KLF8在调控人骨肉瘤细胞增殖和细胞侵袭中的作用

发布时间：2018-04-24 07:03

本文选题：骨肉瘤 + KLF转录因子　；参考：《苏州大学》2015年博士论文

【摘要】：骨肉瘤(osteosarcoma,os)是最常见的原发性恶性骨肿瘤,多发生于儿童和青少年发育时期。通过手术治疗后,发生转移的骨肉瘤病人的术后五年存活率普遍低于20%。骨肉瘤如果不及时治疗,往往会在6至12个月内通过血行转移的方式发生肺转移现象,这已经成为骨肉瘤临床观察中,最为显著的特征之一,也是影响病人预后的主要因素。KLF8(Krüppel-like factor 8)是KLF转录因子家族第一亚群的一员。KLF家族在N末端均具有三个高度保守的锌指蛋白结构,可以特异结合富含GC的启动子区。KLF转录因子在人类生长发育以及各种疾病包括肿瘤发生发展中发挥着重要的调控作用。KLF8被发现在多种肿瘤细胞中高表达。KLF8是调控细胞周期的重要转录因子,其在致癌性转化(oncogenic transformation)和上皮-间质转化(epithelial to mesenchymal transition,EMT)中具有关键作用。虽然关于KLF8在肿瘤中的功能已有不少的研究,但KLF8在人骨肉瘤中的功能还未见详尽报道。本研究的目的就是探讨KLF8在Saos-2骨肉瘤细胞中尤其是细胞侵袭中的生物学作用。首先通过RT-PCR(real-time PCR)和western blotting实验,发现KLF8在骨肉瘤细胞系中的表达远远高于正常成骨细胞中的表达,且恶性越高、侵袭性越强的细胞,KLF8的表达量越高。同时对骨肉瘤病理组织的检测发现,骨肉瘤原发灶KLF8表达高于癌旁组织,但低于肺转移灶。通过构建沉默KLF8基因表达的sh RNA慢病毒载体转染入Saos-2细胞,以随机合成的sh RNA作为阴性对照,QRT-PCR及WB检测发现在高恶性骨肉瘤细胞系Saos-2细胞中成功沉默了KLF8基因的表达,显著下调了KLF8的m RNA和蛋白水平。构建稳定表达KLF8-sh RNA的Saos-2细胞,分析了KLF8基因的沉默对骨肉瘤细胞系生长增殖的影响。首先用MTT方法测定细胞增殖情况。同阴性对照细胞相比,sh RNA-KLF8组的细胞数明显少于阴性对照细胞,说明KLF8的沉默抑制了细胞的增殖。平板克隆形成实验和软琼脂克隆形成实验发现KLF8表达降低的细胞形成克隆数明显少于对照细胞,说明KLF8影响了细胞的群体依赖性和克隆形成能力。流式细胞术对细胞周期和细胞凋亡进行分析发现KLF8的沉默使骨肉瘤细胞在G0/G1期停滞,且细胞凋亡率显著提高。说明KLF8对细胞增殖的影响是通过使细胞周期停滞和增加细胞凋亡两方面来完成的。通过Transwell@细胞侵袭实验检测KLF8沉默后细胞侵袭能力的变化,实验发现KLF8的沉默降低了细胞分解细胞基质并侵袭到下层小室的能力。跟阴性对照比,迁移到下层小室的细胞明显少于对照细胞,说明KLF8的RNA干扰影响了骨肉瘤细胞的侵袭能力。本研究显示KLF8在骨肉瘤的发生以及细胞的侵袭性中具有重要的调控作用,并为KLF8在骨肉瘤治疗中作为可能的药物治疗靶点提供了理论依据。
[Abstract]:Osteosarcomaosarcoma is the most common primary malignant bone tumor occurring in children and adolescents. After surgical treatment, the 5-year survival rate of metastatic osteosarcoma patients was generally lower than 20%. If osteosarcoma is not treated in time, pulmonary metastasis often occurs within 6 to 12 months through blood metastasis, which has become one of the most prominent features in clinical observation of osteosarcoma. KLF8 Kr 眉 ppel-like factor 8 is a member of the first subgroup of KLF transcription factor family. KLF family has three highly conserved zinc finger protein structures at the N-terminal. KLF transcription factor plays an important regulatory role in human growth and development and various diseases, including tumorigenesis. KLF8 has been found to be regulated by overexpression of .KLF8 in various tumor cells. An important transcription factor in cell cycle, It plays a key role in carcinogenic transformation and epithelial-mesenchymal transformation (EMT). Although there have been many studies on the function of KLF8 in human osteosarcoma, the function of KLF8 in human osteosarcoma has not been reported in detail. The purpose of this study was to investigate the biological role of KLF8 in Saos-2 osteosarcoma cells, especially in cell invasion. The expression of KLF8 in osteosarcoma cell line was much higher than that in normal osteoblast cell line, and the higher the malignancy, the higher the expression of KLF8 in the osteosarcoma cell line. The expression of KLF8 in primary tumor was higher than that in paracancerous tissue, but lower than that in lung metastasis. The sh RNA lentivirus vector which silenced the expression of KLF8 gene was constructed and transfected into Saos-2 cells. The expression of KLF8 gene was successfully silenced in high malignant osteosarcoma cell line Saos-2 by RT-PCR and WB detection using the randomly synthesized sh RNA as negative control. The m RNA and protein levels of KLF8 were significantly down-regulated. Saos-2 cells stably expressing KLF8-sh RNA were constructed, and the effects of KLF8 gene silencing on the growth and proliferation of osteosarcoma cell lines were analyzed. Cell proliferation was measured by MTT method. Compared with the negative control cells, the number of cells in the RNA-KLF8 group was significantly lower than that in the negative control cells, indicating that the silencing of KLF8 inhibited the proliferation of the cells. The results of plate clone formation test and soft Agar clone formation test showed that the number of cell forming clones with decreased KLF8 expression was significantly lower than that of the control cells, which indicated that KLF8 affected the colony dependence and clone formation ability of the cells. Cell cycle and apoptosis were analyzed by flow cytometry. It was found that the silencing of KLF8 made osteosarcoma cells arrest in G0/G1 phase and the rate of apoptosis increased significantly. The results showed that the effect of KLF8 on cell proliferation was achieved by arrest of cell cycle and increase of apoptosis. Transwell @ cell invasion assay was used to detect the changes of cell invasion ability after KLF8 silencing. It was found that the silencing of KLF8 decreased the ability of cells to break down the cell matrix and invade the lower chamber. Compared with the negative control, the number of cells migrating to the lower chamber was significantly lower than that of the control cells, indicating that the RNA interference of KLF8 affected the invasion ability of osteosarcoma cells. This study shows that KLF8 plays an important role in the pathogenesis of osteosarcoma and the invasion of osteosarcoma, and provides a theoretical basis for KLF8 as a possible drug target in the treatment of osteosarcoma.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R738.1

【共引文献】