【摘要】：背景食管鱗癌是我國(guó)惡性腫瘤相關(guān)死亡的主要原因之一,雖然有手術(shù)、化療、放療以及其他輔助治療方法,但其遠(yuǎn)期療效令人沮喪,并且部分患者首次診斷時(shí)已是局部晚期或轉(zhuǎn)移性疾病,失去了手術(shù)治療的機(jī)會(huì)。食管鱗癌靶向治療的相關(guān)研究尚處于萌芽階段,目前許多靶向藥物的相關(guān)研究多針對(duì)胃-食管連接部腺癌開展,這些研究結(jié)果對(duì)于食管鱗癌是否適合尚不清楚。近年來RNA在生命系統(tǒng)中的重要作用備受關(guān)注,而DDX蛋白家族RNA解旋酶參與了RNA代謝的全過程。鑒于DDX解旋酶在細(xì)胞生命活動(dòng)中的重要地位,對(duì)該家族成員的功能及其與人類腫瘤關(guān)系的進(jìn)一步研究,探索其上下游效應(yīng)分子,明確其作用機(jī)制和調(diào)控機(jī)理,將為腫瘤的早期診斷和生物靶向治療提供新的途徑。目的本實(shí)驗(yàn)旨在研究DDX蛋白家族RNA解旋酶成員DDX46與食管鱗癌的關(guān)系,闡明其調(diào)控機(jī)制和作用機(jī)理,以期為揭示食管鱗癌新型生物學(xué)標(biāo)記、探索新的個(gè)體化治療策略提供實(shí)驗(yàn)依據(jù)。方法應(yīng)用高內(nèi)涵篩選成像系統(tǒng)(high content screening,HCS)篩選食管鱗癌患者新鮮手術(shù)標(biāo)本(食管鱗癌組織和距腫瘤邊緣5 cm以上食管正常組織)中的差異表達(dá)基因,發(fā)現(xiàn)了一個(gè)先前未被關(guān)注的基因:DDX蛋白家族RNA解旋酶成員DDX46基因。免疫組化染色實(shí)驗(yàn)比較食管鱗癌組織和相對(duì)應(yīng)食管正常組織中DDX46蛋白的表達(dá)情況。以人永生化食管鱗狀上皮細(xì)胞Het-1A為對(duì)照,實(shí)時(shí)定量聚合酶鏈反應(yīng)(quantitative real-time polymerase chain reaction,q RT-PCR)檢測(cè)目的基因m RNA在食管鱗癌細(xì)胞株中的表達(dá)量。應(yīng)用RNAi技術(shù)靶向沉默食管鱗癌細(xì)胞株DDX46基因的表達(dá),進(jìn)行Cellomics細(xì)胞計(jì)數(shù)、MTT實(shí)驗(yàn)、克隆形成、細(xì)胞周期和細(xì)胞凋亡,檢測(cè)DDX46基因靶向沉默對(duì)食管鱗癌細(xì)胞增殖和凋亡的影響。裸鼠成瘤實(shí)驗(yàn)觀察靶向沉默DDX46基因?qū)κ彻荀[癌細(xì)胞裸鼠成瘤的影響。應(yīng)用Affymetrix人類全基因表達(dá)譜芯片檢測(cè)DDX46基因敲減前后兩組食管鱗癌細(xì)胞中全基因m RNA表達(dá)豐度,Ingenuity通路分析(Ingenuity#174;pathway analysi,IPA)差異表達(dá)基因,差異表達(dá)基因?qū)隝PA在線工具進(jìn)行信號(hào)通路富集分析。q RT-PCR驗(yàn)證目的基因敲減后,部分候選下游通路基因m RNA表達(dá)量;Western blot檢測(cè)驗(yàn)證部分候選下游通路基因蛋白表達(dá)量;Stress and Apoptosis Signaling Antibody Array Kit檢測(cè)和比較兩組樣本中信號(hào)通路關(guān)鍵分子的變化,以驗(yàn)證IPA生物信息分析結(jié)果的可靠性。結(jié)果DDX46基因在食管鱗癌組織和細(xì)胞株中高表達(dá),DDX46蛋白表達(dá)主要定位于細(xì)胞核。應(yīng)用DDX46-sh RNA-LV靶向沉默食管鱗癌TE-1(高分化)和Eca-109(低分化)細(xì)胞中DDX46基因,檢測(cè)DDX46基因沉默后食管鱗癌細(xì)胞生物功能學(xué)變化。HCS平臺(tái)連續(xù)檢測(cè)5天,記錄各時(shí)間點(diǎn)的細(xì)胞數(shù)目,繪制出細(xì)胞生長(zhǎng)曲線,與對(duì)照組相比,靶向沉默DDX46基因后TE-1和Eca-109細(xì)胞的生長(zhǎng)均被明顯抑制;MTT檢測(cè)結(jié)果顯示,TE-1和Eca-109細(xì)胞DDX46基因敲減組與對(duì)照組在酶標(biāo)儀對(duì)波長(zhǎng)490 nm的光的吸收率隨時(shí)間變化而逐步降低,表明RNAi靶向沉默DDX46基因使食管鱗癌細(xì)胞活力明顯減弱,顯著抑制了細(xì)胞的增殖能力;細(xì)胞克隆形成能力檢測(cè)顯示,相對(duì)于對(duì)照組,DDX46-sh RNA-LV組TE-1和Eca-109細(xì)胞克隆形成數(shù)顯著減少,食管鱗癌細(xì)胞克隆形成能力受到抑制;流式細(xì)胞儀檢測(cè)DDX46-sh RNA-LV介導(dǎo)的RNAi干預(yù)后TE-1和Eca-109的細(xì)胞周期分布,結(jié)果顯示,與對(duì)照組比較,RNAi沉默DDX46基因?qū)е履康募?xì)胞處于G1期的細(xì)胞增加,而處于S期的細(xì)胞減少,表明RNAi沉默DDX46基因?qū)⑹彻荀[癌細(xì)胞停滯在細(xì)胞周期的G0/G1期;Annexin V-APC單染法流式細(xì)胞儀細(xì)胞凋亡檢測(cè)結(jié)果顯示,相對(duì)于對(duì)照組,RNAi沉默TE-1和Eca-109細(xì)胞中DDX46基因的表達(dá)后,細(xì)胞凋亡率顯著增多。以Eca-109細(xì)胞為成瘤細(xì)胞,裸鼠成瘤瘤體體積增長(zhǎng)曲線顯示RNAi靶向沉默DDX46基因使得裸鼠瘤體生長(zhǎng)減緩,小動(dòng)物活體成像顯示DDX46-sh RNA-LV組熒光區(qū)總熒光表達(dá)量和平均熒光表達(dá)量均明顯低于Control-LV組,靶向沉默DDX46基因明顯抑制Eca-109細(xì)胞裸鼠成瘤。Affymetrix人類全基因表達(dá)譜芯片檢測(cè)顯示,實(shí)驗(yàn)組DDX46-sh RNA-LV相對(duì)于對(duì)照組Control-LV,上調(diào)基因362個(gè),下調(diào)基因644個(gè)。采用IPA生物信息分析,根據(jù)差異基因在經(jīng)典通路中的富集情況,進(jìn)行共表達(dá)網(wǎng)絡(luò)構(gòu)建,結(jié)合實(shí)驗(yàn)數(shù)據(jù)預(yù)測(cè)的信號(hào)通路圖和文獻(xiàn)數(shù)據(jù)支持的激活或抑制狀態(tài)的信號(hào)通路圖顯示有5條抑制的信號(hào)通路和1條激活的信號(hào)通路,PI3K為非正常表達(dá)分子。q RT-PCR檢測(cè)和Western blot檢測(cè)結(jié)果與IPA差異基因經(jīng)典通路分析結(jié)果相符合。RNAi靶向沉默TE-1細(xì)胞中DDX46基因,Western blot檢測(cè)顯示Akt和P-Akt的表達(dá)水平顯著下調(diào);Path Scan#174;Antibody Array檢測(cè)結(jié)果發(fā)現(xiàn)Akt(Ser473,Phosphorylation)和IκBα(Total,N/A)蛋白表達(dá)水平明顯下降,Caspase-3(Asp175,Cleaved)蛋白表達(dá)水平上升,PI3K/Akt/NF-κB信號(hào)轉(zhuǎn)導(dǎo)通路被抑制;Western blot檢測(cè)顯示RNAi靶向沉默TE-1細(xì)胞中DDX46基因后,PI3K蛋白表達(dá)水平顯著下調(diào)。上述結(jié)果驗(yàn)證了IPA生物信息學(xué)分析結(jié)果的可靠性。結(jié)論DDX46基因在食管鱗癌組織和細(xì)胞株中高表達(dá),RNAi靶向沉默食管鱗癌細(xì)胞中的DDX46基因可抑制細(xì)胞增殖,阻滯細(xì)胞周期于G0/G1期,并誘導(dǎo)細(xì)胞凋亡;靶向沉默DDX46基因可明顯抑制裸鼠食管鱗癌細(xì)胞成瘤。DDX46基因沉默可能是通過抑制PI3K的活性,由此參入并下調(diào)PI3K/Akt信號(hào)通路,抑制通路下游的NF-κB信號(hào)通路和m TOR信號(hào)通路,進(jìn)而發(fā)揮抑制腫瘤細(xì)胞增殖、誘導(dǎo)細(xì)胞凋亡的作用,Integrin信號(hào)通路可能通過PI3K與PI3K/Akt信號(hào)通路“Cross Talk”而參與其中。DDX46基因沉默是否通過下調(diào)PI3K活性參與上調(diào)Rho GDI信號(hào)通路,進(jìn)而參與抑制食管鱗癌的轉(zhuǎn)移和浸潤(rùn),是下一步研究的目標(biāo)。
[Abstract]:Background Esophageal squamous cell carcinoma (ESCC) is one of the main causes of cancer-related death in China. Although there are surgery, chemotherapy, radiotherapy and other adjuvant treatment methods, the long-term efficacy of ESCC is frustrating. Some patients are locally advanced or metastatic diseases at the time of the first diagnosis, and the chance of surgical treatment is lost. In recent years, the important role of RNA in the life system has attracted much attention, and the DDX protein family RNA helicase is involved in the whole process of RNA metabolism. The important role of helicase in cell life activities, the further study of its function and its relationship with human tumors, the exploration of its upstream and downstream effector molecules, and the clarification of its mechanism of action and regulation will provide a new way for early diagnosis and targeted therapy of tumors. The relationship between RNA helicase member DDX46 and esophageal squamous cell carcinoma (ESCC) was elucidated, and its regulatory mechanism and mechanism were elucidated in order to provide experimental basis for revealing new biological markers of ESCC and exploring new individualized treatment strategies. Differentially expressed genes were found in the tissues of tubular squamous cell carcinoma (TSCC) and normal esophageal tissues more than 5 cm from the tumor margin. A previously unknown gene, DDX46, a member of the DDX protein family RNA helicase, was identified. Immunohistochemical staining was performed to compare the expression of DDX46 protein in esophageal squamous cell carcinoma (ESCC) and corresponding normal esophageal tissues. Esophageal squamous cell line Het-1A was used as control. The expression of target gene m RNA in esophageal squamous cell carcinoma cell line was detected by real-time quantitative polymerase chain reaction (q RT-PCR). The effects of targeted silencing of DDX46 gene on the proliferation and apoptosis of esophageal squamous cell carcinoma cells were studied. The effects of targeted silencing of DDX46 gene on the tumorigenesis of esophageal squamous cell carcinoma cells in nude mice were observed. The esophageal squamous cells were detected by Affymetrix human gene expression profiling chip before and after DDX46 gene knockdown. Ingenuity pathway analysis (IPA) differentially expressed genes were introduced into the IPA online tool for signal pathway enrichment analysis. Stress and Apoptosis Signaling Antibody Array Kit were used to detect and compare the changes of key molecules of signal pathway in the two groups to verify the reliability of IPA bioinformatics analysis. Nucleus. DDX46-sh RNA-LV was used to silence DDX46 gene in TE-1 (well-differentiated) and Eca-109 (poorly differentiated) esophageal squamous cell carcinoma cells. Biological function of esophageal squamous cell carcinoma cells was detected after DDX46 gene silencing. The number of cells at each time point was recorded by HCS platform for 5 days, and cell growth curve was drawn. Compared with the control group, DDDDX46 was silenced by targeted silencing. The growth of TE-1 and Eca-109 cells was significantly inhibited after gene transfection. MTT assay showed that DDX46 gene knock-down group and control group gradually decreased the absorption rate of light at 490 nm by enzyme-labeled instrument, which indicated that the activity of esophageal squamous cell carcinoma cells was significantly weakened by RNAi targeting DDX46 gene silencing, and the activity of esophageal squamous cell carcinoma cells was significantly inhibited. Compared with the control group, the clone formation of TE-1 and Eca-109 cells in DDX46-sh RNA-LV group was significantly decreased, and the clone formation ability of esophageal squamous cell carcinoma cells was inhibited. The cell cycle distribution of TE-1 and Eca-109 cells after DDX46-sh RNA-LV-mediated RNAi intervention was detected by flow cytometry. Compared with the control group, the silencing of DDX46 gene by RNAi increased the number of cells in G1 phase and decreased the number of cells in S phase, suggesting that the silencing of DDX46 gene by RNAi arrested esophageal squamous cell carcinoma cells in G0/G1 phase of cell cycle; Annexin V-APC single staining flow cytometry showed that compared with the control group, the silencing of TE-1 and TE-1 by RNAi. After DDX46 gene was expressed in Eca-109 cells, the apoptosis rate was significantly increased. Using Eca-109 cells as tumorigenic cells, the tumor volume growth curve of nude mice showed that RNAi targeted silencing of DDX46 gene slowed down tumor growth in nude mice. Small animal imaging showed that the total fluorescent expression and average fluorescent expression of DDX46-sh RNA-LV group were both positive. Targeted silencing of DDX46 gene significantly inhibited the tumorigenesis of Eca-109 cells in nude mice, significantly lower than control-LV group. Affymetrix human whole gene expression profile chip detection showed that DDX46-sh RNA-LV in the experimental group was up-regulated by 362 genes and down-regulated by 644 genes compared with control-LV in the control group. Enrichment, co-expression network construction, combined with experimental data predicted signal pathways and literature data to support the activation or inhibition of the state of signal pathways show that there are five suppressed signal pathways and one activated signal pathway, PI3K is an abnormal expression molecule. Q RT-PCR and Western blot detection results and IPA differential genes The results of classical pathway analysis were consistent. RNAi targeted silencing of DDX46 gene in TE-1 cells, Western blot analysis showed that the expression levels of Akt and P-Akt were significantly down-regulated; Path Scan #174; Antibody Array detection showed that the expression levels of Akt (Ser473, Phosphorylation) and I kappa Balpha (Total, N/A) protein were significantly decreased, and the expression of Caspase-3 (Asp175, Cleaved) protein was significantly decreased. Up-regulation of PI3K/Akt/NF-kappa B signal transduction pathway was inhibited. Western blot analysis showed that the expression of PI3K protein was significantly down-regulated after RNAi targeted silencing of DDX46 gene in TE-1 cells. These results confirmed the reliability of IPA bioinformatics analysis. Conclusion DDX46 gene was highly expressed in esophageal squamous cell carcinoma tissues and cell lines, and RNAi targeted precipitation. DDX46 gene can inhibit cell proliferation, block cell cycle in G0/G1 phase and induce apoptosis in esophageal squamous cell carcinoma cells. Targeted silencing of DDX46 gene can significantly inhibit tumor formation in nude mice esophageal squamous cell carcinoma cells. DDX46 gene silencing may be by inhibiting the activity of PI3K, thus participating in and down-regulating PI3K/Akt signaling pathway, inhibiting the downstream of the pathway of NF-Akt. Integrin signaling pathway may be involved through PI3K and PI3K/Akt signaling pathway "Cross Talk". Does DDX46 gene silencing participate in up-regulation of Rho GDI signaling pathway by down-regulation of PI3K activity, and thus in down-regulation of esophageal squamous cell carcinoma? Metastasis and infiltration are the goals of the next research.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R735.1

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