Myosin Va RNAi慢病毒载体的构建及对肺癌PG细胞运动和迁移能力的影响

目的 构建人非常规肌球蛋白myosin Va基因RNAi慢病毒载体，并探讨其对人肺巨细胞癌PG细胞运动和迁移能力的影响。 方法 针对已经筛选确定的myosin Va基因RNAi有效靶序列，合成靶序列及其阴性对照的寡聚DNA，并连接到pSuper载体获得中间实验质粒pSuper-shM5A及阴性对照pSuper-shCON；然后将H1 promoter-shM5A/shCON表达框重组到慢病毒载体plenti4上，分别得到plenti4-H1-shM5A和shCON 载体，并进行慢病毒包装。随后用病毒上清感染PG细胞，并筛选出zeocin抗性的稳定细胞系PG-shM5A和shCON。通过RT-PCR方法检测PG-shM5A和shCON细胞的myosin Va mRNA表达水平；用创伤愈合和Boyden小室实验测定PG-shM5A和shCON细胞的运动和迁移能力。 结果 限制性内切酶酶切和测序证实，成功构建myosin Va RNAi慢病毒载体plenti4-H1-shM5A。慢病毒包装成功后，感染PG细胞并获得zeocin抗性的细胞；用RT-PCR方法证明，myosin Va mRNA被抑制70%以上。创伤愈合和Boyden小室实验表明，感染携带myosin Va RNAi慢病毒的PG细胞运动和迁移能力显著下调。 结论 成功构建myosin Va RNAi慢病毒载体plenti4-H1-shM5A，它能有效抑制人肺巨细胞癌PG细胞的myosin Va表达并降低其运动和迁移能力，初步揭示了myosin Va与肿瘤细胞恶性行为的相关性。

Construction of a lentivirus vector for RNA interference of myosin Va and its effect on motility and migration of PG cells

Objective To construct a lentivirus vector for RNA interference targeting myosin Va gene and to observe its effect on motility and migration of human pulmonary giant cell carcinoma PG cells. Methods Based on the efficient target sequence for myosin Va RNAi, two pairs of oligo DNA containing myosin Va RNAi target sequence or scramble sequence were synthesized and inserted into pSuper vector, followed by sequence analysis. The expressing cassette H1 promoter-shM5A/shCON from the recombinant pSuper plasmid was then transferred to the lentivirus vector plenti4, and the recombinant lentivirus was packaged. PG cells were transduced with the packaged lentivirus and the positive cells were screened by zeocin selection. RT-PCR was performed to determine the myosin Va RNAi efficiency in zeocin-resistant PG cells, and wounding assay and Boyden chamber assay were utilized to examine the capabilities of motility and migration in myosin Va RNAi PG cells. Results Restriction enzyme digestion and sequencing confirmed the successful construction of the lentivirus vector containing myosin Va RNAi target or scramble sequence. RT-PCR result showed that myosin Va mRNA levels were remarkably reduced in lentivirus-based myosin Va RNAi PG cells. The abilities of motility and migration were also significantly inhibited in lentivirus-based myosin Va RNAi PG cells, as demonstrated in wounding assay and Boyden chamber assay.Conclusion Myosin Va RNAi lentivirus vector was successfully constructed and efficiently repressed myosin Va expression in PG cells. Repression of myosin Va by RNAi led to the inhibition of PG cells motility and migration, indicating that there might exist correlation between the expression of myosin Va and cancer progression.