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目的应用RNA干扰技术抑制大细胞间变性淋巴瘤细胞系(Karpas299)中NPMALK融合基因表达,观察其对肿瘤细胞生长的影响。方法针对NPMALK融合位点设计两个siRNA序列siRNAI与siRNAII,经PCR反应构建含U6启动子siRNA正义和反义线性表达载体,通过脂质体转染Karpas299细胞,应用实时荧光定量RTPCR、Westernblot检测siRNA片段对NPMALKmRNA和蛋白表达的抑制作用,MTT、Hoechst荧光染色检测siRNA对肿瘤细胞生长的影响。结果siRNAI可导致NPMALKmRNA下降约75%(P<0.05),转染72h后可导致蛋白表达下降;转染siRNAII细胞NPMALKmRNA下降为35%(P<0.05),但蛋白水平无明显改变。转染siRNAI的细胞可抑制Karpas299细胞的增殖和诱导凋亡发生,siRNAII则无明显的抑制增殖和诱导凋亡作用。结论含有针对NPMALK融合位点特异siRNA序列的U6表达载体,可特异地抑制NPMALK基因mRNA和蛋白的表达,并能抑制大细胞间变性淋巴瘤肿瘤细胞株Karpas299细胞的增殖,导致肿瘤细胞凋亡增加,提示NPMALK融合基因的异常表达与大细胞间变性淋巴瘤形成密切相关,为研究NPMALK基因功能和大细胞间变性淋巴瘤基因靶向治疗提供了新策略。
Objective To inhibit the expression of NPMALK fusion gene in large cell lymphoma cell line (Karpas299) by RNA interference and observe its effect on tumor cell growth. Methods Two siRNA sequences, siRNAI and siRNAII, were designed according to the NPMALK fusion site. The sense and antisense siRNA expression vector containing U6 promoter was constructed by PCR and transfected into Karpas299 cells by lipofectamine 2000. Real-time fluorescent quantitative PCR and Western blot were used to detect siRNA Fragment of NPMALK mRNA and protein expression inhibition, MTT, Hoechst fluorescent staining to detect the impact of siRNA on tumor cell growth. Results siRNAI could reduce the NPMALK mRNA by about 75% (P <0.05), and decrease the expression of NPMALK mRNA by 72 hours after transfection. The NPMALK mRNA was decreased by 35% (P <0.05) in transfected siRNA II cells, but the protein level did not change significantly. The cells transfected with siRNAI could inhibit the proliferation and induce the apoptosis of Karpas299 cells. SiRNAII did not significantly inhibit the proliferation and induce the apoptosis. Conclusion The U6 expression vector containing NPMALK fusion site-specific siRNA sequence can specifically inhibit the expression of NPMALK gene mRNA and protein, and can inhibit the proliferation of large cell-cell-transplanted lymphoma tumor cell line Karpas299 cells and lead to the increase of tumor cell apoptosis , Suggesting that the abnormal expression of NPMALK fusion gene is closely related to the formation of large cell lymphoma and provides a new strategy for the study of NPMALK gene function and gene targeting therapy of large cell lymphoma.