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目的:构建类表皮生长因子域7(EGFL7)基因RNA干扰(RNAi)重组慢病毒,观察其对喉癌体外侵袭的抑制作用。方法:筛选确定的EGFL7基因RNAi有效靶序列,合成靶序列的Oligo DNA,与含H1启动子和绿色荧光蛋白(GFP)的pLV载体连接产生LV-sh EGFL7慢病毒载体,PCR筛选阳性克隆,测序鉴定。用重组慢病毒体外转导喉癌细胞,Western blot法检测喉癌细胞EGFL7蛋白表达。Boyden侵袭小室法实验观察转导shRNA后的喉癌细胞侵袭能力的变化。利用克隆形成实验检测EGFL7基因沉默对Hep-2细胞集落形成能力的影响。结果:成功构建EGFL7的慢病毒载体LV-sh EGFL7,包装慢病毒,浓缩病毒悬液的滴度为5×108 TU/L。转导siRNA的喉癌细胞EGFL7蛋白表达阴性。EGFL7siRNA转导后喉癌胞体外侵袭能力下降。EGFL7基因沉默组细胞克隆集落形成率与Hep-2组细胞及空载体组细胞比较,细胞克隆集落形成率显著减低。结论:成功构建人EGFL7基因RNAi慢病毒载体,EGFL7基因沉默对喉癌细胞体外侵袭有明显的抑制作用。沉默EGFL7后,Hep-2细胞集落形成能力显著减低,即下调EGFL7基因表达可在一定程度上抑制喉癌细胞的锚着不依赖性增殖能力。
OBJECTIVE: To construct RNA interference (RNAi) recombinant lentivirus of epidermal growth factor domain 7 (EGFL7) gene and observe its inhibitory effect on invasion of laryngeal carcinoma in vitro. Methods: The targeting RNAi of EGFL7 gene was screened. Oligo DNA of target sequence was synthesized and ligated with pLV vector containing H1 promoter and green fluorescent protein (GFP) to generate LV-sh EGFL7 lentiviral vector. The positive clones were screened by PCR and sequenced Identification. The recombinant lentivirus was used to transduce laryngeal carcinoma cells in vitro, and the expression of EGFL7 protein in laryngeal carcinoma cells was detected by Western blot. Boyden invasion chamber method was used to observe the invasion of laryngeal carcinoma cells transduced with shRNA. The colony formation assay was used to detect the effect of EGFL7 gene silencing on the colony formation ability of Hep-2 cells. Results: EGF-7 lentiviral vector LV-sh EGFL7 was successfully constructed and lentivirus was packaged. The titer of concentrated virus suspension was 5 × 108 TU / L. EGFL7 protein was negative in laryngeal cancer cells transduced with siRNA. EGFL7siRNA transduction laryngeal carcinoma cell invasion in vitro decreased. Compared with Hep-2 cells and empty vector cells, the colony formation rate of cells in EGFL7 silencing group was significantly decreased. Conclusion: The RNAi lentiviral vector containing human EGFL7 gene was successfully constructed and the silencing of EGFL7 gene significantly inhibited the invasion of laryngeal carcinoma cells in vitro. After silencing EGFL7, the colony formation ability of Hep-2 cells was significantly reduced. Down-regulation of EGFL7 gene expression could inhibit the anchorage-independent proliferation of laryngeal carcinoma cells to a certain extent.