本文選題：肺癌 + RNA干擾��； 參考：《首都醫(yī)科大學(xué)》2015年博士論文

【摘要】：目的：肺癌是當(dāng)今世界上最常見的惡性腫瘤之一。肺癌的臨床治療包括外科手術(shù)、化學(xué)治療、放射治療及分子靶向治療等多學(xué)科綜合治療。對肺癌發(fā)生發(fā)展分子機(jī)制的研究能為肺癌的治療提供新的靶點(diǎn)。以往的研究表明，TRIM29在多種腫瘤組織中表達(dá)異常增高，但對其具體作用的研究仍較少。本課題旨在研究TRIM29在非小細(xì)胞肺癌增殖、侵襲及順鉑化療過程中的作用，為肺癌的治療提供新的靶點(diǎn)。 方法：(1)采用免疫組化的方法檢測TRIM29在非小細(xì)胞肺癌組織及癌旁正常肺組織中的表達(dá)，并分析TRIM29在兩者之間的差異，及其與臨床病理因素之間的關(guān)系。(2)將針對TRIM29的siRNA導(dǎo)入NCI-H520細(xì)胞；用Real time PCR和Western blotting法檢測TRIM29基因及蛋白的表達(dá)情況；MTT法和Transwell小室法檢測細(xì)胞的增殖和侵襲能力。(3)利用siRNA干擾NCI-H520細(xì)胞中TRIM29的表達(dá)，Western blotting法檢測促凋亡因子Bax、凋亡抑制因子Bcl-2的變化情況；流式細(xì)胞術(shù)檢測細(xì)胞凋亡情況。 結(jié)果：(1) TRIM29在癌旁正常肺組織中呈陰性表達(dá)，而在非小細(xì)胞肺癌組織中的陽性率為63%(63/100)。TRIM29的高表達(dá)在不同年齡及性別之間無統(tǒng)計學(xué)差異(P＞0.05)，而與組織類型、TNM分期以及區(qū)域淋巴結(jié)轉(zhuǎn)移密切相關(guān)，差異具有統(tǒng)計學(xué)意義(P＜0.05)。(2)將特異性siRNA(siRNA1、siRNA2、siRNA3)及陰性對照siRNA(siRNANC)轉(zhuǎn)入NCI-H520細(xì)胞中，RT-PCR和Western blotting分別檢測siRNA干擾TRIM29后在基因水平和蛋白水平的表達(dá)變化。結(jié)果顯示：與空白對照組、NC組相比，siRNA處理組NCI-H520細(xì)胞的TRIM29的mRNA及蛋白表達(dá)量均明顯下降。并且發(fā)現(xiàn)TRIM29siRNA3干擾的特異性及效率更高，我們選擇siRNA3作為TRIM29siRNA進(jìn)行后續(xù)的實驗。我們采用MTT法檢測細(xì)胞在五天內(nèi)的增殖情況，并繪制生長曲線，結(jié)果顯示：與空白組及對照組比較，TRIM29siRNA轉(zhuǎn)染組NCI-H520細(xì)胞生長明顯減慢。采用Transwell小室法檢測細(xì)胞的侵襲能力，結(jié)果顯示：與空白組及對照組比較，TRIM29siRNA轉(zhuǎn)染組NCI-H520細(xì)胞侵襲能力明顯減弱。(3)我們使用siRNA干擾TRIM29的表達(dá)后，用Annexin-V/PI雙凋亡法檢測細(xì)胞的凋亡率，之后再使用濃度為5mg/L的順鉑刺激NCI-H520細(xì)胞24小時，再次用Annexin-V/PI雙凋亡法檢測細(xì)胞的凋亡率。結(jié)果顯示，siRNA干擾TRIM29表達(dá)可誘導(dǎo)NCI-H520細(xì)胞出現(xiàn)凋亡，加入順鉑后，細(xì)胞凋亡明顯增加，NCI-H520細(xì)胞受順鉑作用后的凋亡率為0.22±0.026，使用陰性對照siRNA后NCI-H520細(xì)胞的凋亡率為0.20±0.015，，二者沒有顯著差異；而使用siRNA干擾TRIM29后，加入順鉑，NCI-H520細(xì)胞凋亡率為0.32±0.005，與空白組及對照組相比存在顯著差異。我們運(yùn)用Westernblotting技術(shù)觀察不同處理組細(xì)胞促凋亡因子Bax、凋亡抑制因子Bcl-2的變化情況，進(jìn)一步研究siRNA干擾TRIM29表達(dá)促進(jìn)NCI-H520細(xì)胞凋亡的可能機(jī)制，結(jié)果顯示：TRIM29表達(dá)下降后，Bax的表達(dá)量上調(diào)，Bcl-2表達(dá)量下調(diào)。 結(jié)論：本實驗系統(tǒng)的研究了TRIM29在非小細(xì)胞肺癌中的表達(dá)及其在肺鱗癌NCI-H520細(xì)胞增殖、侵襲和順鉑化療過程中的作用。研究表明：TRIM29在非小細(xì)胞肺癌組織中的過表達(dá)與非小細(xì)胞肺癌的組織類型、臨床分期及淋巴結(jié)轉(zhuǎn)移有關(guān)；siRNA干擾能特異、高效地抑制TRIM29的表達(dá)，在NCI-H520細(xì)胞中，抑制TRIM29的表達(dá)，能降低癌細(xì)胞的增殖及侵襲能力；siRNA干擾TRIM29的表達(dá)，可以使促凋亡因子Bax的表達(dá)量上調(diào)，凋亡抑制因子Bcl-2的表達(dá)量下調(diào)，并提高NCI-H520細(xì)胞對順鉑化療的敏感性。TRIM29可能成為肺癌治療的新靶點(diǎn)。
[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.

【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R734.2

【共引文獻(xiàn)】

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