摘要
目的：研究RNA干扰(RNA interference,RNAi)技术抑制透明质酸受体layilin表达,对透明质酸(hyaluronan,HA)诱导人肺腺癌A549细胞体外增殖的影响。方法：构建能表达针对layilin的短发夹RNA(shRNA)的阳性RNA干扰(posi- tive RNAi,Pi)质粒和表达不针对任何已知mRNA的shRNA的阴性RNAi(negative RNAi,Ni)质粒,分别转染至A549细胞,以RT-PCR和Western blot检测A549细胞转染前后layilin表达,并用新霉素抗性筛选得到layilin表达受抑制的阳性RNAi(Pi)细胞和layilin表达未受影响的阴性RNAi(Ni)细胞。分别在存在或不存在外源性透明质酸的培养条件下,以MTT实验和软琼脂细胞集落形成实验比较正常(control,C)、Pi、Ni组细胞体外增殖能力。结果：在不存在外源性透明质酸的培养条件下,C、Pi、Ni组A549细胞体外增殖能力无显著差异(P＞0．05),在存在外源性透明质酸的培养条件下,Pi和C组相比,体外增殖能力显著降低(P＜0．01),Ni和C组相比,体外增殖能力无显著差异(P＞0．05)。结论：抑制layilin表达能抑制透明质酸诱导人肺腺癌A549细胞体外增殖。
Objective：To inhibit the expression of hyaluronic acid receptor Layilin by RNA interference （RNAi） and investigate its influence on proliferation of human lung carcinoma A549 cells induced by hyaluronic acid in vitro. Methods： RNAi-positire plasmid which expressed short hairpin RNA （shRNA） against layilin and RNAi-negative plasmid which expressed shRNA that does not match any known human coding mRNA was designed, constructed, and transfected into A549 cells. Layilin expression was examined by reverse transcription polymerase chain reaction （RT-PCR） and western blot. RNAi-positive （Pi） and RNAi-negative （Ni） clones were isolated by neomycin selection. The cell proliferation in control, Pi, and Ni group was examined by MTT and soft agar colony formation assay with or without exogenous hyaluronic acid. Results： In the absence of exogenous hyaluronic acid in culture medium, the difference was not significant in proliferation ability between C, Pi, and Ni group（P〈 0.05）. In the presence of exogenous hyaluronic acid in culture medium, the proliferation ability of Pi group was significantly de- creased compared with C group （P〈0.01）. The difference was not significant between Ni group and C group （P〈0.05）. Conclusions： Inhibition of Layilin expression suppressed the proliferation of human lung carcinoma A549 cells induced by hyaluronic acid in vitro.
出处
《肿瘤》
CAS
CSCD
北大核心
2006年第11期990-993,共4页
Tumor
基金
国家自然科学基金(编号:30371586)
第三军医大学校管基金(编号:XG200361)
