DDX46基因在食管鳞癌中的表达及其与食管鳞癌相关性研究
发布时间：2018-08-21 09:50
【摘要】：背景食管鳞癌是我国恶性肿瘤相关死亡的主要原因之一,虽然有手术、化疗、放疗以及其他辅助治疗方法,但其远期疗效令人沮丧,并且部分患者首次诊断时已是局部晚期或转移性疾病,失去了手术治疗的机会。食管鳞癌靶向治疗的相关研究尚处于萌芽阶段,目前许多靶向药物的相关研究多针对胃-食管连接部腺癌开展,这些研究结果对于食管鳞癌是否适合尚不清楚。近年来RNA在生命系统中的重要作用备受关注,而DDX蛋白家族RNA解旋酶参与了RNA代谢的全过程。鉴于DDX解旋酶在细胞生命活动中的重要地位,对该家族成员的功能及其与人类肿瘤关系的进一步研究,探索其上下游效应分子,明确其作用机制和调控机理,将为肿瘤的早期诊断和生物靶向治疗提供新的途径。目的本实验旨在研究DDX蛋白家族RNA解旋酶成员DDX46与食管鳞癌的关系,阐明其调控机制和作用机理,以期为揭示食管鳞癌新型生物学标记、探索新的个体化治疗策略提供实验依据。方法应用高内涵筛选成像系统(high content screening,HCS)筛选食管鳞癌患者新鲜手术标本(食管鳞癌组织和距肿瘤边缘5 cm以上食管正常组织)中的差异表达基因,发现了一个先前未被关注的基因:DDX蛋白家族RNA解旋酶成员DDX46基因。免疫组化染色实验比较食管鳞癌组织和相对应食管正常组织中DDX46蛋白的表达情况。以人永生化食管鳞状上皮细胞Het-1A为对照,实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,q RT-PCR)检测目的基因m RNA在食管鳞癌细胞株中的表达量。应用RNAi技术靶向沉默食管鳞癌细胞株DDX46基因的表达,进行Cellomics细胞计数、MTT实验、克隆形成、细胞周期和细胞凋亡,检测DDX46基因靶向沉默对食管鳞癌细胞增殖和凋亡的影响。裸鼠成瘤实验观察靶向沉默DDX46基因对食管鳞癌细胞裸鼠成瘤的影响。应用Affymetrix人类全基因表达谱芯片检测DDX46基因敲减前后两组食管鳞癌细胞中全基因m RNA表达丰度,Ingenuity通路分析(Ingenuity#174;pathway analysi,IPA)差异表达基因,差异表达基因导入IPA在线工具进行信号通路富集分析。q RT-PCR验证目的基因敲减后,部分候选下游通路基因m RNA表达量;Western blot检测验证部分候选下游通路基因蛋白表达量;Stress and Apoptosis Signaling Antibody Array Kit检测和比较两组样本中信号通路关键分子的变化,以验证IPA生物信息分析结果的可靠性。结果DDX46基因在食管鳞癌组织和细胞株中高表达,DDX46蛋白表达主要定位于细胞核。应用DDX46-sh RNA-LV靶向沉默食管鳞癌TE-1(高分化)和Eca-109(低分化)细胞中DDX46基因,检测DDX46基因沉默后食管鳞癌细胞生物功能学变化。HCS平台连续检测5天,记录各时间点的细胞数目,绘制出细胞生长曲线,与对照组相比,靶向沉默DDX46基因后TE-1和Eca-109细胞的生长均被明显抑制;MTT检测结果显示,TE-1和Eca-109细胞DDX46基因敲减组与对照组在酶标仪对波长490 nm的光的吸收率随时间变化而逐步降低,表明RNAi靶向沉默DDX46基因使食管鳞癌细胞活力明显减弱,显著抑制了细胞的增殖能力;细胞克隆形成能力检测显示,相对于对照组,DDX46-sh RNA-LV组TE-1和Eca-109细胞克隆形成数显著减少,食管鳞癌细胞克隆形成能力受到抑制;流式细胞仪检测DDX46-sh RNA-LV介导的RNAi干预后TE-1和Eca-109的细胞周期分布,结果显示,与对照组比较,RNAi沉默DDX46基因导致目的细胞处于G1期的细胞增加,而处于S期的细胞减少,表明RNAi沉默DDX46基因将食管鳞癌细胞停滞在细胞周期的G0/G1期;Annexin V-APC单染法流式细胞仪细胞凋亡检测结果显示,相对于对照组,RNAi沉默TE-1和Eca-109细胞中DDX46基因的表达后,细胞凋亡率显著增多。以Eca-109细胞为成瘤细胞,裸鼠成瘤瘤体体积增长曲线显示RNAi靶向沉默DDX46基因使得裸鼠瘤体生长减缓,小动物活体成像显示DDX46-sh RNA-LV组荧光区总荧光表达量和平均荧光表达量均明显低于Control-LV组,靶向沉默DDX46基因明显抑制Eca-109细胞裸鼠成瘤。Affymetrix人类全基因表达谱芯片检测显示,实验组DDX46-sh RNA-LV相对于对照组Control-LV,上调基因362个,下调基因644个。采用IPA生物信息分析,根据差异基因在经典通路中的富集情况,进行共表达网络构建,结合实验数据预测的信号通路图和文献数据支持的激活或抑制状态的信号通路图显示有5条抑制的信号通路和1条激活的信号通路,PI3K为非正常表达分子。q RT-PCR检测和Western blot检测结果与IPA差异基因经典通路分析结果相符合。RNAi靶向沉默TE-1细胞中DDX46基因,Western blot检测显示Akt和P-Akt的表达水平显著下调;Path Scan#174;Antibody Array检测结果发现Akt(Ser473,Phosphorylation)和IκBα(Total,N/A)蛋白表达水平明显下降,Caspase-3(Asp175,Cleaved)蛋白表达水平上升,PI3K/Akt/NF-κB信号转导通路被抑制;Western blot检测显示RNAi靶向沉默TE-1细胞中DDX46基因后,PI3K蛋白表达水平显著下调。上述结果验证了IPA生物信息学分析结果的可靠性。结论DDX46基因在食管鳞癌组织和细胞株中高表达,RNAi靶向沉默食管鳞癌细胞中的DDX46基因可抑制细胞增殖,阻滞细胞周期于G0/G1期,并诱导细胞凋亡;靶向沉默DDX46基因可明显抑制裸鼠食管鳞癌细胞成瘤。DDX46基因沉默可能是通过抑制PI3K的活性,由此参入并下调PI3K/Akt信号通路,抑制通路下游的NF-κB信号通路和m TOR信号通路,进而发挥抑制肿瘤细胞增殖、诱导细胞凋亡的作用,Integrin信号通路可能通过PI3K与PI3K/Akt信号通路“Cross Talk”而参与其中。DDX46基因沉默是否通过下调PI3K活性参与上调Rho GDI信号通路,进而参与抑制食管鳞癌的转移和浸润,是下一步研究的目标。
[Abstract]:Background Esophageal squamous cell carcinoma (ESCC) is one of the main causes of cancer-related death in China. Although there are surgery, chemotherapy, radiotherapy and other adjuvant treatment methods, the long-term efficacy of ESCC is frustrating. Some patients are locally advanced or metastatic diseases at the time of the first diagnosis, and the chance of surgical treatment is lost. In recent years, the important role of RNA in the life system has attracted much attention, and the DDX protein family RNA helicase is involved in the whole process of RNA metabolism. The important role of helicase in cell life activities, the further study of its function and its relationship with human tumors, the exploration of its upstream and downstream effector molecules, and the clarification of its mechanism of action and regulation will provide a new way for early diagnosis and targeted therapy of tumors. The relationship between RNA helicase member DDX46 and esophageal squamous cell carcinoma (ESCC) was elucidated, and its regulatory mechanism and mechanism were elucidated in order to provide experimental basis for revealing new biological markers of ESCC and exploring new individualized treatment strategies. Differentially expressed genes were found in the tissues of tubular squamous cell carcinoma (TSCC) and normal esophageal tissues more than 5 cm from the tumor margin. A previously unknown gene, DDX46, a member of the DDX protein family RNA helicase, was identified. Immunohistochemical staining was performed to compare the expression of DDX46 protein in esophageal squamous cell carcinoma (ESCC) and corresponding normal esophageal tissues. Esophageal squamous cell line Het-1A was used as control. The expression of target gene m RNA in esophageal squamous cell carcinoma cell line was detected by real-time quantitative polymerase chain reaction (q RT-PCR). The effects of targeted silencing of DDX46 gene on the proliferation and apoptosis of esophageal squamous cell carcinoma cells were studied. The effects of targeted silencing of DDX46 gene on the tumorigenesis of esophageal squamous cell carcinoma cells in nude mice were observed. The esophageal squamous cells were detected by Affymetrix human gene expression profiling chip before and after DDX46 gene knockdown. Ingenuity pathway analysis (IPA) differentially expressed genes were introduced into the IPA online tool for signal pathway enrichment analysis. Stress and Apoptosis Signaling Antibody Array Kit were used to detect and compare the changes of key molecules of signal pathway in the two groups to verify the reliability of IPA bioinformatics analysis. Nucleus. DDX46-sh RNA-LV was used to silence DDX46 gene in TE-1 (well-differentiated) and Eca-109 (poorly differentiated) esophageal squamous cell carcinoma cells. Biological function of esophageal squamous cell carcinoma cells was detected after DDX46 gene silencing. The number of cells at each time point was recorded by HCS platform for 5 days, and cell growth curve was drawn. Compared with the control group, DDDDX46 was silenced by targeted silencing. The growth of TE-1 and Eca-109 cells was significantly inhibited after gene transfection. MTT assay showed that DDX46 gene knock-down group and control group gradually decreased the absorption rate of light at 490 nm by enzyme-labeled instrument, which indicated that the activity of esophageal squamous cell carcinoma cells was significantly weakened by RNAi targeting DDX46 gene silencing, and the activity of esophageal squamous cell carcinoma cells was significantly inhibited. Compared with the control group, the clone formation of TE-1 and Eca-109 cells in DDX46-sh RNA-LV group was significantly decreased, and the clone formation ability of esophageal squamous cell carcinoma cells was inhibited. The cell cycle distribution of TE-1 and Eca-109 cells after DDX46-sh RNA-LV-mediated RNAi intervention was detected by flow cytometry. Compared with the control group, the silencing of DDX46 gene by RNAi increased the number of cells in G1 phase and decreased the number of cells in S phase, suggesting that the silencing of DDX46 gene by RNAi arrested esophageal squamous cell carcinoma cells in G0/G1 phase of cell cycle; Annexin V-APC single staining flow cytometry showed that compared with the control group, the silencing of TE-1 and TE-1 by RNAi. After DDX46 gene was expressed in Eca-109 cells, the apoptosis rate was significantly increased. Using Eca-109 cells as tumorigenic cells, the tumor volume growth curve of nude mice showed that RNAi targeted silencing of DDX46 gene slowed down tumor growth in nude mice. Small animal imaging showed that the total fluorescent expression and average fluorescent expression of DDX46-sh RNA-LV group were both positive. Targeted silencing of DDX46 gene significantly inhibited the tumorigenesis of Eca-109 cells in nude mice, significantly lower than control-LV group. Affymetrix human whole gene expression profile chip detection showed that DDX46-sh RNA-LV in the experimental group was up-regulated by 362 genes and down-regulated by 644 genes compared with control-LV in the control group. Enrichment, co-expression network construction, combined with experimental data predicted signal pathways and literature data to support the activation or inhibition of the state of signal pathways show that there are five suppressed signal pathways and one activated signal pathway, PI3K is an abnormal expression molecule. Q RT-PCR and Western blot detection results and IPA differential genes The results of classical pathway analysis were consistent. RNAi targeted silencing of DDX46 gene in TE-1 cells, Western blot analysis showed that the expression levels of Akt and P-Akt were significantly down-regulated; Path Scan #174; Antibody Array detection showed that the expression levels of Akt (Ser473, Phosphorylation) and I kappa Balpha (Total, N/A) protein were significantly decreased, and the expression of Caspase-3 (Asp175, Cleaved) protein was significantly decreased. Up-regulation of PI3K/Akt/NF-kappa B signal transduction pathway was inhibited. Western blot analysis showed that the expression of PI3K protein was significantly down-regulated after RNAi targeted silencing of DDX46 gene in TE-1 cells. These results confirmed the reliability of IPA bioinformatics analysis. Conclusion DDX46 gene was highly expressed in esophageal squamous cell carcinoma tissues and cell lines, and RNAi targeted precipitation. DDX46 gene can inhibit cell proliferation, block cell cycle in G0/G1 phase and induce apoptosis in esophageal squamous cell carcinoma cells. Targeted silencing of DDX46 gene can significantly inhibit tumor formation in nude mice esophageal squamous cell carcinoma cells. DDX46 gene silencing may be by inhibiting the activity of PI3K, thus participating in and down-regulating PI3K/Akt signaling pathway, inhibiting the downstream of the pathway of NF-Akt. Integrin signaling pathway may be involved through PI3K and PI3K/Akt signaling pathway "Cross Talk". Does DDX46 gene silencing participate in up-regulation of Rho GDI signaling pathway by down-regulation of PI3K activity, and thus in down-regulation of esophageal squamous cell carcinoma? Metastasis and infiltration are the goals of the next research.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1
本文编号：2195333
[Abstract]:Background Esophageal squamous cell carcinoma (ESCC) is one of the main causes of cancer-related death in China. Although there are surgery, chemotherapy, radiotherapy and other adjuvant treatment methods, the long-term efficacy of ESCC is frustrating. Some patients are locally advanced or metastatic diseases at the time of the first diagnosis, and the chance of surgical treatment is lost. In recent years, the important role of RNA in the life system has attracted much attention, and the DDX protein family RNA helicase is involved in the whole process of RNA metabolism. The important role of helicase in cell life activities, the further study of its function and its relationship with human tumors, the exploration of its upstream and downstream effector molecules, and the clarification of its mechanism of action and regulation will provide a new way for early diagnosis and targeted therapy of tumors. The relationship between RNA helicase member DDX46 and esophageal squamous cell carcinoma (ESCC) was elucidated, and its regulatory mechanism and mechanism were elucidated in order to provide experimental basis for revealing new biological markers of ESCC and exploring new individualized treatment strategies. Differentially expressed genes were found in the tissues of tubular squamous cell carcinoma (TSCC) and normal esophageal tissues more than 5 cm from the tumor margin. A previously unknown gene, DDX46, a member of the DDX protein family RNA helicase, was identified. Immunohistochemical staining was performed to compare the expression of DDX46 protein in esophageal squamous cell carcinoma (ESCC) and corresponding normal esophageal tissues. Esophageal squamous cell line Het-1A was used as control. The expression of target gene m RNA in esophageal squamous cell carcinoma cell line was detected by real-time quantitative polymerase chain reaction (q RT-PCR). The effects of targeted silencing of DDX46 gene on the proliferation and apoptosis of esophageal squamous cell carcinoma cells were studied. The effects of targeted silencing of DDX46 gene on the tumorigenesis of esophageal squamous cell carcinoma cells in nude mice were observed. The esophageal squamous cells were detected by Affymetrix human gene expression profiling chip before and after DDX46 gene knockdown. Ingenuity pathway analysis (IPA) differentially expressed genes were introduced into the IPA online tool for signal pathway enrichment analysis. Stress and Apoptosis Signaling Antibody Array Kit were used to detect and compare the changes of key molecules of signal pathway in the two groups to verify the reliability of IPA bioinformatics analysis. Nucleus. DDX46-sh RNA-LV was used to silence DDX46 gene in TE-1 (well-differentiated) and Eca-109 (poorly differentiated) esophageal squamous cell carcinoma cells. Biological function of esophageal squamous cell carcinoma cells was detected after DDX46 gene silencing. The number of cells at each time point was recorded by HCS platform for 5 days, and cell growth curve was drawn. Compared with the control group, DDDDX46 was silenced by targeted silencing. The growth of TE-1 and Eca-109 cells was significantly inhibited after gene transfection. MTT assay showed that DDX46 gene knock-down group and control group gradually decreased the absorption rate of light at 490 nm by enzyme-labeled instrument, which indicated that the activity of esophageal squamous cell carcinoma cells was significantly weakened by RNAi targeting DDX46 gene silencing, and the activity of esophageal squamous cell carcinoma cells was significantly inhibited. Compared with the control group, the clone formation of TE-1 and Eca-109 cells in DDX46-sh RNA-LV group was significantly decreased, and the clone formation ability of esophageal squamous cell carcinoma cells was inhibited. The cell cycle distribution of TE-1 and Eca-109 cells after DDX46-sh RNA-LV-mediated RNAi intervention was detected by flow cytometry. Compared with the control group, the silencing of DDX46 gene by RNAi increased the number of cells in G1 phase and decreased the number of cells in S phase, suggesting that the silencing of DDX46 gene by RNAi arrested esophageal squamous cell carcinoma cells in G0/G1 phase of cell cycle; Annexin V-APC single staining flow cytometry showed that compared with the control group, the silencing of TE-1 and TE-1 by RNAi. After DDX46 gene was expressed in Eca-109 cells, the apoptosis rate was significantly increased. Using Eca-109 cells as tumorigenic cells, the tumor volume growth curve of nude mice showed that RNAi targeted silencing of DDX46 gene slowed down tumor growth in nude mice. Small animal imaging showed that the total fluorescent expression and average fluorescent expression of DDX46-sh RNA-LV group were both positive. Targeted silencing of DDX46 gene significantly inhibited the tumorigenesis of Eca-109 cells in nude mice, significantly lower than control-LV group. Affymetrix human whole gene expression profile chip detection showed that DDX46-sh RNA-LV in the experimental group was up-regulated by 362 genes and down-regulated by 644 genes compared with control-LV in the control group. Enrichment, co-expression network construction, combined with experimental data predicted signal pathways and literature data to support the activation or inhibition of the state of signal pathways show that there are five suppressed signal pathways and one activated signal pathway, PI3K is an abnormal expression molecule. Q RT-PCR and Western blot detection results and IPA differential genes The results of classical pathway analysis were consistent. RNAi targeted silencing of DDX46 gene in TE-1 cells, Western blot analysis showed that the expression levels of Akt and P-Akt were significantly down-regulated; Path Scan #174; Antibody Array detection showed that the expression levels of Akt (Ser473, Phosphorylation) and I kappa Balpha (Total, N/A) protein were significantly decreased, and the expression of Caspase-3 (Asp175, Cleaved) protein was significantly decreased. Up-regulation of PI3K/Akt/NF-kappa B signal transduction pathway was inhibited. Western blot analysis showed that the expression of PI3K protein was significantly down-regulated after RNAi targeted silencing of DDX46 gene in TE-1 cells. These results confirmed the reliability of IPA bioinformatics analysis. Conclusion DDX46 gene was highly expressed in esophageal squamous cell carcinoma tissues and cell lines, and RNAi targeted precipitation. DDX46 gene can inhibit cell proliferation, block cell cycle in G0/G1 phase and induce apoptosis in esophageal squamous cell carcinoma cells. Targeted silencing of DDX46 gene can significantly inhibit tumor formation in nude mice esophageal squamous cell carcinoma cells. DDX46 gene silencing may be by inhibiting the activity of PI3K, thus participating in and down-regulating PI3K/Akt signaling pathway, inhibiting the downstream of the pathway of NF-Akt. Integrin signaling pathway may be involved through PI3K and PI3K/Akt signaling pathway "Cross Talk". Does DDX46 gene silencing participate in up-regulation of Rho GDI signaling pathway by down-regulation of PI3K activity, and thus in down-regulation of esophageal squamous cell carcinoma? Metastasis and infiltration are the goals of the next research.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.1
【参考文献】
相关期刊论文 前8条
1 Wanqing Chen;Rongshou Zheng;Hongmei Zeng;Siwei Zhang;Jie He;;Annual report on status of cancer in China, 2011[J];Chinese Journal of Cancer Research;2015年01期
2 李凤;翁文浩;龙吟;李智;;RNA解旋酶DDX39对肾透明细胞癌增殖的影响[J];同济大学学报(医学版);2014年05期
3 王丙剑;徐标;;整合素连接激酶的研究进展[J];中华生物医学工程杂志;2012年04期
4 钱帅伟;漆正堂;丁树哲;;哺乳动物雷帕霉素靶蛋白通路与细胞自噬[J];生命科学;2011年08期
5 田芳;柴玉荣;江亚南;张晓艳;;姜黄素通过下调IκBα磷酸化抑制食管鳞癌细胞的体外增殖[J];基础医学与临床;2011年07期
6 牛慧彦;李慧;王佳贺;张萌;张毅;何平;;RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌细胞中的相关性研究[J];中国组织化学与细胞化学杂志;2011年02期
7 孟艳;米蕊芳;赵春华;;PI3K-AKT-mTOR信号通路在细胞分化中的作用[J];基础医学与临床;2007年12期
8 孙晓杰;黄常志;;PI3K-Akt信号通路与肿瘤[J];世界华人消化杂志;2006年03期
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