非小細胞肺癌DLC-1、ROCK1的表達及生物學作用機制的研究
發(fā)布時間：2018-07-26 14:37
【摘要】：肺癌是我國乃至全世界發(fā)病率及死亡率均較高的惡性腫瘤之一,而非小細胞肺癌NSCLC (non small cell lung cancer,NSCLC)占全部肺癌的80%以上。雖然人們目前對于非小細胞肺癌發(fā)病、早期診斷和治療已經(jīng)進行了非常深入的研究,但其確切發(fā)生、發(fā)展的分子機制尚不明確,有待于進一步研究以期在非小細胞肺癌早期診斷和治療上取得進一步的突破。肝癌缺失基因(deleted in liver cancer 1,DLC-1)是在肝癌研究中被首次發(fā)現(xiàn)的一種抑癌基因,它位于人體第8號染色體的21.3-22區(qū)域。DLC-1基因在乳腺癌、鼻咽癌、胰腺癌、大腸癌、胃癌等多種惡性腫瘤中的表達及相關機制是當今研究的熱點之一。當前研究表明,在大多數(shù)惡性腫瘤中DLC-1呈現(xiàn)出低水平表達甚至是表達缺失,恢復DLC-1的表達能夠顯著地抑制腫瘤細胞的增殖能力,促進腫瘤細胞的凋亡。可見,研究DLC-1在惡性腫瘤中的表達機制,對于發(fā)現(xiàn)新的腫瘤治療靶點和新的腫瘤治療方法具有十分重要的意義。目前國內外對于DLC-1在肺癌中的研究相對較少,DLC-1與患者的生存率方面的研究尚屬空白,DLC-1在肺癌中發(fā)揮作用的機制并不明確。本課題通過研究DLC-1在非小細胞肺癌組織和癌旁組織中的表達差異,分析DLC-1與患者性別、吸煙史及腫瘤病理類型、分化程度、淋巴結是否轉移、TNM分期的相關性,探討DLC-1與肺癌發(fā)生發(fā)展的關系；并構建了pcDNA3.1-DLC-1質粒,在非小細胞肺癌細胞株中過表達該質粒,探索DLC-1對非小細胞肺癌細胞生長、侵襲的影響；有報道表明,DLC-1可能通過RhoA/ROCK信號通路調控腫瘤的發(fā)生及生長,因此本研究也探索了DLC-1對于ROCK1的影響,探索DLC-1與RhoA/ROCK信號通路的關系。對于DLC-1在肺癌中發(fā)揮作用的機制進行研究,為發(fā)現(xiàn)新的腫瘤治療靶點和新的腫瘤治療方法提供新的思路。本研究分為以下三個部分：第一部分非小細胞肺癌中DLC-1、ROCK1蛋白表達水平的檢測及臨床意義目的探索非小細胞肺癌組織中肝癌缺失基因DLC-1基因和Rho相關卷曲螺旋形成蛋白(Rho-kinase 1,ROCK1)的蛋白表達水平,分析兩種蛋白與各項臨床指標的相關性。方法1.本研究回顧性分析了2011年1月至2012年6月在濱州醫(yī)學院附屬醫(yī)院胸外科進行外科手術治療的68例非小細胞肺癌病人的臨床資料,所有病人均經(jīng)病理檢查證實,且術前未行放化療等抗腫瘤治療。收集68對非小細胞肺癌的癌組織及對應的癌旁組織石蠟包埋塊。2.采用Envision免疫組織化學技術檢測DLC-1基因與ROCK1基因在癌組織及對應的癌旁組織中的表達水平。3.分析DLC-1、ROCK1與各項臨床指標的相關性,并研究兩種蛋白表達水平的相關性。4.所有患者的隨訪截止到2015年3月。5.生存期計算從手術日期至死亡或2015年3月末次隨訪結束。根據(jù)隨訪結果比較DLC-1不同表達組之間生存期的差異。結果1.免疫組化結果顯示：DLC-1蛋白在非小細胞肺癌組織中呈現(xiàn)低水平表達甚至是表達缺失,其陽性表達率為38.24%(26/68),明顯低于癌旁組織75%(51/68),二者的表達差異有統(tǒng)計學意義(X2=14.256, P0.01): ROCKI蛋白在非小細胞肺癌中的陽性表達率為57.35%(39/68),在癌旁組織呈表達缺失,二者的表達差異有統(tǒng)計學意義(X2=45.378,P0.01)；2.在非小細胞肺癌組織中,DLC-1、ROCKI蛋白的表達與性別、吸煙史有無及腫瘤的病理學類型無關,而與腫瘤分化程度、淋巴結轉移及TNM分期有關,差異有統(tǒng)計學意義；非小細胞肺癌DLC-1陽性表達組中ROCK1的陽性表達率為34.62%(9/26),DLC-1陰性表達組中ROCK1的陽性表達率為71.43%(30/42),相關分析提示,DLC-1與ROCK1的表達中呈負相關(r=-2.359,P=0.04)；3.DLC-1蛋白高表達組的3年生存率(88%)高于低表達組(57.2%),差異有統(tǒng)計學意義(P=0.031)。結論DLC-1蛋白在非小細胞肺癌組織中呈現(xiàn)低表達水平甚至是表達缺失,與ROCK1蛋白呈現(xiàn)明顯的負相關性,這表明DLC-1在非小細胞肺癌中發(fā)揮作用的機制可能是通過調控ROCK1信號通路。DLC-1蛋白可以作為檢測非小細胞肺癌患者預后水平的潛在指標之一。第二部分DLC-1、ROCK1在非小細胞肺癌組織及細胞株中的表達及其相關性研究目的研究非小細胞肺癌新鮮組織及細胞株中DLC-1和ROCK1的蛋白及mRNA水平的表達情況及二者的相關性。方法1.收集新鮮非小細胞肺癌組織及癌旁組織各23例,肺癌細胞株H1299、A549及Calu6。2.運用逆轉錄聚合酶鏈反應(Real time RT-PCR)檢測新鮮肺癌組織中DLC-1及ROCK1基因mRNA水平的表達情況。3.運用逆轉錄聚合酶鏈反應(Real time RT-PCR)及Western Blot檢測肺癌細胞株中的DLC-1及ROCK1基因mRNA水平及蛋白水平的表達情況。結果1.共收集肺癌及癌旁組織各23例,結果提示在肺癌組織中DLC-1 mRNA的表達量明顯低于癌旁組織,ROCK1則在肺癌組織中表達量高于癌旁組織,二者亦存在負相關的關系。2.在肺癌H1299細胞株中,DLC-1基因mRNA水平及蛋白水平表達呈陽性,而在A549、Calu6細胞株中則呈陰性；ROCK1的結果與之相反。結論DLC-1在肺癌新鮮組織及細胞株中呈低表達或表達缺失,ROCK1則相反,進一步證實DLC-1在非小細胞肺癌中發(fā)揮作用的機制可能是通過對于ROCK1信號通路的調控。第三部分pcDNA3.1-DLC-1重組載體的構建及DLC-1對肺癌細胞的生物學功能的影響目的構建DLC-1基因的表達載體pcDNA3.1-DLC-1,在肺癌細胞系中過表達DLC-1蛋白,探索DLC-1蛋白對于肺癌細胞株生長、侵襲及凋亡的影響,對ROCK1表達的影響,進一步探討DLC-1蛋白在非小細胞肺癌中發(fā)揮作用的機制,為非小細胞肺癌的診斷及治療提供新的臨床思路及理論依據(jù)。方法1.DLC-1表達載體的構建：提取肺癌細胞株H1299總RNA,通過逆轉錄酶合成cDNA;應用RT-PCR擴增DLC-1基因全序列ORF,插入真核表達載體pcDNA3.1.2.DCL-1對ROCK1表達的影響：用無內毒素抽提方法抽取已構建好的表達載體,脂質體瞬時轉染A549、Calu6細胞,48小時后抽取細胞RNA,逆轉錄cDNA,鑒定RNA和cDNA質量,設計ROCK1引物,RT-PCR鑒定ROCK1基因表達變化。以質�？蛰dpcDNA3.1轉染的A549、Calu6為陰性對照。3.DLC-1對非小細胞肺癌侵襲能力的影響：用無內毒素抽提方法抽取已構建好表達載體,脂質體瞬時轉染A549細胞,利用transwell小室實驗評估其對腫瘤細胞侵襲能力的影響。4.DCL-1對非小細胞肺癌生長能力的影響：用無內毒素抽提方法抽取已構建好表達載體,脂質體瞬時轉染A549、Calu6細胞,稀釋至200cells/10ml,接種入6孔板,待克隆生長完成,以結晶紫染色,觀察細胞生長情況。以質粒空載pcDNA3.1轉染的A549、Calu6為陰性對照。5.DCL-1對非小細胞肺癌細胞周期及凋亡的影響：用無內毒素抽提方法抽取已構建好表達載體,脂質體瞬時轉染A549細胞,DAPI染色,FACS檢測其對細胞周期和凋亡的影響。以質粒空載pcDNA3.1轉染的A549為陰性對照。結果1.應用基因克隆技術成功構建DLC-1基因真核表達載體pcDNA3.1-DLC-1.2.肺癌細胞株A549、Calu6中過表達DLC-1對ROCK1轉錄水平有著負性調控的作用。轉染了pcDNA3.1-DLC-1后DLC-1在mRNA水平上有較高的過表達效果,結果具有顯著性差異,P0.05。對其中的ROCK1的mRNA水平進行檢測可知,過表達DLC-1后,ROCK1的mRNA水平受到了顯著的抑制,A549、Calu6中過表達DLC-1后的ROCK1的mRNA水平下降分別為對照組的33%、47%,結果均具有顯著性差異,P0.05。3.肺癌細胞株A549、Calu6細胞中過表達的DLC-1后,在體外侵襲實驗中對實驗組和對照組的穿膜細胞進行統(tǒng)計分別為26.8±1.3、67.8±5.2,結果具有顯著性差異,P0.05�？寺⌒纬蓪嶒炋崾続549、Calu6細胞的克隆形成能力顯著下降；A549細胞株的對照組的克隆形成率為38.9±2.6%,實驗組的克隆形成率為21.8±5.2%,結果具有顯著性差異,P0.05; Calu6細胞株的對照組的克隆形成率為35.8±1.2%,實驗組的克隆形成率為19.6±6.9%,結果具有顯著性差異,P0.05。流式結果顯示過表達DLC-1后,可以顯著抑制細胞周期,降低S期細胞和G2期細胞數(shù),提高G1期細胞數(shù),并可促進癌細胞的凋亡。結論在肺癌細胞中DLC-1的表達與ROCK1呈明顯的負相關,且DLC-1能夠顯著地抑制肺癌細胞的增殖和侵襲能力,對肺癌細胞的細胞周期也具有明顯的抑制作用,并促進了肺癌細胞的凋亡,因此,DLC-1可能是通過作用ROCK1調控肺癌的發(fā)生和發(fā)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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2
本文編號：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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2
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