NDNF在肾癌中的表达及生物学功能研究
发布时间：2018-07-31 20:39
【摘要】：研究背景:肾细胞癌简称肾癌,是泌尿系统最常见的恶性肿瘤之一,它的发病率很高,且近年来呈现出逐年增长的趋势,因为对其发生发展的分子机制的研究并不十分清楚,给它的早期诊断以及治疗带来了很多困扰。神经元衍生的神经营养因子(NDNF)基因,又名C4orf31。目前的研究发现其在神经系统中分布广泛,起着营养支持神经元的作用,它还能维持内皮细胞生存和血管形成,有利于血管的重建。我们课题组通过前期的转录组测序工作发现NDNF在肾癌中低表达,但是,NDNF与肾癌发生发展的关系到底如何,目前尚无相关研究报道进行说明,因此,本研究主要针对NDNF在肾癌中的表达以及一些生物学功能进行了初步的研究。研究目的:1、检测NDNF在肾癌组织及肾癌细胞系中的表达;2、了解NDNF对肾癌细胞ACHN和786-0增殖、迁移以及侵袭能力的影响;3、检测 NDNF 对肾癌细胞 ACHN 和 786-0 中 EMT 标记物 E-cadherin、N-cadherin和Vimentin蛋白表达的影响,初步探索NDNF影响肾癌细胞转移的分子机制。研究方法:首先,收集未经放化疗的69例临床手术切除的肾癌组织及相应的癌旁正常组织,采用RT-qPCR和Western blot检测NDNF在肾癌组织及相应的癌旁正常组织中的mRNA和蛋白表达水平;其次,采用相同的实验方法检测NDNF在肾癌细胞系ACHN、OS-RC-2、786-0和769-P中的表达情况;然后,构建NDNF稳定表达的重组慢病毒表达细胞模型,采用CCK-8法和细胞平板克隆形成实验评估肾癌细胞的增殖能力;Transwell细胞迁移和侵袭实验检测肾癌细胞的迁移和侵袭能力;最后,采用Western blot检测EMT标记物E-cadherin、N-cadherin和Vimentin蛋白在肾癌细胞中的表达水平,初步探索NDNF影响肾癌细胞迁移的分子机制。实验数据均采用SPSS22.0统计软件进行数据分析处理,所有实验都重复至少3次以上,实验结果数据用平均值±标准差(mean±S.D.)表示。实验结果的比较采用Independent sample t-test检验。P0.05表示差异具有显著的统计学意义。研究结果:1、NDNF在肾癌组织和肾癌细胞系中的mRNA和蛋白表达水平均下调;2、肾癌细胞ACHN和786-0转染过表达病毒后,NDNF的蛋白表达水平明显高于相应的阴性对照组细胞;3、NDNF抑制肾癌细胞增殖:CCK8细胞增殖试验中,肾癌细胞ACHN和786-0的NDNF过表达组的细胞生长曲线均明显低于相应的阴性对照组(P0.05)。细胞平板克隆形成实验中,ACHN细胞的NDNF过表达组细胞克隆数为89,阴性对照组为265,克隆形成率分别为9.8%、36.4% (P0.05) ; 786-0细胞的NDNF过表达组细胞克隆数为115,阴性对照组为158,克隆形成率分别为16.4%、22.7% (P0.05),即NDNF抑制了肾癌细胞ACHN和786-0的细胞增殖能力;4、NDNF抑制肾癌细胞迁移:NDNF过表达组的肾癌细胞穿过Transwell小室至其下侧的细胞数量显著少于阴性对照组的肾癌细胞。33%醋酸洗脱小室下侧的细胞后测其平均吸光值,ACHN细胞株:0.55 (NDNF过表达组)、1.00 (阴性对照组)(P0.05) ; 786-0细胞株:0.68 (NDNF过表达组)、1.00 (阴性对照组)(P0.05)。即NDNF抑制了肾癌细胞ACHN和786-0的细胞迁移能力;5、NDNF抑制肾癌细胞侵袭:NDNF过表达组的肾癌细胞穿过Transwell小室至其下侧的细胞数量显著少于阴性对照组肾癌细胞。33%醋酸洗脱小室下侧的细胞后测其平均吸光值,ACHN细胞株:0.47 (NDNF过表达组)、1.00 (阴性对照组)(P0.05) ; 786-0细胞株:0.71 (NDNF过表达组)、1.00 (阴性对照组)(P0.05)。即NDNF抑制了肾癌细胞ACHN和786-0的细胞侵袭能力;6、在肾癌细胞ACHN和786-0中,NDNF过表达组中的EMT的间质表型标记物N-cadherin和Vimentin蛋白的表达水平相较于阴性对照组均显著下调,但是上皮表型标记物E-cadherin的蛋白表达水平显著上调。研究结论:NDNF在肾癌组织及肾癌细胞系中的表达下调;NDNF抑制肾癌细胞ACHN和786-0的增殖、迁移以及侵袭能力;NDNF下调肾癌细胞ACHN和786-0中N-cadherin和Vimentin蛋白的表达水平并上调E-cadherin蛋白的表达水平,这提示我们NDNF可能是通过EMT相关的通路来抑制肾癌细胞的迁移侵袭能力的。
[Abstract]:Background: renal cell carcinoma (RCC), called renal carcinoma, is one of the most common malignant tumors of the urinary system. Its incidence is very high, and in recent years it has been increasing year by year, because the molecular mechanism of its development is not very clear, which has brought a lot of trouble to its early diagnosis and treatment. The current study of the NDNF gene, also known as C4orf31., has found that it is widely distributed in the nervous system and plays a role in nourishing the neuron. It can also maintain endothelial cell survival and angiogenesis and facilitate the reconstruction of blood vessels. Our group has found that NDNF is low expression in renal cancer through the previous transcriptional sequencing work, but, NDNF and The relationship between the development of renal cancer and the development of renal cancer has not been reported. Therefore, this study is mainly aimed at the preliminary study of the expression of NDNF in renal carcinoma and some biological functions. 1, the expression of NDNF in renal carcinoma and renal cell carcinoma cell lines was detected. 2, NDNF was used to understand the ACHN and 786-0 of renal cell carcinoma cells. The effects of proliferation, migration and invasiveness; 3, to detect the effects of NDNF on the expression of E-cadherin, N-cadherin and Vimentin in renal cell carcinoma cells, ACHN and EMT, and to explore the molecular mechanism of NDNF affecting the metastasis of renal cell carcinoma. RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of NDNF in the renal carcinoma tissue and the corresponding normal tissues. Secondly, the same experimental method was used to detect the expression of NDNF in the renal cell line ACHN, OS-RC-2786-0 and 769-P. Then, the recombinant lentivirus, which was expressed steadily in NDNF, was constructed. The cell model was expressed, the proliferation ability of renal cell carcinoma cells was evaluated by CCK-8 method and cell plate clone, and migration and invasion test of Transwell cells was used to detect the migration and invasion ability of renal cell carcinoma cells. Finally, the expression level of EMT marker E-cadherin, N-cadherin and Vimentin protein in renal cell carcinoma cells was detected by Western blot. The molecular mechanism of renal cell migration by NDNF was explored step by step. The experimental data were analyzed by SPSS22.0 software, all experiments were repeated more than 3 times, and the experimental data were expressed with mean standard deviation (mean + S.D.). The comparison of experimental results using Independent sample t-test test.P0.05 indicated that the difference was Significant statistical significance. 1, the mRNA and protein expression levels of NDNF in renal and renal carcinoma cells were all downregulated; 2, after ACHN and 786-0 transfected in renal cell carcinoma cells, the protein expression level of NDNF was significantly higher than that of the corresponding negative control group; 3, NDNF inhibited the proliferation of renal cell carcinoma cells: in CCK8 cell proliferation test, kidney The cell growth curves of the ACHN and 786-0 NDNF over expression groups were significantly lower than that in the corresponding negative control group (P0.05). In the cell plate clone formation experiment, the number of cell clones in the NDNF overexpression group of ACHN cells was 89, the negative control group was 265, the clone formation rate was 9.8%, 36.4% (P0.05), and the NDNF overexpression group of 786-0 cells was the cell gram. The number of augmentation was 115 and the negative control group was 158, and the clone formation rate was 16.4%, 22.7% (P0.05), that is, NDNF inhibited the proliferation of ACHN and 786-0 of renal cell carcinoma cells; 4, NDNF inhibited the migration of renal cell carcinoma cells: the number of cells passing through the NDNF overexpression group from the Transwell cell to its lower side was significantly less than that of the negative control group of renal cell carcinoma cell.33% The average absorbance of the cells in the lower side of the chamber was measured by acetic acid, ACHN cell lines: 0.55 (NDNF overexpressed group), 1 (negative control group) (P0.05); 786-0 cell lines: 0.68 (NDNF overexpression group), 1 (negative control group) (P0.05). That is, NDNF inhibited the cell migration ability of ACHN and 786-0 in renal cell carcinoma cells; 5, NDNF inhibited the invasion of renal cell carcinoma cells: NDNF over The number of cells passing through the Transwell cell to the lower side of the renal cell carcinoma cells in the expression group was significantly less than that in the negative control group of renal cancer cells.33% acetic acid eluting the lower side of the cell. The ACHN cell line: 0.47 (NDNF overexpression group), 1 (negative control group) (P0.05); 786-0 cell lines: 0.71 (NDNF overexpression group), 1 (negative pairs) (P0.05). That is, NDNF inhibited the cell invasiveness of ACHN and 786-0 of renal cell carcinoma cells; 6, in the ACHN and 786-0 of renal cell carcinoma cells, the expression level of the interstitial phenotypic markers N-cadherin and Vimentin protein in the NDNF overexpression group was significantly lower than that in the negative control group, but the protein expression level of the epithelial phenotype marker E-cadherin. The expression of NDNF in renal carcinoma and renal carcinoma cell lines is down regulated; NDNF inhibits the proliferation, migration and invasiveness of ACHN and 786-0 of renal cell carcinoma cells; NDNF downregulates the expression level of ACHN and N-cadherin and Vimentin protein in renal cell carcinoma cells and up regulation of E-cadherin protein expression, which suggests that NDNF may be The migration and invasion ability of renal cell carcinoma cells was inhibited by EMT related pathways.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11
本文编号：2156765
[Abstract]:Background: renal cell carcinoma (RCC), called renal carcinoma, is one of the most common malignant tumors of the urinary system. Its incidence is very high, and in recent years it has been increasing year by year, because the molecular mechanism of its development is not very clear, which has brought a lot of trouble to its early diagnosis and treatment. The current study of the NDNF gene, also known as C4orf31., has found that it is widely distributed in the nervous system and plays a role in nourishing the neuron. It can also maintain endothelial cell survival and angiogenesis and facilitate the reconstruction of blood vessels. Our group has found that NDNF is low expression in renal cancer through the previous transcriptional sequencing work, but, NDNF and The relationship between the development of renal cancer and the development of renal cancer has not been reported. Therefore, this study is mainly aimed at the preliminary study of the expression of NDNF in renal carcinoma and some biological functions. 1, the expression of NDNF in renal carcinoma and renal cell carcinoma cell lines was detected. 2, NDNF was used to understand the ACHN and 786-0 of renal cell carcinoma cells. The effects of proliferation, migration and invasiveness; 3, to detect the effects of NDNF on the expression of E-cadherin, N-cadherin and Vimentin in renal cell carcinoma cells, ACHN and EMT, and to explore the molecular mechanism of NDNF affecting the metastasis of renal cell carcinoma. RT-qPCR and Western blot were used to detect the mRNA and protein expression levels of NDNF in the renal carcinoma tissue and the corresponding normal tissues. Secondly, the same experimental method was used to detect the expression of NDNF in the renal cell line ACHN, OS-RC-2786-0 and 769-P. Then, the recombinant lentivirus, which was expressed steadily in NDNF, was constructed. The cell model was expressed, the proliferation ability of renal cell carcinoma cells was evaluated by CCK-8 method and cell plate clone, and migration and invasion test of Transwell cells was used to detect the migration and invasion ability of renal cell carcinoma cells. Finally, the expression level of EMT marker E-cadherin, N-cadherin and Vimentin protein in renal cell carcinoma cells was detected by Western blot. The molecular mechanism of renal cell migration by NDNF was explored step by step. The experimental data were analyzed by SPSS22.0 software, all experiments were repeated more than 3 times, and the experimental data were expressed with mean standard deviation (mean + S.D.). The comparison of experimental results using Independent sample t-test test.P0.05 indicated that the difference was Significant statistical significance. 1, the mRNA and protein expression levels of NDNF in renal and renal carcinoma cells were all downregulated; 2, after ACHN and 786-0 transfected in renal cell carcinoma cells, the protein expression level of NDNF was significantly higher than that of the corresponding negative control group; 3, NDNF inhibited the proliferation of renal cell carcinoma cells: in CCK8 cell proliferation test, kidney The cell growth curves of the ACHN and 786-0 NDNF over expression groups were significantly lower than that in the corresponding negative control group (P0.05). In the cell plate clone formation experiment, the number of cell clones in the NDNF overexpression group of ACHN cells was 89, the negative control group was 265, the clone formation rate was 9.8%, 36.4% (P0.05), and the NDNF overexpression group of 786-0 cells was the cell gram. The number of augmentation was 115 and the negative control group was 158, and the clone formation rate was 16.4%, 22.7% (P0.05), that is, NDNF inhibited the proliferation of ACHN and 786-0 of renal cell carcinoma cells; 4, NDNF inhibited the migration of renal cell carcinoma cells: the number of cells passing through the NDNF overexpression group from the Transwell cell to its lower side was significantly less than that of the negative control group of renal cell carcinoma cell.33% The average absorbance of the cells in the lower side of the chamber was measured by acetic acid, ACHN cell lines: 0.55 (NDNF overexpressed group), 1 (negative control group) (P0.05); 786-0 cell lines: 0.68 (NDNF overexpression group), 1 (negative control group) (P0.05). That is, NDNF inhibited the cell migration ability of ACHN and 786-0 in renal cell carcinoma cells; 5, NDNF inhibited the invasion of renal cell carcinoma cells: NDNF over The number of cells passing through the Transwell cell to the lower side of the renal cell carcinoma cells in the expression group was significantly less than that in the negative control group of renal cancer cells.33% acetic acid eluting the lower side of the cell. The ACHN cell line: 0.47 (NDNF overexpression group), 1 (negative control group) (P0.05); 786-0 cell lines: 0.71 (NDNF overexpression group), 1 (negative pairs) (P0.05). That is, NDNF inhibited the cell invasiveness of ACHN and 786-0 of renal cell carcinoma cells; 6, in the ACHN and 786-0 of renal cell carcinoma cells, the expression level of the interstitial phenotypic markers N-cadherin and Vimentin protein in the NDNF overexpression group was significantly lower than that in the negative control group, but the protein expression level of the epithelial phenotype marker E-cadherin. The expression of NDNF in renal carcinoma and renal carcinoma cell lines is down regulated; NDNF inhibits the proliferation, migration and invasiveness of ACHN and 786-0 of renal cell carcinoma cells; NDNF downregulates the expression level of ACHN and N-cadherin and Vimentin protein in renal cell carcinoma cells and up regulation of E-cadherin protein expression, which suggests that NDNF may be The migration and invasion ability of renal cell carcinoma cells was inhibited by EMT related pathways.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11
【参考文献】
相关期刊论文 前3条
1 张辉;洪洋;徐然;宋永胜;;KISS-1与MMP-9在肾癌中的表达及其调控关系的研究[J];现代肿瘤医学;2011年03期
2 孙长成;孔垂泽;李泽良;王侠;杨春明;;肾癌组织中Pten/MMAC1/Tep1的表达及临床意义[J];中华肿瘤防治杂志;2007年11期
3 李金雨,曹润福,陈江,汪鸿,谢庆祥,缪友仁;PTEN蛋白在肾癌组织中的表达及意义[J];中华泌尿外科杂志;2003年02期
，本文编号：2156765
本文链接：https://www.wllwen.com/yixuelunwen/mjlw/2156765.html
最近更新
教材专著
