非小细胞肺癌DLC-1、ROCK1的表达及生物学作用机制的研究
发布时间：2018-07-26 14:37
【摘要】：肺癌是我国乃至全世界发病率及死亡率均较高的恶性肿瘤之一,而非小细胞肺癌NSCLC (non small cell lung cancer,NSCLC)占全部肺癌的80%以上。虽然人们目前对于非小细胞肺癌发病、早期诊断和治疗已经进行了非常深入的研究,但其确切发生、发展的分子机制尚不明确,有待于进一步研究以期在非小细胞肺癌早期诊断和治疗上取得进一步的突破。肝癌缺失基因(deleted in liver cancer 1,DLC-1)是在肝癌研究中被首次发现的一种抑癌基因,它位于人体第8号染色体的21.3-22区域。DLC-1基因在乳腺癌、鼻咽癌、胰腺癌、大肠癌、胃癌等多种恶性肿瘤中的表达及相关机制是当今研究的热点之一。当前研究表明,在大多数恶性肿瘤中DLC-1呈现出低水平表达甚至是表达缺失,恢复DLC-1的表达能够显著地抑制肿瘤细胞的增殖能力,促进肿瘤细胞的凋亡。可见,研究DLC-1在恶性肿瘤中的表达机制,对于发现新的肿瘤治疗靶点和新的肿瘤治疗方法具有十分重要的意义。目前国内外对于DLC-1在肺癌中的研究相对较少,DLC-1与患者的生存率方面的研究尚属空白,DLC-1在肺癌中发挥作用的机制并不明确。本课题通过研究DLC-1在非小细胞肺癌组织和癌旁组织中的表达差异,分析DLC-1与患者性别、吸烟史及肿瘤病理类型、分化程度、淋巴结是否转移、TNM分期的相关性,探讨DLC-1与肺癌发生发展的关系；并构建了pcDNA3.1-DLC-1质粒,在非小细胞肺癌细胞株中过表达该质粒,探索DLC-1对非小细胞肺癌细胞生长、侵袭的影响；有报道表明,DLC-1可能通过RhoA/ROCK信号通路调控肿瘤的发生及生长,因此本研究也探索了DLC-1对于ROCK1的影响,探索DLC-1与RhoA/ROCK信号通路的关系。对于DLC-1在肺癌中发挥作用的机制进行研究,为发现新的肿瘤治疗靶点和新的肿瘤治疗方法提供新的思路。本研究分为以下三个部分：第一部分非小细胞肺癌中DLC-1、ROCK1蛋白表达水平的检测及临床意义目的探索非小细胞肺癌组织中肝癌缺失基因DLC-1基因和Rho相关卷曲螺旋形成蛋白(Rho-kinase 1,ROCK1)的蛋白表达水平,分析两种蛋白与各项临床指标的相关性。方法1.本研究回顾性分析了2011年1月至2012年6月在滨州医学院附属医院胸外科进行外科手术治疗的68例非小细胞肺癌病人的临床资料,所有病人均经病理检查证实,且术前未行放化疗等抗肿瘤治疗。收集68对非小细胞肺癌的癌组织及对应的癌旁组织石蜡包埋块。2.采用Envision免疫组织化学技术检测DLC-1基因与ROCK1基因在癌组织及对应的癌旁组织中的表达水平。3.分析DLC-1、ROCK1与各项临床指标的相关性,并研究两种蛋白表达水平的相关性。4.所有患者的随访截止到2015年3月。5.生存期计算从手术日期至死亡或2015年3月末次随访结束。根据随访结果比较DLC-1不同表达组之间生存期的差异。结果1.免疫组化结果显示：DLC-1蛋白在非小细胞肺癌组织中呈现低水平表达甚至是表达缺失,其阳性表达率为38.24%(26/68),明显低于癌旁组织75%(51/68),二者的表达差异有统计学意义(X2=14.256, P0.01): ROCKI蛋白在非小细胞肺癌中的阳性表达率为57.35%(39/68),在癌旁组织呈表达缺失,二者的表达差异有统计学意义(X2=45.378,P0.01)；2.在非小细胞肺癌组织中,DLC-1、ROCKI蛋白的表达与性别、吸烟史有无及肿瘤的病理学类型无关,而与肿瘤分化程度、淋巴结转移及TNM分期有关,差异有统计学意义；非小细胞肺癌DLC-1阳性表达组中ROCK1的阳性表达率为34.62%(9/26),DLC-1阴性表达组中ROCK1的阳性表达率为71.43%(30/42),相关分析提示,DLC-1与ROCK1的表达中呈负相关(r=-2.359,P=0.04)；3.DLC-1蛋白高表达组的3年生存率(88%)高于低表达组(57.2%),差异有统计学意义(P=0.031)。结论DLC-1蛋白在非小细胞肺癌组织中呈现低表达水平甚至是表达缺失,与ROCK1蛋白呈现明显的负相关性,这表明DLC-1在非小细胞肺癌中发挥作用的机制可能是通过调控ROCK1信号通路。DLC-1蛋白可以作为检测非小细胞肺癌患者预后水平的潜在指标之一。第二部分DLC-1、ROCK1在非小细胞肺癌组织及细胞株中的表达及其相关性研究目的研究非小细胞肺癌新鲜组织及细胞株中DLC-1和ROCK1的蛋白及mRNA水平的表达情况及二者的相关性。方法1.收集新鲜非小细胞肺癌组织及癌旁组织各23例,肺癌细胞株H1299、A549及Calu6。2.运用逆转录聚合酶链反应(Real time RT-PCR)检测新鲜肺癌组织中DLC-1及ROCK1基因mRNA水平的表达情况。3.运用逆转录聚合酶链反应(Real time RT-PCR)及Western Blot检测肺癌细胞株中的DLC-1及ROCK1基因mRNA水平及蛋白水平的表达情况。结果1.共收集肺癌及癌旁组织各23例,结果提示在肺癌组织中DLC-1 mRNA的表达量明显低于癌旁组织,ROCK1则在肺癌组织中表达量高于癌旁组织,二者亦存在负相关的关系。2.在肺癌H1299细胞株中,DLC-1基因mRNA水平及蛋白水平表达呈阳性,而在A549、Calu6细胞株中则呈阴性；ROCK1的结果与之相反。结论DLC-1在肺癌新鲜组织及细胞株中呈低表达或表达缺失,ROCK1则相反,进一步证实DLC-1在非小细胞肺癌中发挥作用的机制可能是通过对于ROCK1信号通路的调控。第三部分pcDNA3.1-DLC-1重组载体的构建及DLC-1对肺癌细胞的生物学功能的影响目的构建DLC-1基因的表达载体pcDNA3.1-DLC-1,在肺癌细胞系中过表达DLC-1蛋白,探索DLC-1蛋白对于肺癌细胞株生长、侵袭及凋亡的影响,对ROCK1表达的影响,进一步探讨DLC-1蛋白在非小细胞肺癌中发挥作用的机制,为非小细胞肺癌的诊断及治疗提供新的临床思路及理论依据。方法1.DLC-1表达载体的构建：提取肺癌细胞株H1299总RNA,通过逆转录酶合成cDNA;应用RT-PCR扩增DLC-1基因全序列ORF,插入真核表达载体pcDNA3.1.2.DCL-1对ROCK1表达的影响：用无内毒素抽提方法抽取已构建好的表达载体,脂质体瞬时转染A549、Calu6细胞,48小时后抽取细胞RNA,逆转录cDNA,鉴定RNA和cDNA质量,设计ROCK1引物,RT-PCR鉴定ROCK1基因表达变化。以质粒空载pcDNA3.1转染的A549、Calu6为阴性对照。3.DLC-1对非小细胞肺癌侵袭能力的影响：用无内毒素抽提方法抽取已构建好表达载体,脂质体瞬时转染A549细胞,利用transwell小室实验评估其对肿瘤细胞侵袭能力的影响。4.DCL-1对非小细胞肺癌生长能力的影响：用无内毒素抽提方法抽取已构建好表达载体,脂质体瞬时转染A549、Calu6细胞,稀释至200cells/10ml,接种入6孔板,待克隆生长完成,以结晶紫染色,观察细胞生长情况。以质粒空载pcDNA3.1转染的A549、Calu6为阴性对照。5.DCL-1对非小细胞肺癌细胞周期及凋亡的影响：用无内毒素抽提方法抽取已构建好表达载体,脂质体瞬时转染A549细胞,DAPI染色,FACS检测其对细胞周期和凋亡的影响。以质粒空载pcDNA3.1转染的A549为阴性对照。结果1.应用基因克隆技术成功构建DLC-1基因真核表达载体pcDNA3.1-DLC-1.2.肺癌细胞株A549、Calu6中过表达DLC-1对ROCK1转录水平有着负性调控的作用。转染了pcDNA3.1-DLC-1后DLC-1在mRNA水平上有较高的过表达效果,结果具有显著性差异,P0.05。对其中的ROCK1的mRNA水平进行检测可知,过表达DLC-1后,ROCK1的mRNA水平受到了显著的抑制,A549、Calu6中过表达DLC-1后的ROCK1的mRNA水平下降分别为对照组的33%、47%,结果均具有显著性差异,P0.05。3.肺癌细胞株A549、Calu6细胞中过表达的DLC-1后,在体外侵袭实验中对实验组和对照组的穿膜细胞进行统计分别为26.8±1.3、67.8±5.2,结果具有显著性差异,P0.05。克隆形成实验提示A549、Calu6细胞的克隆形成能力显著下降；A549细胞株的对照组的克隆形成率为38.9±2.6%,实验组的克隆形成率为21.8±5.2%,结果具有显著性差异,P0.05; Calu6细胞株的对照组的克隆形成率为35.8±1.2%,实验组的克隆形成率为19.6±6.9%,结果具有显著性差异,P0.05。流式结果显示过表达DLC-1后,可以显著抑制细胞周期,降低S期细胞和G2期细胞数,提高G1期细胞数,并可促进癌细胞的凋亡。结论在肺癌细胞中DLC-1的表达与ROCK1呈明显的负相关,且DLC-1能够显著地抑制肺癌细胞的增殖和侵袭能力,对肺癌细胞的细胞周期也具有明显的抑制作用,并促进了肺癌细胞的凋亡,因此,DLC-1可能是通过作用ROCK1调控肺癌的发生和发展。
[Abstract]:Lung cancer is one of the malignant tumors with high morbidity and mortality in our country and in the world, but the non small cell lung cancer NSCLC (non small cell lung cancer, NSCLC) accounts for more than 80% of all lung cancer. Although people are now studying the early diagnosis and treatment of non-small cell lung cancer, the early diagnosis and treatment have been carried out very deeply, but it is exact, The molecular mechanism of development is still unclear, and further research is needed to make further breakthroughs in the early diagnosis and treatment of non-small cell lung cancer. Deleted in liver cancer 1 (DLC-1) is a tumor suppressor gene found in the study of liver cancer for the first time. It is located in the.DLC-1 base of the 21.3-22 region of chromosome eighth of human body. The expression and related mechanisms in many kinds of malignant tumors such as breast cancer, nasopharyngeal carcinoma, pancreatic cancer, large intestine cancer and gastric cancer are one of the hotspots of current research. The current research shows that the expression of DLC-1 in most malignant tumors is low level or even expression, and the expression of DLC-1 can significantly inhibit the proliferation of tumor cells. To promote the apoptosis of tumor cells, it is obvious that the study of the expression mechanism of DLC-1 in malignant tumor is of great significance for the discovery of new tumor treatment targets and new tumor treatment methods. At present, there are relatively few studies on DLC-1 in lung cancer at home and abroad. The research on the survival rate of DLC-1 and patients is still a blank, and DLC-1 is in the lung cancer. The role of the mechanism is not clear. By studying the difference in expression of DLC-1 in non-small cell lung cancer tissues and adjacent tissues, the relationship between DLC-1 and the sex of the patients, the history of smoking and tumor pathology, the degree of differentiation, the metastasis of lymph nodes, the correlation of TNM staging, and the relationship between the DLC-1 and the development of lung cancer, and the construction of pcDN A3.1-DLC-1 plasmid, overexpressing this plasmid in non small cell lung cancer cell lines, explores the effect of DLC-1 on the growth and invasion of non-small cell lung cancer cells. It is reported that DLC-1 may regulate the occurrence and growth of tumor through the RhoA/ROCK signaling pathway. Therefore, this study also explored the effect of DLC-1 on ROCK1, and explored the DLC-1 and RhoA/ROCK signals. The mechanism of the role of DLC-1 in lung cancer is studied to provide new ideas for the discovery of new tumor treatment targets and new tumor treatment methods. This study is divided into three parts: the first part is the detection of DLC-1, ROCK1 protein expression level in non small cell lung cancer and its clinical significance to explore non small cells The protein expression level of DLC-1 gene deletion gene DLC-1 and Rho related curl spironoforming protein (Rho-kinase 1, ROCK1) in lung cancer tissue was used to analyze the correlation between the two proteins and various clinical indexes. Methods the surgical treatment in Department of thoracic surgery of Affiliated Hospital of Binzhou Medical College from January 2011 to June 2012 was retrospectively analyzed. The clinical data of 68 non small cell lung cancer patients, all patients were confirmed by pathological examination, and were not treated with chemotherapy and radiotherapy before operation. 68 pairs of non small cell lung cancer tissues and paraffin paraffin paraffin embedded.2. were collected by Envision immunohistochemical technique to detect the DLC-1 gene and ROCK1 gene in the cancer tissue and to the other .3. analysis of the expression level in the para cancerous tissue, DLC-1, the correlation of ROCK1 with various clinical indicators, and the correlation of the expression level of two proteins. All patients with.4. were followed up to the.5. survival period in March 2015 from the date of operation to death or in the end of 3 month follow-up in 2015. According to the follow-up results, the DLC-1 different expression groups were compared. Results the difference of survival time between the two groups. Results 1. immunohistochemical results showed that DLC-1 protein showed low level expression or even expression deletion in non-small cell lung cancer, and the positive expression rate was 38.24% (26/68), obviously lower than that of paracancerous tissue 75% (51/68). The difference in expression of the two was of general significance (X2=14.256, P0.01): ROCKI protein in non small cells The positive expression rate in lung cancer was 57.35% (39/68), the expression was absent in the para cancer tissues, and the difference in the expression of the two was statistically significant (X2=45.378, P0.01). 2. in non-small cell lung cancer, the expression of DLC-1, ROCKI protein was related to the sex, the history of smoking, and the degree of differentiation, lymph node metastasis and TNM. The positive expression rate of ROCK1 in the positive expression group of non small cell lung cancer was 34.62% (9/26), and the positive expression rate of ROCK1 in the negative expression group of DLC-1 was 71.43% (30/42). The correlation analysis suggested that the expression of DLC-1 and ROCK1 was negatively correlated (r=-2.359, P=0.04), and the 3 year survival of the high expression group of 3.DLC-1 protein. The rate (88%) was higher than that of low expression group (57.2%), and the difference was statistically significant (P=0.031). Conclusion DLC-1 protein showed low expression level or even expression deletion in non-small cell lung cancer, which showed a significant negative correlation with ROCK1 protein, which indicated that the mechanism of DLC-1 play a role in non-small cell lung cancer may be through the regulation of ROCK1 signaling pathway.D. LC-1 protein can be used as one of the potential indicators for detecting the prognosis of non small cell lung cancer patients. Expression and correlation of second part DLC-1, ROCK1 in non-small cell lung cancer tissues and cell lines and their correlation study to study the expression of protein and mRNA levels of DLC-1 and ROCK1 in non small cell lung cancer fresh tissues and cells and the expression of the level of mRNA and the level of ROCK1 Method 1. a total of 23 fresh non-small cell lung cancer tissues and 23 para cancerous tissues were collected. The expression of DLC-1 and ROCK1 in fresh lung cancer tissues was detected by reverse transcriptase polymerase chain reaction (Real time RT-PCR). The expression of DLC-1 and ROCK1 in fresh lung cancer tissues was detected by reverse transcriptase polymerase chain reaction (Real time) and Calu6.2.. The expression of DLC-1 and ROCK1 gene mRNA level and protein level in lung cancer cell lines was detected by tern Blot. Results 1. all 23 cases of lung cancer and para cancerous tissue were collected. The results suggested that the expression of DLC-1 mRNA in lung cancer tissues was significantly lower than that of para cancerous tissue, ROCK1 in lung cancer tissues was higher than that of para cancer tissue, and negative correlation existed between the two. .2. was positive in the mRNA level and protein level of the DLC-1 gene in the H1299 cell line of lung cancer, but negative in A549 and Calu6 cell lines. The result of ROCK1 was contrary to that. Conclusion DLC-1 in the fresh tissues and cell lines of lung cancer showed low expression or loss of expression, and ROCK1 phase was opposite, further confirmed DLC-1 in non small cell lung cancer. The mechanism of the volatiles may be through the regulation of the ROCK1 signaling pathway. Third the construction of pcDNA3.1-DLC-1 recombinant vector and the effect of DLC-1 on the biological function of lung cancer cells aim to construct the expression vector pcDNA3.1-DLC-1 of the DLC-1 gene, and to express the DLC-1 protein in the lung cancer cell lines, and to explore the DLC-1 protein for the lung cancer cell strains. The effect of length, invasion and apoptosis on the expression of ROCK1, further explore the mechanism of DLC-1 protein in non-small cell lung cancer, and provide new clinical ideas and theoretical basis for the diagnosis and treatment of non-small cell lung cancer. Method construction of 1.DLC-1 expression vector: extracting H1299 total RNA of lung cancer cell line and synthesizing C through reverse transcriptase DNA, the whole sequence ORF of DLC-1 gene was amplified by RT-PCR, and the effect of pcDNA3.1.2.DCL-1 on the expression of ROCK1 was inserted into the eukaryotic expression vector pcDNA3.1.2.DCL-1: the expressed vector was extracted without endotoxin extraction, and the liposomes were transiently transfected to A549, Calu6 cells. After 48 hours, the cell RNA was extracted, and the reverse transcript cDNA was used to identify the RNA and cDNA quality. The expression of ROCK1 gene expression was identified. The effect of negative control.3.DLC-1 on invasive ability of non-small cell lung cancer was detected by plasmid empty pcDNA3.1 transfected A549 and Calu6 as negative control.3.DLC-1. The transfected A549 cells were transiently transfected by liposome without endotoxin extraction. The impact of tumor cells on the invasion ability of tumor cells was evaluated by Transwell chamber test. Effect of.4.DCL-1 on the growth ability of non-small cell lung cancer: extracting the constructed expression vector without endotoxin extraction, transfection of liposome into A549, Calu6 cells, diluted to 200cells/10ml, inoculated into 6 orifice plates, completing the clone growth and observing the cell growth with crystal violet staining, A549, Calu transfected with plasmid empty pcDNA3.1, Calu 6 the effect of negative control.5.DCL-1 on the cell cycle and apoptosis of non-small cell lung cancer: extracting the constructed expression vector with non endotoxin extraction, transiently transfecting A549 cells into liposomes, DAPI staining, and FACS detection of its effect on cell cycle and apoptosis. A549 as negative control by plasmid empty pcDNA3.1 transfection. Results 1. application gene The cloning technology successfully constructed the DLC-1 gene eukaryotic expression vector pcDNA3.1-DLC-1.2. lung cancer cell line A549, and the overexpression of DLC-1 in Calu6 had a negative regulation on the ROCK1 transcriptional level. After transfection of pcDNA3.1-DLC-1, DLC-1 had a higher overexpression on mRNA level. The results showed significant difference, P0.05. was the ROCK1 mRNA level. It was found that after overexpression of DLC-1, the mRNA level of ROCK1 was significantly inhibited. The mRNA level of ROCK1 after overexpression of DLC-1 in Calu6 was 33%, 47%, respectively, and the results were significantly different. The P0.05.3. lung cancer cell line, A549, Calu6 cells expressed the DLC-1 in the experiment, and the experiment group and the experiment group in the invasion experiment in vitro. The results were 26.8 + 1.3,67.8 + 5.2 in the control group, and the results showed significant difference. The P0.05. clone formation experiment suggested that the clone formation ability of the Calu6 cells decreased significantly, the clone formation rate of the A549 cell line was 38.9 + 2.6%, and the clonogenic rate of the experimental group was 21.8 + 5.2%, and the results had significant difference. The clone formation rate of the control group of P0.05 Calu6 cell line was 35.8 + 1.2%, the clone formation rate of the experimental group was 19.6 + 6.9%, and the results showed significant difference. The P0.05. flow results showed that after the expression of DLC-1, the cell cycle could be inhibited, the number of cells in S stage and G2 phase, the number of G1 phase cells, and the apoptosis of the cancer cells were increased. Conclusion the expression of DLC-1 in lung cancer cells has a significant negative correlation with ROCK1, and DLC-1 can significantly inhibit the proliferation and invasion of lung cancer cells, also significantly inhibit the cell cycle of lung cancer cells, and promote the apoptosis of lung cancer cells. Therefore, DLC-1 can regulate the occurrence and development of lung cancer through the action of ROCK1.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
本文编号：2146330
[Abstract]:Lung cancer is one of the malignant tumors with high morbidity and mortality in our country and in the world, but the non small cell lung cancer NSCLC (non small cell lung cancer, NSCLC) accounts for more than 80% of all lung cancer. Although people are now studying the early diagnosis and treatment of non-small cell lung cancer, the early diagnosis and treatment have been carried out very deeply, but it is exact, The molecular mechanism of development is still unclear, and further research is needed to make further breakthroughs in the early diagnosis and treatment of non-small cell lung cancer. Deleted in liver cancer 1 (DLC-1) is a tumor suppressor gene found in the study of liver cancer for the first time. It is located in the.DLC-1 base of the 21.3-22 region of chromosome eighth of human body. The expression and related mechanisms in many kinds of malignant tumors such as breast cancer, nasopharyngeal carcinoma, pancreatic cancer, large intestine cancer and gastric cancer are one of the hotspots of current research. The current research shows that the expression of DLC-1 in most malignant tumors is low level or even expression, and the expression of DLC-1 can significantly inhibit the proliferation of tumor cells. To promote the apoptosis of tumor cells, it is obvious that the study of the expression mechanism of DLC-1 in malignant tumor is of great significance for the discovery of new tumor treatment targets and new tumor treatment methods. At present, there are relatively few studies on DLC-1 in lung cancer at home and abroad. The research on the survival rate of DLC-1 and patients is still a blank, and DLC-1 is in the lung cancer. The role of the mechanism is not clear. By studying the difference in expression of DLC-1 in non-small cell lung cancer tissues and adjacent tissues, the relationship between DLC-1 and the sex of the patients, the history of smoking and tumor pathology, the degree of differentiation, the metastasis of lymph nodes, the correlation of TNM staging, and the relationship between the DLC-1 and the development of lung cancer, and the construction of pcDN A3.1-DLC-1 plasmid, overexpressing this plasmid in non small cell lung cancer cell lines, explores the effect of DLC-1 on the growth and invasion of non-small cell lung cancer cells. It is reported that DLC-1 may regulate the occurrence and growth of tumor through the RhoA/ROCK signaling pathway. Therefore, this study also explored the effect of DLC-1 on ROCK1, and explored the DLC-1 and RhoA/ROCK signals. The mechanism of the role of DLC-1 in lung cancer is studied to provide new ideas for the discovery of new tumor treatment targets and new tumor treatment methods. This study is divided into three parts: the first part is the detection of DLC-1, ROCK1 protein expression level in non small cell lung cancer and its clinical significance to explore non small cells The protein expression level of DLC-1 gene deletion gene DLC-1 and Rho related curl spironoforming protein (Rho-kinase 1, ROCK1) in lung cancer tissue was used to analyze the correlation between the two proteins and various clinical indexes. Methods the surgical treatment in Department of thoracic surgery of Affiliated Hospital of Binzhou Medical College from January 2011 to June 2012 was retrospectively analyzed. The clinical data of 68 non small cell lung cancer patients, all patients were confirmed by pathological examination, and were not treated with chemotherapy and radiotherapy before operation. 68 pairs of non small cell lung cancer tissues and paraffin paraffin paraffin embedded.2. were collected by Envision immunohistochemical technique to detect the DLC-1 gene and ROCK1 gene in the cancer tissue and to the other .3. analysis of the expression level in the para cancerous tissue, DLC-1, the correlation of ROCK1 with various clinical indicators, and the correlation of the expression level of two proteins. All patients with.4. were followed up to the.5. survival period in March 2015 from the date of operation to death or in the end of 3 month follow-up in 2015. According to the follow-up results, the DLC-1 different expression groups were compared. Results the difference of survival time between the two groups. Results 1. immunohistochemical results showed that DLC-1 protein showed low level expression or even expression deletion in non-small cell lung cancer, and the positive expression rate was 38.24% (26/68), obviously lower than that of paracancerous tissue 75% (51/68). The difference in expression of the two was of general significance (X2=14.256, P0.01): ROCKI protein in non small cells The positive expression rate in lung cancer was 57.35% (39/68), the expression was absent in the para cancer tissues, and the difference in the expression of the two was statistically significant (X2=45.378, P0.01). 2. in non-small cell lung cancer, the expression of DLC-1, ROCKI protein was related to the sex, the history of smoking, and the degree of differentiation, lymph node metastasis and TNM. The positive expression rate of ROCK1 in the positive expression group of non small cell lung cancer was 34.62% (9/26), and the positive expression rate of ROCK1 in the negative expression group of DLC-1 was 71.43% (30/42). The correlation analysis suggested that the expression of DLC-1 and ROCK1 was negatively correlated (r=-2.359, P=0.04), and the 3 year survival of the high expression group of 3.DLC-1 protein. The rate (88%) was higher than that of low expression group (57.2%), and the difference was statistically significant (P=0.031). Conclusion DLC-1 protein showed low expression level or even expression deletion in non-small cell lung cancer, which showed a significant negative correlation with ROCK1 protein, which indicated that the mechanism of DLC-1 play a role in non-small cell lung cancer may be through the regulation of ROCK1 signaling pathway.D. LC-1 protein can be used as one of the potential indicators for detecting the prognosis of non small cell lung cancer patients. Expression and correlation of second part DLC-1, ROCK1 in non-small cell lung cancer tissues and cell lines and their correlation study to study the expression of protein and mRNA levels of DLC-1 and ROCK1 in non small cell lung cancer fresh tissues and cells and the expression of the level of mRNA and the level of ROCK1 Method 1. a total of 23 fresh non-small cell lung cancer tissues and 23 para cancerous tissues were collected. The expression of DLC-1 and ROCK1 in fresh lung cancer tissues was detected by reverse transcriptase polymerase chain reaction (Real time RT-PCR). The expression of DLC-1 and ROCK1 in fresh lung cancer tissues was detected by reverse transcriptase polymerase chain reaction (Real time) and Calu6.2.. The expression of DLC-1 and ROCK1 gene mRNA level and protein level in lung cancer cell lines was detected by tern Blot. Results 1. all 23 cases of lung cancer and para cancerous tissue were collected. The results suggested that the expression of DLC-1 mRNA in lung cancer tissues was significantly lower than that of para cancerous tissue, ROCK1 in lung cancer tissues was higher than that of para cancer tissue, and negative correlation existed between the two. .2. was positive in the mRNA level and protein level of the DLC-1 gene in the H1299 cell line of lung cancer, but negative in A549 and Calu6 cell lines. The result of ROCK1 was contrary to that. Conclusion DLC-1 in the fresh tissues and cell lines of lung cancer showed low expression or loss of expression, and ROCK1 phase was opposite, further confirmed DLC-1 in non small cell lung cancer. The mechanism of the volatiles may be through the regulation of the ROCK1 signaling pathway. Third the construction of pcDNA3.1-DLC-1 recombinant vector and the effect of DLC-1 on the biological function of lung cancer cells aim to construct the expression vector pcDNA3.1-DLC-1 of the DLC-1 gene, and to express the DLC-1 protein in the lung cancer cell lines, and to explore the DLC-1 protein for the lung cancer cell strains. The effect of length, invasion and apoptosis on the expression of ROCK1, further explore the mechanism of DLC-1 protein in non-small cell lung cancer, and provide new clinical ideas and theoretical basis for the diagnosis and treatment of non-small cell lung cancer. Method construction of 1.DLC-1 expression vector: extracting H1299 total RNA of lung cancer cell line and synthesizing C through reverse transcriptase DNA, the whole sequence ORF of DLC-1 gene was amplified by RT-PCR, and the effect of pcDNA3.1.2.DCL-1 on the expression of ROCK1 was inserted into the eukaryotic expression vector pcDNA3.1.2.DCL-1: the expressed vector was extracted without endotoxin extraction, and the liposomes were transiently transfected to A549, Calu6 cells. After 48 hours, the cell RNA was extracted, and the reverse transcript cDNA was used to identify the RNA and cDNA quality. The expression of ROCK1 gene expression was identified. The effect of negative control.3.DLC-1 on invasive ability of non-small cell lung cancer was detected by plasmid empty pcDNA3.1 transfected A549 and Calu6 as negative control.3.DLC-1. The transfected A549 cells were transiently transfected by liposome without endotoxin extraction. The impact of tumor cells on the invasion ability of tumor cells was evaluated by Transwell chamber test. Effect of.4.DCL-1 on the growth ability of non-small cell lung cancer: extracting the constructed expression vector without endotoxin extraction, transfection of liposome into A549, Calu6 cells, diluted to 200cells/10ml, inoculated into 6 orifice plates, completing the clone growth and observing the cell growth with crystal violet staining, A549, Calu transfected with plasmid empty pcDNA3.1, Calu 6 the effect of negative control.5.DCL-1 on the cell cycle and apoptosis of non-small cell lung cancer: extracting the constructed expression vector with non endotoxin extraction, transiently transfecting A549 cells into liposomes, DAPI staining, and FACS detection of its effect on cell cycle and apoptosis. A549 as negative control by plasmid empty pcDNA3.1 transfection. Results 1. application gene The cloning technology successfully constructed the DLC-1 gene eukaryotic expression vector pcDNA3.1-DLC-1.2. lung cancer cell line A549, and the overexpression of DLC-1 in Calu6 had a negative regulation on the ROCK1 transcriptional level. After transfection of pcDNA3.1-DLC-1, DLC-1 had a higher overexpression on mRNA level. The results showed significant difference, P0.05. was the ROCK1 mRNA level. It was found that after overexpression of DLC-1, the mRNA level of ROCK1 was significantly inhibited. The mRNA level of ROCK1 after overexpression of DLC-1 in Calu6 was 33%, 47%, respectively, and the results were significantly different. The P0.05.3. lung cancer cell line, A549, Calu6 cells expressed the DLC-1 in the experiment, and the experiment group and the experiment group in the invasion experiment in vitro. The results were 26.8 + 1.3,67.8 + 5.2 in the control group, and the results showed significant difference. The P0.05. clone formation experiment suggested that the clone formation ability of the Calu6 cells decreased significantly, the clone formation rate of the A549 cell line was 38.9 + 2.6%, and the clonogenic rate of the experimental group was 21.8 + 5.2%, and the results had significant difference. The clone formation rate of the control group of P0.05 Calu6 cell line was 35.8 + 1.2%, the clone formation rate of the experimental group was 19.6 + 6.9%, and the results showed significant difference. The P0.05. flow results showed that after the expression of DLC-1, the cell cycle could be inhibited, the number of cells in S stage and G2 phase, the number of G1 phase cells, and the apoptosis of the cancer cells were increased. Conclusion the expression of DLC-1 in lung cancer cells has a significant negative correlation with ROCK1, and DLC-1 can significantly inhibit the proliferation and invasion of lung cancer cells, also significantly inhibit the cell cycle of lung cancer cells, and promote the apoptosis of lung cancer cells. Therefore, DLC-1 can regulate the occurrence and development of lung cancer through the action of ROCK1.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2
【参考文献】
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