摘要
背景与目的
甲状腺癌是威胁人们健康的常见头颈部恶性肿瘤,近年其发病率有上升趋势。虽然各种诊治措施不断进展,但是甲状腺癌的远期生存率仍然没有得到明显的提高,肿瘤细胞的侵袭和远处转移是其治疗失败的主要原因。肿瘤的转移是个复杂的过程,基本步骤包括肿瘤细胞侵袭、突破细胞基质,进入血液,经血流到达远处器官,突破血管进入靶器官定居、增殖。
癌细胞对细胞外基质的黏附性降低是肿瘤细胞迁移、侵袭、转移的重要原因之一。研究表明:粘着斑激酶(focal adhesion kinase,FAK)作为胞内重要的信号分子,介导细胞内信号网络系统的交联,在细胞的移动和侵袭中起关键的作用。FAK在甲状腺原发癌及转移癌中均较正常甲状腺组织表达增高。因此,如果人为干预FAK的表达,有望为甲状腺癌的治疗提供一条新的途径。RNA interference(RNAi)具有很强的抑制目的基因作用,较反义寡核苷酸抑制目的基因的效果更强,RNAi被认为是继PCR后又一划时代的基因工程方法。本课题利用RNA干扰(RNA interference)沉默FAK表达研究其对甲状腺癌SW579细胞株的增殖、侵袭及转移能力的影响,为新的治疗方法提供依据。
研究方法
1.构建FAK RNAi慢病毒载体,对体外培养甲状腺癌SW579细胞进行感染,建立稳定转染细胞株。通过报告基因GFP检测细胞转染状态。
2. MTT法检测SW579细胞沉默FAK表达前后增殖活性的变化。运用Western blot方法鉴定FAK被抑制的情况。
3.制备Transwell小室,检测沉默FAK基因表达对SW579细胞侵袭及转移能力的影响。
结果
1.成功构建了靶向FAK的RNAi慢病毒载体,获得稳定转染SW579细胞。
2.MTT检测结果:绘制生长曲线,可见干扰组细胞与未处理组及阴性对照组相比,干扰组细胞存活率明显减低(P<0.05),未处理组及阴性对照组无明显差别(P>0.05)。Western blot方法检测FAK靶向RNAi后,与未处理组及阴性质粒对照组相比,干扰组FAK的表达均明显降低。
3.体外侵袭与转移实验:对比未处理组及阴性对照组细胞,干扰组细胞侵袭及转移能力明显下降(P<0.05),正常组及阴性对照组细胞之间无明显差别(P>0.05)。
结论
1.成功构建FAK的RNAi慢病毒载体能稳定感染甲状腺癌SW579细胞;
2.FAK RNAi能有效抑制转染甲状腺癌SW579细胞增殖能力;
3.沉默FAK基因表达能有效抑制甲状腺癌细胞SW579侵袭及转移能力。
Background and Objectives
Thyroid cancer is common malignant tumor of head and neck, incidence of the cases increased gradually. Despite recent advancements in diagnosis and treatment, the long-term survival rate has undergone little improvement in the recent years, which is attributed mainly to tumor invasion and metastasis. Tumor metastasis is a complex process.The tumor cell invade and break through the extracellular matrix is the basic step,then enter blood,reach distant organs through the blood,break blood vessels into the target organ for settlement、proliferation.
Loss of adhesion of the malignant cells to the extracellular matrix is one of the fundamental pathways that promote tumor cell migration, invasion, and metastasis. Evidences have suggested that as an important intracellular signaling molecule,focal adhesion kinase(FAK) mediate intracellular signaling network of cross-linking, play a key role in cells’movement and invasion.The expression of FAK in primary cancer and metastasis cancer are more than in normal thyroid tissues.Therefore, interventing the expression of FAK could provide a new way to treat the thyroid cancer.
RNA interference(RNAi) has a strong role in inhibition of the targeted gene,which is more than antisense oligonucleotides.RNAi is considered as a landmark after the PCR method of genetic engineering.
This study was to evaluate the ability of proliferation、invasion and metastasis in transfected SW579 cells after FAK silenced by RNA interference and provide evidence to a new therapeutic method.
Methods
1. Construct FAK RNAi lentivirus vector, transfect thyroid cancer SW579 cells in vitro to establish a stable transfected cell line. Cell transfection efficiency was detected by GFP.
2. To use MTT assay to detect the proliferative activity of SW579 before and after the interference with FAK expression.The situation of suppressed FAK are identified with Western blot.
3. Prepare Transwell chambers to detect the effect of FAK silencing on invasion and metastasis of SW579 Cells.
Results
1. To construct RNAi lentiviral vector with targeting FAK successfully, obtain transfection SW579 Cells.
2. Compared with the untreated group and group of cells in negative control group, the interference group cells’survival rate were significantly decreased (P <0.05), but the normal group and the negative control group had no significant difference (P>0.05). And FAK expressions in the interference group were significantly reduced than other groups.
3. Invasion and metastasis in vitro experiments: compared with the normal group and negative control group cells, the ability of invasion and metastasis had decreased significantly (P<0.05) in the interference group cells, while the normal group and negative control group had no significant difference (P>0.05).
Conclusions
1. Successfully construct FAK RNAi lentivirus vector could stably transfected thyroid cancer cells SW579.
2. FAK RNAi can effectively inhibit the proliferation activity of transfected SW579 cells.
3. Silencing FAK gene expression can significantly inhibit the capacity of invasion and metastasis of SW579 cells.
引文
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