Annexin A2 on biological behavior of human lung adencarcinoma cells A549, which provides experiment evidence for further illuminating molecule mechanism about malignant progression of lung cancer and searching potential target for prevention and treatment.
Methods:(1) Human lung adencarcinoma cell line A549 as the research object, the shRNA plasmid targeting Annexin A2 was constructed in vitro and transiently transfected into A549 via lipofectamine 2000 mediation, at equal pace independence sequence control, keno-carrier control, blank control were set up, then transfection efficiency was observed by fluorescence microscope at 24 h posttransfection and shRNA plasmid interfering cells were harvested at 48 h posttransfection. (2) The mRNA expression difference of Annexin A2 was detected by semi-quantitative RT-PCR in every group cell. (3) The expression difference of Annexin A2 protein was examined by Western blotting and immuocytochemistry in every group cell. (4) The proliferation capability was determined by MTT assay. (5) Invasion capability of every group A549 cell in vitro was evaluated by using transwell chamber model. (6) The concentration difference of matrix metalloproteinase-2 and cathepsin B was measured by ELISA in the supernatant of every group cell.
Results:(1) The shRNA plasmid targeting Annexin A2 was successfully transfected into human lung adencarcinoma cell line A549, and transfection efficiency was 60%. (2) At 48 h posttransfection, the expression of Annexin A2 mRNA and protein in shRNA plasmid transfection group was down-regulated significantly (p<0.05), (p<0.05), the proliferation capability of A549 cells descended extremely (p<0.05), the invasion capability of A549 cells in vitro decreased substantially (p<0.05), the concentration of matrix metalloproteinase-2 and cathepsin B in the supernatant reduced significantly (p<0.05), (p<0.05).
Conclusions:(1) RNA interfering to Annexin A2 effectively degrades Annexin A2 mRNA and inhibits the expression of protein, and predominantly decreases proliferation and invasion capability of A549 cells in vitro. (2) Down-regulation of Annexin A2 gene expression can reduce the expression of downstream target genes including matrix metalloproteinase-2 and cathepsin B. (3) It implys that Annexin A2 might play an important role in the progression, invasion and metastasis of lung cancer, and regulating the expression of matrix metalloproteinase-2 and cathepsin B could be one of pathways for the effect of Annexin A2 on biological behavior of human lung adencarcinoma cells A549.
方法：(1)以人肺腺癌A549细胞株为研究对象,体外构建Annexin A2序列特异性双链短发卡RNA (short hairpin RNA, shRNA)质粒,脂质体介导转染A549细胞,同时设无关序列组、空载体组、空白组为对照组,于转染后24小时荧光显微镜观察转染效率和转染后48小时收集shRNA质粒干扰细胞。(2)采用半定量RT-PCR方法检测各组细胞Annexin A2 mRNA的表达。(3)用Western blot和免疫细胞化学方法检测各组细胞Annexin A2蛋白质表达。(4)通过MTT法测定各组细胞增殖能力。(5)用Transwell小室模型检测各组细胞体外侵袭能力。(6)利用ELISA方法检测各组培养细胞上清中基质金属蛋白酶-2(matrix metalloproteinase-2, MMP-2)和组织蛋白酶B(cathepsin B, CB)的浓度。
结果：(1)AnnexinA2 shRNA质粒成功转染人肺腺癌A549细胞,转染效率达60%。(2)转染48小时后,shRNA转染组Annexin A2的mRNA及蛋白质表达水平均明显下调(p<0.05)、(p<0.05),细胞增殖活性显著降低(p<0.05),细胞的体外侵袭能力明显减弱(p<0.05),其培养上清中MMP-2和CB的浓度显著减少(p<0.05)、(p<0.05)。
结论：(1)采用RNA干扰技术能有效降解Annexin A2 mRNA,抑制其蛋白的表达,显著降低人肺癌细胞增殖、体外侵袭能力。(2)Annexin A2基因表达的下调能引起下游靶基因MMP-2及CB的表达下降。(3)表明Annexin A2在肺癌的发生发展及侵袭转移中起着重要的作用,调控MMP-2及CB的表达可能是Annexin A2影响人肺腺癌A549细胞的生物学行为的途径之一。
Background and Objective:Lung cancer is a kind of malignant tumor which has higher level of incidence and worse therapeutic efficacy in the world, and has become the first cause of malignancy-related deaths in most countries. Malignant proliferation, invasion and metastasis are the essence for malignant phenotype of lung cancer, and the main factor impacting on survival rate and case fatality. Annexin A2 is a calcium-dependent phosphnolipid-binding protein that possesses many biology functions and is related to many diseases. A number of studies have discovered that Annexin A2 participates in tumor cells DNA synthesis, invasion, metastasis and relates to development of majority tumors. In previous work, we utilized serum and comparison proteome system to study the normal-metaplasia-dysplasia-carcinoma tissue of human bronchial epithelium, lung squamous cancer and normal tissue beside lung cancer, then discovered that the expression of Annexin A2 was obviously ascended in carcinoma tissue and possessed antigenicity. However the mechanism was unknown. In order to further investigate the relationship between Annexin A2 and lung cancer, our study is to apply RNA interference technique for researching the effect of silencing of Annexin A2 on biological behavior of human lung adencarcinoma cells A549, which provides experiment evidence for further illuminating molecule mechanism about malignant progression of lung cancer and searching potential target for prevention and treatment.
Methods:(1) Human lung adencarcinoma cell line A549 as the research object, the shRNA plasmid targeting Annexin A2 was constructed in vitro and transiently transfected into A549 via lipofectamine 2000 mediation, at equal pace independence sequence control, keno-carrier control, blank control were set up, then transfection efficiency was observed by fluorescence microscope at 24 h posttransfection and shRNA plasmid interfering cells were harvested at 48 h posttransfection. (2) The mRNA expression difference of Annexin A2 was detected by semi-quantitative RT-PCR in every group cell. (3) The expression difference of Annexin A2 protein was examined by Western blotting and immuocytochemistry in every group cell. (4) The proliferation capability was determined by MTT assay. (5) Invasion capability of every group A549 cell in vitro was evaluated by using transwell chamber model. (6) The concentration difference of matrix metalloproteinase-2 and cathepsin B was measured by ELISA in the supernatant of every group cell.
Results:(1) The shRNA plasmid targeting Annexin A2 was successfully transfected into human lung adencarcinoma cell line A549, and transfection efficiency was 60%. (2) At 48 h posttransfection, the expression of Annexin A2 mRNA and protein in shRNA plasmid transfection group was down-regulated significantly (p<0.05), (p<0.05), the proliferation capability of A549 cells descended extremely (p<0.05), the invasion capability of A549 cells in vitro decreased substantially (p<0.05), the concentration of matrix metalloproteinase-2 and cathepsin B in the supernatant reduced significantly (p<0.05), (p<0.05).
Conclusions:(1) RNA interfering to Annexin A2 effectively degrades Annexin A2 mRNA and inhibits the expression of protein, and predominantly decreases proliferation and invasion capability of A549 cells in vitro. (2) Down-regulation of Annexin A2 gene expression can reduce the expression of downstream target genes including matrix metalloproteinase-2 and cathepsin B. (3) It implys that Annexin A2 might play an important role in the progression, invasion and metastasis of lung cancer, and regulating the expression of matrix metalloproteinase-2 and cathepsin B could be one of pathways for the effect of Annexin A2 on biological behavior of human lung adencarcinoma cells A549.
引文
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