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目的:构建针对NOB1基因的shRNA慢病毒载体,并在卵巢癌SKOV3细胞上鉴定其沉默效率,观察其对细胞增殖能力的影响。方法:将筛选的NOB1基因特异性siRNA靶点,合成短发卡结构shRNA,并与pLVTHM慢病毒载体重组形成shRNA表达载体,包装shRNA慢病毒颗粒,随后将其感染卵巢癌SKOV3细胞;采用Real-timePCR和Westernblot的方法检测靶基因在mRNA和蛋白水平的沉默效率,MTT和克隆形成实验观察NOB1基因沉默对SKOV3细胞增殖的作用。结果:构建的shRNA慢病毒颗粒感染SKOV3细胞后,NOB1基因的mRNA表达量较阴性对照载体慢病毒感染组下降73.5%,蛋白表达显著抑制,同时MTT实验显示细胞增殖能力显著降低,克隆形成实验表明细胞克隆形成能力明显减弱。结论:成功构建的NOB1基因shRNA慢病毒表达载体能够在卵巢癌SKOV3细胞上有效沉默靶基因,验证了NOB1基因具有影响卵巢癌细胞恶性增殖的能力。
OBJECTIVE: To construct shRNA lentivirus vector targeting NOB1 gene and identify its silencing efficiency on SKOV3 ovarian cancer cells and observe its effect on cell proliferation. Methods: The specific siRNA target of NOB1 gene was screened, short hairpin shRNA was synthesized and recombined with pLVTHM lentiviral vector to form shRNA expression vector. The shRNA lentivirus particles were packaged and then infected into ovarian cancer SKOV3 cells. Real-time PCR And Western blot were used to detect the silencing efficiency of target gene at mRNA and protein level. The effect of NOB1 gene silencing on the proliferation of SKOV3 cells was observed by MTT and clonogenic assay. Results: The expression of NOB1 mRNA in SKOV3 cells was decreased by 73.5% compared with that of the negative control lentiviral vector, and the protein expression was significantly inhibited. The results of MTT assay showed that the proliferation of SKOV3 cells was significantly decreased Cell cloning ability was significantly weakened. Conclusion: The constructed NOB1 shRNA lentiviral vector can effectively silence the target gene in ovarian cancer SKOV3 cells, and the NOB1 gene has the ability to affect the malignant proliferation of ovarian cancer cells.