肺癌組織與細(xì)胞中Cullin7的表達(dá)及臨床意義的研究
發(fā)布時(shí)間：2018-07-17 19:28
【摘要】：肺癌已成為全世界癌癥死亡的首要原因,每年超過(guò)一百萬(wàn)的人從這種疾病死亡,肺癌死亡率40年間上升近10倍。全世界肺癌每年新增病例約180萬(wàn),其中三分之二發(fā)現(xiàn)時(shí)已處于晚期,肺癌確診后患者5年生存率只有約10%。肺癌最主要的治療手段是以手術(shù)、放療、化療和分子靶向治療為主的綜合治療。其中65-70%發(fā)現(xiàn)已為晚期失去手術(shù)機(jī)會(huì),對(duì)于此類(lèi)患者如果可以采用化療或放療,5年生存率僅為7-17%,如果患者不能耐受或失去放化療機(jī)會(huì),5年生存率僅為2%。因此,探尋更為有效的肺癌治療策略尤其是肺癌早期診斷早期治療具有重要意義。對(duì)了解新的發(fā)病機(jī)制和確定新的腫瘤標(biāo)志物方面將有很多空間。最新研究提出,如能及早發(fā)現(xiàn)肺癌,把它扼殺在萌芽狀態(tài)阻止其沿多條進(jìn)化路徑發(fā)展,情況可能會(huì)變得樂(lè)觀,將會(huì)有更多肺癌患者存活。人體內(nèi)Cullin7(一種多功能泛素連接酶亞基)的高表達(dá)是否就是肺癌發(fā)生的眾多機(jī)制之一,肺癌的產(chǎn)生是否就與人體內(nèi)或肺癌組織中的Cullin7的異常表達(dá)高度相關(guān),值得肯定的是Cullin7也許會(huì)是探索肺癌發(fā)病原因及治療研究中的一個(gè)新方向。Cullin7(CUL7)是將包含F(xiàn)-box蛋白Fbw8、Skpl和ROC1 RING指形蛋白的E3泛素連接酶組織在一起的分子骨架。CUL7 E3連接酶失調(diào)與人類(lèi)遺傳疾病直接相關(guān),因?yàn)樵诔Ｈ旧w隱性遺傳3M綜合征和雅庫(kù)特人矮小綜合征患者中觀察到Cullin7生殖細(xì)胞系突變,其特征為嚴(yán)重的出生前和出生后生長(zhǎng)遲緩。另外,小鼠Cullin7基因切除會(huì)導(dǎo)致子宮內(nèi)生長(zhǎng)遲緩以及圍產(chǎn)期幼仔死亡,強(qiáng)調(diào)了其對(duì)于生長(zhǎng)調(diào)節(jié)的重要意義。最近識(shí)別出胰島素受體底物1-種胰島素和胰島素樣生長(zhǎng)因子-1信號(hào)傳導(dǎo)的重要介質(zhì),是CUL7 E3連接酶的蛋白水解靶標(biāo),這揭示了Cullin7與充分確立的生長(zhǎng)調(diào)節(jié)途徑間的分子聯(lián)系。該結(jié)果連同其它證明CUL7與p53腫瘤抑制蛋白,以及猴病毒40大T抗原腫瘤蛋白間存在相互作用的研究均進(jìn)一步提示了CUL7是生長(zhǎng)控制中的一個(gè)新角色。曾有研究表明Cullin7對(duì)于癌癥萌發(fā)過(guò)程中的血管生成至關(guān)重要,最新的研究表明Cullin7可在人和小鼠腫瘤內(nèi)皮細(xì)胞中表達(dá)。通過(guò)Guo Hongsheng等人研究Cullin7在乳腺癌細(xì)胞中的表達(dá),認(rèn)為HCC1937細(xì)胞中的Cullin7在細(xì)胞增殖中異位表達(dá)明顯增加,而敲除BT474細(xì)胞中的Cullin7會(huì)導(dǎo)致生長(zhǎng)速率減緩。這些結(jié)果表明Cullin7會(huì)刺激乳腺癌細(xì)胞增殖。同時(shí)首次證明Cullin7可在蛋白水平上(而非mRNA水平上)調(diào)節(jié)乳腺癌細(xì)胞中的p53表達(dá),這表明Cullin7采用替代機(jī)制來(lái)提高乳腺癌細(xì)胞的侵襲性。然而,Cullin7對(duì)其他原癌基因和抑癌基因水平的影響需要進(jìn)一步的研究才能確定。第一部分我們通過(guò)免疫組織化學(xué)染色檢測(cè)人正常肺組織以及肺癌組織中Cullin7的表達(dá)差異。染色結(jié)果顯示在正常肺組織中Cullin7蛋白表達(dá)僅見(jiàn)于少量細(xì)胞胞質(zhì)中,且著色較淺；肺癌組織中Cullin7蛋白在多數(shù)細(xì)胞的胞質(zhì)和胞核中呈強(qiáng)著色,且胞核著色相比胞質(zhì)顏色更深。顯微鏡下觀察發(fā)現(xiàn)癌組織中Cullin7蛋白染色陽(yáng)性細(xì)胞明顯多于正常組織,計(jì)數(shù)結(jié)果t檢驗(yàn)顯示差異具有統(tǒng)計(jì)學(xué)意義(P0.01),該結(jié)果表明肺癌組織Cullin7蛋白表達(dá)明顯高于正常肺組織。我們采用Cullin7-siRNA干擾肺癌A427、H460、A549、H1299細(xì)胞中Cullin7的表達(dá),并通過(guò)半定量RT-PCR檢測(cè)siRNA干擾效率。瓊脂糖凝膠電泳結(jié)果顯示A427、H460、A549對(duì)照組細(xì)胞中Cullin7基因表達(dá)量較高,而H1299細(xì)胞中Cullin7基因表達(dá)量稍低。干擾后的A427細(xì)胞樣品中Cullin7基因RNA表達(dá)量弱于對(duì)照組,干擾效率相對(duì)較低；H460、A549、H1299田胞樣品中Cullin7基因RNA表達(dá)量明顯弱于對(duì)照組,siRNA的干擾效率在A549、H1299細(xì)胞中尤其顯著。為了確定Cullin7對(duì)肺癌細(xì)胞增殖的影響,我們對(duì)轉(zhuǎn)染Cullin7-siRNA后的A427、H460、A549、H1299四種肺癌細(xì)胞進(jìn)行MTT檢測(cè),實(shí)驗(yàn)結(jié)果顯示,A427、H460、A549、H1299四種肺癌細(xì)胞在干擾Cullin7表達(dá)后,吸光度(OD值)比對(duì)照組分別降低30%、39%、57%和60%,降低幅度與其基因干擾效率高低趨勢(shì)一致,且具有統(tǒng)計(jì)學(xué)意義(P0.05)。實(shí)驗(yàn)結(jié)果提示干擾Cullin7表達(dá)之后對(duì)肺癌細(xì)胞的增殖能力具有抑制作用。為了進(jìn)一步闡明Cullin7對(duì)肺癌細(xì)胞增殖能力的影響,我們對(duì)轉(zhuǎn)染后的A427、H460、A549和H1299四種細(xì)胞進(jìn)行克隆形成實(shí)驗(yàn),顯微鏡下觀察,最終實(shí)驗(yàn)結(jié)果表明,A427、H460、A549和H1299四種肺癌細(xì)胞在干擾Cullin7表達(dá)后,其克隆形成數(shù)目與對(duì)照組相比均有顯著減少。結(jié)果表明,干擾Cullin7表達(dá)可以抑制肺癌細(xì)胞形成克隆。為驗(yàn)證Cullin7表達(dá)降低對(duì)肺癌細(xì)胞體外增殖能力的影響,我們采取異種移植瘤模型實(shí)驗(yàn)(裸鼠成瘤實(shí)驗(yàn))進(jìn)一步檢測(cè)Cullin7表達(dá)降低是否能夠影響肺癌細(xì)胞體外增殖能力。裸鼠成瘤實(shí)驗(yàn)結(jié)果表明,在肺癌細(xì)胞中低表達(dá)Cullin7能夠抑制體內(nèi)腫瘤的形成,表現(xiàn)在成瘤的體積和成瘤的重量均明顯低于肺癌細(xì)胞對(duì)照組,t檢驗(yàn)差異具有統(tǒng)計(jì)學(xué)意義(P0.01),上述體內(nèi)實(shí)驗(yàn)證實(shí)了Cullin7低表達(dá)能夠抑制肺癌細(xì)胞的成瘤能力。第二部分為進(jìn)一步確定Cullin7參與的調(diào)控通路,我們采用Western-B lot檢測(cè)干擾Cullin7表達(dá)后相關(guān)重要調(diào)控蛋白表達(dá)量的變化。結(jié)果顯示在A427、H460、A549和H1299四種肺癌細(xì)胞系中干擾Cullin7基因表達(dá)后,Cullin7蛋白量顯著下降,并且p53、p27和p21三種蛋白的表達(dá)量與對(duì)照組相比均有上升,并且在A427與A549兩種細(xì)胞中三種蛋白表達(dá)量的上升水平與對(duì)照組相比更加顯著。實(shí)驗(yàn)結(jié)果提示Cullin7蛋白可能是p53、p27和p21蛋白表達(dá)的一個(gè)負(fù)調(diào)控因子。在腫瘤細(xì)胞中,p27與p21是p53調(diào)控的下游基因,所以我們進(jìn)一步驗(yàn)證了Cullin7與p53的調(diào)控關(guān)系。由于A549肺癌細(xì)胞株中p53、p27和p21三種蛋白表達(dá)量變化水平與對(duì)照組相比更加顯著,我們選取A549用于后續(xù)實(shí)驗(yàn),對(duì)該細(xì)胞株進(jìn)行C ullin7siRNA的干擾及Cullin7siRNA與p53siRNA的共同干擾,觀察A549細(xì)胞的克隆形成數(shù)目變化。實(shí)驗(yàn)結(jié)果表明,在Cullin7siRNA干擾Cullin7的表達(dá)后,A549細(xì)胞的克隆形成數(shù)目顯著減少；同時(shí)干擾Cullin7與p53的表達(dá)時(shí),A549細(xì)胞的克隆形成效率有顯著恢復(fù)。實(shí)驗(yàn)結(jié)果提示Cullin7可能通過(guò)抑制p53表達(dá)來(lái)調(diào)控肺癌細(xì)胞增殖。為進(jìn)一步確定Cullin7作用于p53基因的調(diào)控機(jī)制,在A549細(xì)胞中,我們通過(guò)Western-Blot實(shí)驗(yàn)檢測(cè)p53調(diào)控的H2AX磷酸化(γ-H2 X)蛋白的表達(dá)。Western-Blot實(shí)驗(yàn)結(jié)果顯示,當(dāng)干擾Cullin7的表達(dá)時(shí),p53蛋白表達(dá)量升高,同時(shí)γ-H2AX蛋白相對(duì)于對(duì)照組出現(xiàn)明顯增加,p21和p27蛋白表達(dá)量亦有明顯上升。然而同時(shí)干擾Cullin7與p53表達(dá)時(shí),γ-H2AX蛋白表達(dá)相對(duì)于對(duì)照組變化并不明顯,p21和p27蛋白表達(dá)量亦未有明顯變化。免疫熒光染色結(jié)果也表明,干擾Cullin7表達(dá)會(huì)導(dǎo)致細(xì)胞γ-H2AX蛋白表達(dá)量增加,而同時(shí)干擾Cullin7及p53表達(dá)時(shí)并不會(huì)造成γ-H2AX蛋白表達(dá)量出現(xiàn)顯著變化。上述實(shí)驗(yàn)結(jié)果提示,Cullin7可能是通過(guò)p53蛋白參與到γ-H2AX修飾及相關(guān)的基因修復(fù)途徑對(duì)肺癌細(xì)胞進(jìn)行調(diào)控。
[Abstract]:Lung cancer has become the leading cause of cancer death in the world. More than one million of the people are dying from this disease every year, and the mortality rate of lung cancer has risen by nearly 10 times in 40 years. The number of new cases of lung cancer in the world is about 1 million 800 thousand a year, of which 2/3 has been found at a late stage. The 5 year survival rate of patients with lung cancer is only about 10%.'s most important treatment hand for lung cancer. The segment is a comprehensive treatment based on surgery, radiotherapy, chemotherapy and molecular targeting. 65-70% has been found to be a late loss of operation. If chemotherapy or radiotherapy can be used for such patients, the 5 year survival rate is only 7-17%. If the patient is unable to tolerate or lose the chemoradiation machine, the 5 year survival rate is only 2%., so the search is more effective. The treatment strategy of lung cancer, especially early diagnosis of lung cancer, is of great significance. There will be a lot of room for understanding the new pathogenesis and identifying new tumor markers. The latest research suggests that if we can detect lung cancer early and stifle it in the bud to prevent it from developing along multiple pathways, the situation may become optimistic. The high expression of Cullin7 (a multifunction ubiquitin ligase subunit) in the human body is one of the many mechanisms of lung cancer. Whether the production of lung cancer is highly related to the abnormal expression of Cullin7 in the human body or lung tissue, it is worth affirming that Cullin7 may be the cause of exploring the cause of lung cancer and A new direction in the treatment study,.Cullin7 (CUL7), is a direct correlation between the.CUL7 E3 ligase dysfunction of the E3 ubiquitin ligase containing the F-box protein Fbw8, Skpl and ROC1 RING finger proteins, which is directly related to human genetic disease, because it is observed in the autosomal recessive hereditary 3M syndrome and the ikustan dwarf syndrome The mutation of the Cullin7 germ cell line is characterized by severe pre birth and postnatal growth retardation. In addition, the Cullin7 gene removal in mice will lead to intrauterine growth retardation and perinatal infant death, emphasizing its importance for growth regulation. Recently, the insulin receptor substrate 1- species insulin and insulin like growth are identified. The important medium for the conduction of the sub-1 signal is the proteolysis target of the CUL7 E3 ligase, which reveals the molecular linkage between Cullin7 and the fully established pathway of growth regulation. The results, together with other studies demonstrating the interaction between CUL7 and p53 tumor suppressor, and the 40 big T antigen tumor proteins of monkey virus, further hint CUL7 It is a new role in growth control. Some studies have shown that Cullin7 is essential for angiogenesis during the course of cancer germination. The latest research shows that Cullin7 can be expressed in human and mouse tumor endothelial cells. The expression of Cullin7 in breast cancer cells is studied by Guo Hongsheng et al., and the Cullin7 in HCC1937 cells is thinner. Ectopic expression in cell proliferation is significantly increased, while Cullin7 in BT474 cells may lead to a slow growth rate. These results suggest that Cullin7 stimulates the proliferation of breast cancer cells. At the same time, it is the first time that Cullin7 can regulate the expression of p53 in breast cancer cells at the protein level (not mRNA level), suggesting that Cullin7 is used as an alternative mechanism to improve the expression of p53 in breast cancer cells. The invasiveness of high breast cancer cells. However, the effect of Cullin7 on other proto oncogenes and tumor suppressor genes needs further study. Part 1 we detected the difference in Cullin7 expression in normal lung tissue and lung cancer tissues by immunohistochemical staining. The color staining results showed that Cullin7 eggs were in normal lung tissue. The white expression was found only in a small number of cytoplasm and shallower. The Cullin7 protein in the lung cancer tissues was strongly colored in the cytoplasm and nucleus of most of the cells, and the coloration of the nucleus was deeper than that of the cytoplasm. Under the microscope, it was found that the Cullin7 protein staining positive cells in the cancer tissues were much more than those of the normal tissues. The results of the t test showed the difference. The results showed that the expression of Cullin7 protein in lung cancer tissues was significantly higher than that of normal lung tissue. We used Cullin7-siRNA to interfere with the expression of Cullin7 in A427, H460, A549, H1299 cells in lung cancer and detected siRNA interference efficiency by semi quantitative RT-PCR. The results of agar gel electrophoresis showed A427, H460, and control cells. The expression of Cullin7 gene was higher and the expression of Cullin7 gene in H1299 cells was slightly lower. The RNA expression of Cullin7 gene in the A427 cell samples after interference was weaker than that of the control group, and the interference efficiency was relatively low. The RNA expression of Cullin7 gene in H460, A549, H1299 field cell samples was obviously weaker than that of the control group. To determine the effect of Cullin7 on the proliferation of lung cancer cells, we performed MTT tests on four lung cancer cells of A427, H460, A549 and H1299 after transfection of Cullin7-siRNA. The results showed that the absorbance of A427, H460, A549, H1299 four lung cancer cells was 30%, 39%, 57% and 60% lower than the control group after interference of Cullin7. The experimental results suggest that the interference of Cullin7 expression can inhibit the proliferation of lung cancer cells. In order to further clarify the effect of Cullin7 on the proliferation of lung cancer cells, we cloned four cells of A427, H460, A549 and H1299 after transfection. The results showed that the number of four lung cancer cells in A427, H460, A549 and H1299 decreased significantly compared with the control group after interfering Cullin7 expression. The results showed that the interference of Cullin7 expression could inhibit the cell form cloning of lung cancer. The results showed that the expression of Cullin7 was reduced to the lung cancer cells in vitro. The effect of proliferation ability, we take the xenograft tumor model test (nude mouse tumorigenesis experiment) to further detect whether the decrease of Cullin7 expression can affect the proliferation ability of lung cancer cells in vitro. The results of tumor formation in nude mice show that the low expression of Cullin7 in lung cancer cells can inhibit the formation of tumor in vivo, and is manifested in the volume and tumor formation of the tumor. The weight of the t test was significantly lower than that of the lung cancer cell control group, and the difference of the t test was statistically significant (P0.01). The above experiments confirmed that the low expression of Cullin7 could inhibit the tumor formation of lung cancer cells. The second part was to further determine the regulatory pathway of Cullin7 participation, and we used Western-B lot to detect the important regulation after interference of Cullin7 expression. The changes in protein expression showed that after the interference of Cullin7 gene expression in four lung cancer cell lines of A427, H460, A549 and H1299, the amount of Cullin7 protein decreased significantly, and the expression of the three proteins of p53, p27 and p21 increased compared with those of the control group, and the increased levels of the three protein expressions in the A427 and A549 two cells were compared with those of the control. The experimental results suggest that Cullin7 protein may be a negative regulator of the expression of p53, p27 and p21. In tumor cells, p27 and p21 are downstream genes of p53 regulation, so we further verified the regulatory relationship between Cullin7 and p53. P27 and three protein expressions of A549 changes water due to p53 in the A549 lung cancer cell lines. A549 was more significant compared with the control group. We selected A549 for subsequent experiments. The interference of C ullin7siRNA and the common interference between Cullin7siRNA and p53siRNA were used to observe the changes in the number of cloning and formation of A549 cells. The experimental results showed that the number of the clone formation of A549 cells decreased significantly after the Cullin7siRNA interfered with the expression of Cullin7. Less; when the expression of Cullin7 and p53 is interfered with the expression of the A549 cells, the cloning efficiency of the A549 cells can be recovered significantly. The experimental results suggest that Cullin7 may regulate the proliferation of lung cancer cells by inhibiting the expression of p53. To further determine the regulation mechanism of the Cullin7 action on the p53 gene, we detect p53 H2AX by Western-Blot in A549 cells. The expression of phosphorylation (gamma -H2 X) protein expression.Western-Blot showed that when the expression of Cullin7 was disturbed, the expression of p53 protein increased, and the expression of gamma -H2AX protein increased significantly compared to the control group, and the expression of p21 and p27 protein increased significantly. However, when the expression of Cullin7 and p53 was disturbed, the expression of gamma -H2AX protein was relative to the control group. There was no obvious change in the expression of p21 and p27 protein. The results of immunofluorescence staining also showed that interference of Cullin7 expression could lead to increased expression of gamma -H2AX protein, while interference of Cullin7 and p53 expression did not cause significant changes in the expression of gamma -H2AX protein at the same time. The results suggested that Cullin7 may be passed. P53 protein is involved in gamma -H2AX modification and related gene repair pathways to regulate lung cancer cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R734.2
本文編號(hào)：2130695
[Abstract]:Lung cancer has become the leading cause of cancer death in the world. More than one million of the people are dying from this disease every year, and the mortality rate of lung cancer has risen by nearly 10 times in 40 years. The number of new cases of lung cancer in the world is about 1 million 800 thousand a year, of which 2/3 has been found at a late stage. The 5 year survival rate of patients with lung cancer is only about 10%.'s most important treatment hand for lung cancer. The segment is a comprehensive treatment based on surgery, radiotherapy, chemotherapy and molecular targeting. 65-70% has been found to be a late loss of operation. If chemotherapy or radiotherapy can be used for such patients, the 5 year survival rate is only 7-17%. If the patient is unable to tolerate or lose the chemoradiation machine, the 5 year survival rate is only 2%., so the search is more effective. The treatment strategy of lung cancer, especially early diagnosis of lung cancer, is of great significance. There will be a lot of room for understanding the new pathogenesis and identifying new tumor markers. The latest research suggests that if we can detect lung cancer early and stifle it in the bud to prevent it from developing along multiple pathways, the situation may become optimistic. The high expression of Cullin7 (a multifunction ubiquitin ligase subunit) in the human body is one of the many mechanisms of lung cancer. Whether the production of lung cancer is highly related to the abnormal expression of Cullin7 in the human body or lung tissue, it is worth affirming that Cullin7 may be the cause of exploring the cause of lung cancer and A new direction in the treatment study,.Cullin7 (CUL7), is a direct correlation between the.CUL7 E3 ligase dysfunction of the E3 ubiquitin ligase containing the F-box protein Fbw8, Skpl and ROC1 RING finger proteins, which is directly related to human genetic disease, because it is observed in the autosomal recessive hereditary 3M syndrome and the ikustan dwarf syndrome The mutation of the Cullin7 germ cell line is characterized by severe pre birth and postnatal growth retardation. In addition, the Cullin7 gene removal in mice will lead to intrauterine growth retardation and perinatal infant death, emphasizing its importance for growth regulation. Recently, the insulin receptor substrate 1- species insulin and insulin like growth are identified. The important medium for the conduction of the sub-1 signal is the proteolysis target of the CUL7 E3 ligase, which reveals the molecular linkage between Cullin7 and the fully established pathway of growth regulation. The results, together with other studies demonstrating the interaction between CUL7 and p53 tumor suppressor, and the 40 big T antigen tumor proteins of monkey virus, further hint CUL7 It is a new role in growth control. Some studies have shown that Cullin7 is essential for angiogenesis during the course of cancer germination. The latest research shows that Cullin7 can be expressed in human and mouse tumor endothelial cells. The expression of Cullin7 in breast cancer cells is studied by Guo Hongsheng et al., and the Cullin7 in HCC1937 cells is thinner. Ectopic expression in cell proliferation is significantly increased, while Cullin7 in BT474 cells may lead to a slow growth rate. These results suggest that Cullin7 stimulates the proliferation of breast cancer cells. At the same time, it is the first time that Cullin7 can regulate the expression of p53 in breast cancer cells at the protein level (not mRNA level), suggesting that Cullin7 is used as an alternative mechanism to improve the expression of p53 in breast cancer cells. The invasiveness of high breast cancer cells. However, the effect of Cullin7 on other proto oncogenes and tumor suppressor genes needs further study. Part 1 we detected the difference in Cullin7 expression in normal lung tissue and lung cancer tissues by immunohistochemical staining. The color staining results showed that Cullin7 eggs were in normal lung tissue. The white expression was found only in a small number of cytoplasm and shallower. The Cullin7 protein in the lung cancer tissues was strongly colored in the cytoplasm and nucleus of most of the cells, and the coloration of the nucleus was deeper than that of the cytoplasm. Under the microscope, it was found that the Cullin7 protein staining positive cells in the cancer tissues were much more than those of the normal tissues. The results of the t test showed the difference. The results showed that the expression of Cullin7 protein in lung cancer tissues was significantly higher than that of normal lung tissue. We used Cullin7-siRNA to interfere with the expression of Cullin7 in A427, H460, A549, H1299 cells in lung cancer and detected siRNA interference efficiency by semi quantitative RT-PCR. The results of agar gel electrophoresis showed A427, H460, and control cells. The expression of Cullin7 gene was higher and the expression of Cullin7 gene in H1299 cells was slightly lower. The RNA expression of Cullin7 gene in the A427 cell samples after interference was weaker than that of the control group, and the interference efficiency was relatively low. The RNA expression of Cullin7 gene in H460, A549, H1299 field cell samples was obviously weaker than that of the control group. To determine the effect of Cullin7 on the proliferation of lung cancer cells, we performed MTT tests on four lung cancer cells of A427, H460, A549 and H1299 after transfection of Cullin7-siRNA. The results showed that the absorbance of A427, H460, A549, H1299 four lung cancer cells was 30%, 39%, 57% and 60% lower than the control group after interference of Cullin7. The experimental results suggest that the interference of Cullin7 expression can inhibit the proliferation of lung cancer cells. In order to further clarify the effect of Cullin7 on the proliferation of lung cancer cells, we cloned four cells of A427, H460, A549 and H1299 after transfection. The results showed that the number of four lung cancer cells in A427, H460, A549 and H1299 decreased significantly compared with the control group after interfering Cullin7 expression. The results showed that the interference of Cullin7 expression could inhibit the cell form cloning of lung cancer. The results showed that the expression of Cullin7 was reduced to the lung cancer cells in vitro. The effect of proliferation ability, we take the xenograft tumor model test (nude mouse tumorigenesis experiment) to further detect whether the decrease of Cullin7 expression can affect the proliferation ability of lung cancer cells in vitro. The results of tumor formation in nude mice show that the low expression of Cullin7 in lung cancer cells can inhibit the formation of tumor in vivo, and is manifested in the volume and tumor formation of the tumor. The weight of the t test was significantly lower than that of the lung cancer cell control group, and the difference of the t test was statistically significant (P0.01). The above experiments confirmed that the low expression of Cullin7 could inhibit the tumor formation of lung cancer cells. The second part was to further determine the regulatory pathway of Cullin7 participation, and we used Western-B lot to detect the important regulation after interference of Cullin7 expression. The changes in protein expression showed that after the interference of Cullin7 gene expression in four lung cancer cell lines of A427, H460, A549 and H1299, the amount of Cullin7 protein decreased significantly, and the expression of the three proteins of p53, p27 and p21 increased compared with those of the control group, and the increased levels of the three protein expressions in the A427 and A549 two cells were compared with those of the control. The experimental results suggest that Cullin7 protein may be a negative regulator of the expression of p53, p27 and p21. In tumor cells, p27 and p21 are downstream genes of p53 regulation, so we further verified the regulatory relationship between Cullin7 and p53. P27 and three protein expressions of A549 changes water due to p53 in the A549 lung cancer cell lines. A549 was more significant compared with the control group. We selected A549 for subsequent experiments. The interference of C ullin7siRNA and the common interference between Cullin7siRNA and p53siRNA were used to observe the changes in the number of cloning and formation of A549 cells. The experimental results showed that the number of the clone formation of A549 cells decreased significantly after the Cullin7siRNA interfered with the expression of Cullin7. Less; when the expression of Cullin7 and p53 is interfered with the expression of the A549 cells, the cloning efficiency of the A549 cells can be recovered significantly. The experimental results suggest that Cullin7 may regulate the proliferation of lung cancer cells by inhibiting the expression of p53. To further determine the regulation mechanism of the Cullin7 action on the p53 gene, we detect p53 H2AX by Western-Blot in A549 cells. The expression of phosphorylation (gamma -H2 X) protein expression.Western-Blot showed that when the expression of Cullin7 was disturbed, the expression of p53 protein increased, and the expression of gamma -H2AX protein increased significantly compared to the control group, and the expression of p21 and p27 protein increased significantly. However, when the expression of Cullin7 and p53 was disturbed, the expression of gamma -H2AX protein was relative to the control group. There was no obvious change in the expression of p21 and p27 protein. The results of immunofluorescence staining also showed that interference of Cullin7 expression could lead to increased expression of gamma -H2AX protein, while interference of Cullin7 and p53 expression did not cause significant changes in the expression of gamma -H2AX protein at the same time. The results suggested that Cullin7 may be passed. P53 protein is involved in gamma -H2AX modification and related gene repair pathways to regulate lung cancer cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R734.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 劉民培,吳祖澤;腫瘤基因的一種新的功能分類(lèi)與概念[J];中國(guó)腫瘤臨床;2004年21期
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