SiRNA沉默LRP-5基因对胃癌细胞生物学行为及差异蛋白质组的影响

发布时间：2018-11-16 18:17

【摘要】：低密度脂蛋白受体相关蛋白5(LRP-5)是低密度脂蛋白受体家族成员之一,该蛋白质属于单跨膜受体,由1615个氨基酸残基组成,分为胞内区、跨膜区、胞外区三个区。LRP-5与其家族中另一个成员LRP-6高度同源。研究表明,LRP-5/LRP-6与七跨膜受体蛋白质FZD共同构成了WNT/β-catenin信号转导通路的受体系统。LRP-5/LRP-6是配体WNTs蛋白质结合的辅受体,当配体WNTs与FZD和LRP-5/LRP-6同时结合时,该信号通路才能被激活。而WNT/β-catenin信号转导通路异常调控与癌症的关系是目前研究的一个热点问题。本研究以WNT/β-catenin信号通路中辅受体LRP-5为切入点,应用RNAi技术沉默LRP-5基因表达,探讨WNT/β-catenin信号转导通路改变对胃癌细胞增殖、凋亡、侵袭、迁移等生物学行为及差异蛋白质组学的影响,以期探讨胃癌发病机制,为胃癌治疗的可能药物靶点提供实验依据。首先,用qRT-PCR和免疫组化的方法检测了不同分化程度的胃癌组织中LRP-5基因的表达状态,为下一步开展RNAi实验奠定可行性基础研究。结果显示,中分化、低分化的胃癌组织中LRP-5高表达,与正常胃组织比较差异有统计学意义(P㩳0.05),而且伴有淋巴细胞转移的胃癌组织中也呈现LRP-5高表达。这提示LRP-5与胃癌的发生、发展、转移和恶性程度相关。其次,构建了4个针对LRP-5基因不同位点的pGPU6/GFP/Neo-LRP-5干扰质粒,进行转染效率和干扰效率的评价后,成功筛选出了pGPU6/GFP/Neo-LRP-5-Homo-4868(L68)干扰质粒。经瞬时转染48h沉默MGC-803中LRP-5基因表达后,观察其对细胞增殖、粘附、凋亡、周期、侵袭、迁移的影响。结果显示:1、CCK8法检测,LRP-5基因干扰组转染MGC-803细胞48h,相对生长速度显著低于空白对照组和阴性对照组(P0.05);2、LRP-5基因干扰组MGC-803细胞的粘附细胞数为155.00±18.33,较空白对照组和阴性对照组升高,差异有统计学意义(P0.05);3、流式细胞仪双染法检测细胞凋亡,在LRP-5干扰质粒转染MGC-803细胞48h后,活细胞数量减少、凋亡细胞增多,与阴性对照组比较差异有统计学意义(P㩳0.05);4、流式细胞仪PI单染检测细胞周期,在LRP-5干扰质粒转染MGC-803细胞48h后,LRP-5干扰组的S期比例升高、G2期比例降低,发生了S期阻滞现象;5、Transwell小室基底膜实验表明,LRP-5干扰组侵袭迁移到Transwell基底膜下层的细胞计数为(38.67±3.51),与空白对照组(52.67±4.51)、Mock组(50.67±3.79)、阴性对照组(47.00±4.36)比较,细胞的侵袭能力有所下降,差异有统计学意义(P㩳0.05);6、划痕实验显示,LRP-5干扰组MGC-803细胞的迁移愈合率为0.7509±0.0108,与空白对照组和阴性对照组比较,愈合率下降,迁移能力下降,差异有统计学意义(P0.05)。最后,本研究采用RNAi技术沉默人胃癌MGC-803细胞中LRP-5基因表达,在转染MGC-803细胞48h后,抽提干扰组和阴性对照组细胞总蛋白。应用2D电泳分离纯化蛋白质、PDQuest软件分析2D电泳胶图,结果显示,两组之间共有29个差异蛋白质点。选择其中15个点进行质谱鉴定,成功12个。与阴性对照组比较,L68干扰组中有10个点上调,2个点下调。这些蛋白质包括:细胞骨架、分子伴侣、细胞周期、细胞增殖、糖代谢相关的蛋白质。本研究首次应用RNAi技术沉默LRP-5基因,对胃癌MGC-803细胞的生物学行为及差异蛋白质的影响进行深入研究,初步发现了LRP-5的低表达可抑制胃癌MGC-803细胞的增殖、迁移和侵袭,为探讨胃癌发病机制和开发胃癌治疗的可能药物靶点提供了实验依据。
[Abstract]:The low-density lipoprotein receptor-related protein 5 (LRP-5) is one of the members of the low-density lipoprotein receptor family, which belongs to a single-transmembrane receptor and consists of 1615 amino acid residues, and is divided into three regions of an intracellular region, a transmembrane region and an extracellular region. The LRP-5 is highly homologous to the other member LRP-6 in the family. The study shows that the LRP-5/ LRP-6 and the seven-transmembrane receptor protein FZD form the receptor system of the WNT/ HCO3-catenin signal transduction pathway. LRP-5/ LRP-6 is an auxiliary receptor for ligand WNTs protein binding, which can be activated when ligand WNTs is combined with FZD and LRP-5/ LRP-6. The relationship between the abnormal regulation and the cancer of the WNT/ P-catenin signal transduction pathway is a hot issue in the current research. In this study, the LRP-5 gene expression in WNT/ HCO3-cattenin signal pathway was used to silence the expression of LRP-5 gene, and the effects of the change of WNT/ P-catenin signal transduction pathway on the proliferation, apoptosis, invasion and migration of gastric cancer cells and the differential proteomics were discussed. Objective To study the mechanism of gastric cancer and provide experimental basis for possible drug target for gastric cancer treatment. First, the expression status of LRP-5 gene in gastric cancer tissues with different degree of differentiation was detected by qRT-PCR and immunohistochemistry, and the feasibility of the next step was studied. The results showed that the high expression of LRP-5 in the middle and low-differentiated gastric cancer tissues was significantly different from that of the normal gastric tissues (P <0.05), and the LRP-5 high expression was also present in the gastric cancer tissues with the lymphocyte metastasis. This suggests that LRP-5 is associated with the occurrence, development, metastasis and malignancy of gastric cancer. The pGPU6/ GFP/ Neo-LRP-5 interference plasmid was constructed and the pGU6/ GFP/ Neo-LRP-5-Homo-4868 (L68) interference plasmid was successfully screened. After transient transfection of the LRP-5 gene in MGC-803, the effects of the expression of LRP-5 on cell proliferation, adhesion, apoptosis, cycle, invasion and migration were observed. The results showed that: 1, CCK8 method, LRP-5 gene interference group was transfected into MGC-803 cells for 48h, and the relative growth rate was significantly lower than that of blank control group and negative control group (P0.05). The number of adherent cells of MGC-803 cells in LRP-5 gene interference group was 155. 00-18.33, and the control group and negative control group increased. The difference was significant (P0.05); 3, the cell apoptosis was detected by flow cytometry double-staining method, and after the LRP-5 interference plasmid was transfected into the MGC-803 cells for 48h, the number of living cells was reduced, the number of apoptotic cells increased, and the difference with the negative control group was statistically significant (P 0.05); and 4, The cell cycle of the cell cycle was detected by flow cytometry PI. The S-phase ratio of LRP-5 interference group was increased and the proportion of G2 phase was decreased after 48 h of the LRP-5 interference plasmid. The cell count of LRP-5 interference group invasion and migration to the lower layer of Transwell basement membrane was (38. 67-3.51), compared with the blank control group (52. 67-4.51), the Mock group (50. 67-3.79) and the negative control group (47. 00-4.36), the invasion ability of the cells decreased and the difference was statistically significant (P-0.05); The results showed that the rate of migration and healing of MGC-803 cells in LRP-5 interference group was 0. 7509-0. 0108, compared with the blank control group and the negative control group, the healing rate decreased, the migration ability decreased, and the difference was statistically significant (P0.05). Finally, the LRP-5 gene expression in the MGC-803 cell of human gastric cancer was silenced by RNAi, and the total protein of the interfering group and the negative control group was extracted after the MGC-803 cell was transfected for 48h. The protein and PDQuest software were separated and purified by 2D electrophoresis. The results showed that there were 29 different protein points between the two groups. 15 of them were selected for mass spectrometry and 12 were successful. Compared with the negative control group, 10 of the L68 interference groups were up-regulated and 2 points were down-regulated. These proteins include a cytoskeleton, a molecular chaperone, a cell cycle, cell proliferation, and sugar metabolism-related proteins. In this study, the LRP-5 gene was used to silence the effects of LRP-5 gene on the biological behavior and differential protein of MGC-803 cells in gastric cancer. The low expression of LRP-5 was found to inhibit the proliferation, migration and invasion of MGC-803 cells in gastric cancer. In order to study the mechanism of gastric cancer and the possible drug target for the development of gastric cancer, the experimental basis is provided.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2

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