RNAi沉默Nanog基因对人结肠癌干细胞生物学行为的影响

发布时间：2018-04-13 07:12

本文选题：肿瘤干细胞 + 结肠癌　；参考：《吉林大学》2017年硕士论文

【摘要】：结肠癌(Colorectal cancer)是世界上最常见的消化道恶性肿瘤之一,其死亡率居肿瘤相关死亡率的第三位。在我国,结肠癌的发病率及死亡率也呈逐年上升的趋势。目前针对结肠癌的治疗手段仍以外科手术切除联合放化疗为主,但是患者仍会出现术后肿瘤复发和转移,严重危害患者的健康与生命。因此,急需寻找更合适及更有效的治疗结肠癌的方法。近年来,随着对恶性肿瘤的深入研究及肿瘤干细胞理论的提出,人们发现,肿瘤中存在极少数具有“干性”,能够自我更新并具有多向分化潜能的肿瘤干细胞(Cancer stem cell,CSC),与肿瘤发生发展、转移及复发密切相关。肿瘤干细胞理论认为,肿瘤干细胞是肿瘤的启动细胞,可能是恶性肿瘤形成的根源,只有靶向治疗肿瘤干细胞,才能最终治愈肿瘤。干性基因在干细胞的自我更新和分化中发挥关键作用,其中干细胞转录因子Nanog不仅在维持胚胎干细胞多能性和自我更新方面发挥重要作用,而且在多种肿瘤中异常表达。研究发现,Nanog的高表达与肿瘤不良预后密切相关,提示Nanog可能通过维持肿瘤干细胞的干性促进肿瘤的发生发展。因此,为了探究Nanog对结肠癌干细胞的生物学影响,本实验以Ep CAM和CD44为肿瘤干细胞筛选标志,通过免疫磁珠法从人结肠癌细胞系HCT-116中筛选出Ep CAM+CD44+HCT-116的人结肠癌干细胞(Colorectal cancer stem cell,CCSC),利用RNA干扰(RNA interference,RNAi)沉默结肠癌干细胞中Nanog的表达,探究其对结肠癌干细胞生物学行为的影响。结果显示,筛选获得的Ep CAM+CD44+HCT-116细胞在无血清DMEM/F12培养基中悬浮生长,由单个细胞逐渐长成肿瘤微球;经含血清培养基诱导后,细胞微球呈现贴壁分化状态。Real-time PCR检测结果显示:Ep CAM+CD44+HCT-116细胞中Nanog的m RNA表达显著高于未分选的HCT-116细胞(P0.01);Ep CAM+CD44+HCT-116经Nanog si RNA作用48h之后,细胞中Nanog基因的m RNA和蛋白表达水平均显著下降(P0.01,P0.05)。MTS增殖实验显示Nanog基因沉默可显著抑制Ep CAM+CD44+HCT-116增殖(P0.01)。Annexin V-FITC/PI双染法从细胞膜的完整程度上区分早、晚期凋亡细胞,结果显示:Ep CAM+CD44+HCT-116中Nanog基因沉默后,早、晚期凋亡细胞比例增加;JC-1法通过细胞膜电位的变化反映细胞凋亡情况,结果显示:Nanog基因沉默后细胞膜电位大幅度降低,凋亡细胞比例增加;Real-time PCR检测结果显示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中抗凋亡基因Bcl-2的m RNA表达水平显著降低(P0.05),而促凋亡基因Bax及Caspase-3的m RNA表达水平均显著升高(P0.01);Western blot结果显示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中cleaved-caspase3蛋白表达量显著增加(P0.05)。Transwell小室结果显示:沉默Nanog基因可显著抑制Ep CAM+CD44+HCT-116的侵袭(P0.01);同时Real-time PCR结果显示:Nanog基因沉默后,Ep CAM+CD44+HCT-116中促进侵袭基因MMP-2及MMP-9的m RNA表达水平显著降低(P0.05,P0.01),而抑制侵袭基因Timp-1的m RNA表达水平显著升高(P0.01)。动物实验结果显示,Nanog si RNA组肿瘤体积及瘤重显著低于对照组及空白组(P0.05),并且Nanog si RNA组荷瘤鼠的生存期也显著长于对照组及空白组(P0.05)。综上所述,Nanog沉默能够抑制结肠癌干细胞的增殖和侵袭,促进结肠癌干细胞的凋亡,并降低结肠癌干细胞的成瘤能力,这为针对结肠癌干细胞的靶向治疗提供了实验依据。
[Abstract]:Colon cancer (Colorectal cancer) is one of the most common malignant tumor of digestive tract, the mortality rate was third. The tumor related mortality in our country, the incidence and mortality of colorectal cancer also showed an increasing trend. At present for colon cancer treatment is still outside the surgery resection combined with radiotherapy and chemotherapy, but there would still be in patients with postoperative tumor recurrence and metastasis, patients with serious harm to health and life. Therefore, the urgent need to find a more suitable method and treatment of colon cancer is more effective. In recent years, with the proposed cell theory on malignant tumor research and tumor stem were found, with a very small number of "dry" the tumor, capable of self-renewal and multi-directional differentiation of cancer stem cells (Cancer stem cell, CSC), and the occurrence and development of tumor, metastasis and recurrence are closely related. The cancer stem cell theory, tumor stem Cells are tumor initiating cells, may be the root cause of the formation of malignant tumors, only targeted therapy of cancer stem cells to cure the tumor. Dry gene plays a key role in the self-renewal and differentiation of stem cells, including stem cell transcription factor Nanog in maintenance of embryonic stem cells can play an important role and the self the update, and the abnormal expression in a variety of tumors. The study found that high expression of Nanog and tumor prognosis is closely related to the occurrence and development of Nanog may be prompted by maintaining the tumor stem cells promote tumor dry. Therefore, in order to explore the biological effects of Nanog stem cells for colon cancer, in this experiment, Ep CAM and CD44 as tumor stem cell marker screening, by immunomagnetic beads from human colon cells screened human colon cancer stem Ep CAM+CD44+HCT-116 colorectal cancer cell line HCT-116 in Colorectal (cancer stem cell, CCS C (RNA), using RNA interference interference, RNAi) silencing of colon cancer stem cells expression of Nanog, to explore its effect on colon cancer stem cells. The results showed that the Ep screened CAM+CD44+HCT-116 cells in serum-free suspension culture medium DMEM/F12 growth, from a single cell into tumor by serum containing microspheres; medium after induction, cell adherence and differentiation state of.Real-time microspheres showed PCR showed that the expression of M RNA Nanog Ep in CAM+CD44+HCT-116 cells was significantly higher than that of unsorted HCT-116 cells (P0.01); Ep CAM+CD44+ HCT-116 Nanog Si RNA by 48h, the expression level of M protein and RNA Nanog gene in the cells were significantly decreased (P0.01, P0.05.MTS) proliferation assay showed that Nanog gene silencing can significantly inhibit the proliferation of CAM+CD44+HCT-116 Ep (P0.01).Annexin V-FITC/PI double staining method from the integrity of the cell membrane to distinguish early, The results of late apoptotic cells showed that Ep in CAM+CD44+HCT-116 after Nanog gene silencing, the percentage of apoptotic cells increased early and late; JC-1 method by changing the membrane potential reflect the cell apoptosis, the results showed that Nanog gene silencing cell membrane potential decreased, apoptotic cells increased; Real-time PCR results showed that Nanog gene after the silence, the expression level of M RNA Ep anti apoptosis gene Bcl-2 in CAM+CD44+HCT-116 decreased significantly (P0.05), the expression level of M RNA and apoptosis gene Bax and Caspase-3 were significantly increased (P0.01); Western blot showed that Nanog gene silencing, cleaved-caspase3 protein expression was significantly increased in Ep CAM+CD44+HCT-116 (P0.05).Transwell chamber the results showed that silencing Nanog gene can inhibit the invasion of Ep CAM+CD44+HCT-116 (P0.01); and Real-time PCR results showed that: after Nanog gene silencing, Ep CAM+CD 44+HCT-116 m RNA MMP-2 to promote invasion gene and MMP-9 expression level were significantly decreased (P0.05, P0.01), and the inhibition of M RNA invasion gene Timp-1 expression level increased significantly (P0.01). The results of animal experiments showed that the Nanog Si RNA group tumor volume and tumor weight was significantly lower than the control group and blank control group (P0.05), and Nanog Si RNA group, the survival time of tumor bearing mice was significantly longer than that of the control group and blank control group (P0.05). In conclusion, Nanog silencing inhibits the proliferation and invasion of colon cancer stem cells and promote the apoptosis of colon cancer stem cells, and reduce colon cancer stem cell tumorigenic ability, this is for colon cancer stem cells. Targeted therapy provides an experimental basis.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35

【参考文献】