TRIM29蛋白在非小细胞肺癌增殖、侵袭及顺铂化疗过程中的作用

发布时间：2018-05-10 16:00

本文选题：肺癌 + RNA干扰　；参考：《首都医科大学》2015年博士论文

【摘要】：目的：肺癌是当今世界上最常见的恶性肿瘤之一。肺癌的临床治疗包括外科手术、化学治疗、放射治疗及分子靶向治疗等多学科综合治疗。对肺癌发生发展分子机制的研究能为肺癌的治疗提供新的靶点。以往的研究表明，TRIM29在多种肿瘤组织中表达异常增高，但对其具体作用的研究仍较少。本课题旨在研究TRIM29在非小细胞肺癌增殖、侵袭及顺铂化疗过程中的作用，为肺癌的治疗提供新的靶点。方法：(1)采用免疫组化的方法检测TRIM29在非小细胞肺癌组织及癌旁正常肺组织中的表达，并分析TRIM29在两者之间的差异，及其与临床病理因素之间的关系。(2)将针对TRIM29的siRNA导入NCI-H520细胞；用Real time PCR和Western blotting法检测TRIM29基因及蛋白的表达情况；MTT法和Transwell小室法检测细胞的增殖和侵袭能力。(3)利用siRNA干扰NCI-H520细胞中TRIM29的表达，Western blotting法检测促凋亡因子Bax、凋亡抑制因子Bcl-2的变化情况；流式细胞术检测细胞凋亡情况。结果：(1) TRIM29在癌旁正常肺组织中呈阴性表达，而在非小细胞肺癌组织中的阳性率为63%(63/100)。TRIM29的高表达在不同年龄及性别之间无统计学差异(P＞0.05)，而与组织类型、TNM分期以及区域淋巴结转移密切相关，差异具有统计学意义(P＜0.05)。(2)将特异性siRNA(siRNA1、siRNA2、siRNA3)及阴性对照siRNA(siRNANC)转入NCI-H520细胞中，RT-PCR和Western blotting分别检测siRNA干扰TRIM29后在基因水平和蛋白水平的表达变化。结果显示：与空白对照组、NC组相比，siRNA处理组NCI-H520细胞的TRIM29的mRNA及蛋白表达量均明显下降。并且发现TRIM29siRNA3干扰的特异性及效率更高，我们选择siRNA3作为TRIM29siRNA进行后续的实验。我们采用MTT法检测细胞在五天内的增殖情况，并绘制生长曲线，结果显示：与空白组及对照组比较，TRIM29siRNA转染组NCI-H520细胞生长明显减慢。采用Transwell小室法检测细胞的侵袭能力，结果显示：与空白组及对照组比较，TRIM29siRNA转染组NCI-H520细胞侵袭能力明显减弱。(3)我们使用siRNA干扰TRIM29的表达后，用Annexin-V/PI双凋亡法检测细胞的凋亡率，之后再使用浓度为5mg/L的顺铂刺激NCI-H520细胞24小时，再次用Annexin-V/PI双凋亡法检测细胞的凋亡率。结果显示，siRNA干扰TRIM29表达可诱导NCI-H520细胞出现凋亡，加入顺铂后，细胞凋亡明显增加，NCI-H520细胞受顺铂作用后的凋亡率为0.22±0.026，使用阴性对照siRNA后NCI-H520细胞的凋亡率为0.20±0.015，，二者没有显著差异；而使用siRNA干扰TRIM29后，加入顺铂，NCI-H520细胞凋亡率为0.32±0.005，与空白组及对照组相比存在显著差异。我们运用Westernblotting技术观察不同处理组细胞促凋亡因子Bax、凋亡抑制因子Bcl-2的变化情况，进一步研究siRNA干扰TRIM29表达促进NCI-H520细胞凋亡的可能机制，结果显示：TRIM29表达下降后，Bax的表达量上调，Bcl-2表达量下调。结论：本实验系统的研究了TRIM29在非小细胞肺癌中的表达及其在肺鳞癌NCI-H520细胞增殖、侵袭和顺铂化疗过程中的作用。研究表明：TRIM29在非小细胞肺癌组织中的过表达与非小细胞肺癌的组织类型、临床分期及淋巴结转移有关；siRNA干扰能特异、高效地抑制TRIM29的表达，在NCI-H520细胞中，抑制TRIM29的表达，能降低癌细胞的增殖及侵袭能力；siRNA干扰TRIM29的表达，可以使促凋亡因子Bax的表达量上调，凋亡抑制因子Bcl-2的表达量下调，并提高NCI-H520细胞对顺铂化疗的敏感性。TRIM29可能成为肺癌治疗的新靶点。
[Abstract]:Objective: lung cancer is one of the most common malignant tumors in the world. The clinical treatment of lung cancer includes surgery, chemotherapy, radiation therapy and molecular targeting therapy. The study of the molecular mechanism of the development of lung cancer can provide new targets for the treatment of lung cancer. Previous studies have shown that TRIM29 is in a variety of swollen. There is an abnormal increase in the expression of the tumor, but the specific role of the tumor is still less. The purpose of this study is to study the role of TRIM29 in the proliferation, invasion and cisplatin chemotherapy of non-small cell lung cancer, and provide new targets for the treatment of lung cancer.
Methods: (1) the expression of TRIM29 in non-small cell lung cancer tissues and adjacent normal lung tissues was detected by immunohistochemical method, and the difference between TRIM29 and clinicopathological factors was analyzed. (2) NCI-H520 fine cell was introduced to siRNA of TRIM29; TRIM29 Real time PCR and Western blotting method were used to detect TRIM29. The expression of gene and protein; MTT method and Transwell chamber method to detect cell proliferation and invasion ability. (3) using siRNA to interfere with the expression of TRIM29 in NCI-H520 cells, Western blotting method to detect the change of apoptotic factor Bax, the apoptosis inhibitory factor Bcl-2, and flow cytometry to detect the cell apoptosis.
Results: (1) TRIM29 was negative in normal lung tissue adjacent to cancer, but the positive rate of 63% (63/100).TRIM29 in non-small cell lung cancer was not statistically significant (P > 0.05) in different ages and sexes (P > 0.05), and was closely related to tissue type, TNM staging and regional lymph node metastasis (P 0.05). (2) the specific siRNA (siRNA1, siRNA2, siRNA3) and negative control siRNA (siRNANC) were transferred into NCI-H520 cells. RT-PCR and Western blotting detected the changes in the expression of siRNA interference TRIM29 at the gene level and protein level respectively. The protein expression was significantly decreased and the specificity and efficiency of TRIM29siRNA3 interference were higher. We selected siRNA3 as TRIM29siRNA for subsequent experiments. We used MTT to detect the proliferation of cells within five days and draw the growth curve. The results showed that TRIM29siRNA transfected group NCI-H52 was compared with the empty white group and the control group. The growth of 0 cells was significantly slowed down. The invasion ability of cells was detected by Transwell chamber method. The results showed that the invasion ability of NCI-H520 cells in TRIM29siRNA transfected group was significantly decreased compared with the blank group and the control group. (3) after the expression of TRIM29 was interfered with siRNA, the apoptosis rate of cells was detected by the double apoptosis method of Annexin-V/PI, then the concentration of the cells was detected by the double apoptosis method. Cisplatin, which was 5mg/L, stimulated NCI-H520 cells for 24 hours. The apoptosis rate of cells was detected by Annexin-V/PI double apoptosis. The results showed that siRNA interference with TRIM29 expression induced apoptosis of NCI-H520 cells. After adding cisplatin, apoptosis increased obviously. The apoptosis rate of NCI-H520 cells was 0.22 + 0.026 after cisplatin, and negative control Si was used. The apoptosis rate of NCI-H520 cells after RNA was 0.20 + 0.015, and there was no significant difference between two and two. After adding cisplatin to TRIM29, the apoptosis rate of NCI-H520 cells was 0.32 + 0.005, and there was a significant difference compared with the blank group and the control group. We used Westernblotting technique to observe the cell apoptosis factor Bax and the inhibitory cause of apoptosis in the dissimilar treatment group. The possible mechanism of siRNA interference with TRIM29 expression to promote apoptosis of NCI-H520 cells was further studied by the change of subBcl-2. The results showed that after the decrease of TRIM29 expression, the expression of Bax was up-regulated and the expression of Bcl-2 was down regulated.
Conclusion: the expression of TRIM29 in non-small cell lung cancer and its role in NCI-H520 cell proliferation, invasion and cisplatin chemotherapy in lung squamous cell carcinoma are systematically studied. The study shows that the overexpression of TRIM29 in non-small cell lung cancer is related to the histological type, clinical stage and lymph node metastasis of non-small cell lung cancer; siRNA Interference can inhibit the expression of TRIM29 highly efficiently and inhibit the expression of TRIM29 in NCI-H520 cells, which can reduce the proliferation and invasion ability of cancer cells. SiRNA interference with TRIM29 expression can increase the expression of apoptotic factor Bax, decrease the expression of apoptosis inhibitory factor Bcl-2, and improve the sensitivity of NCI-H520 cells to cisplatin chemotherapy. Sex.TRIM29 may be a new target for the treatment of lung cancer.

【学位授予单位】：首都医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R734.2

【共引文献】