抑制人PC4基因表达的siRNA载体的构建与筛选

Construction of siRNA vector for inhibiting expression of human positive co-activator 4

目的构建和筛选能高效、特异性抑制人转录辅助调控因子4(positive coactivator 4,PC4)基因表达的siRNA载体。方法设计并合成人PC4基因特异性的4对siRNA干扰靶点,分别克隆入pSES-HUS腺病毒穿梭质粒,构建重组pSES-HUS-PCi1,pSES-HUS-PCi2,pSES-HUS-PCi3和pSES-HUS-PCi4质粒。使用脂质体包裹,瞬时转染293T细胞,qRT-PCR和Western blot检测其对PC4 mRNA和蛋白表达的影响,筛选最佳的重组质粒及干扰靶点,同时探讨其对上皮来源的293T细胞增殖和迁徙的影响。结果构建的4对重组质粒载体经酶切鉴定和基因测序分析,其基因大小、序列与预期相符。将4对重组质粒及pSES-HUS空质粒瞬时转染293T细胞,发现pSES-HUS-PCi1重组质粒组较pSES-HUS空质粒组和正常293T细胞组中PC4 mRNA和蛋白的表达低,且差异具有统计学意义(P<0.01)。可见pSES-HUS-PCi1重组质粒的PC4靶点序列(正义,5'-AACAGAGCAGCAGCAGCAGATTTT-3’;反义,5'-ATCTGCTGCTGCTGCTCTGTTTT-3')能抑制PC4mRNA的表达和蛋白的合成,其抑制率分别是85.30%和80.57%。干扰PC4表达后293T细胞的体外增殖和迁徙能力下降,且以pSES-HUS-PCi1重组质粒组最为明显(P<0.05)。结论成功构建并筛选出高效、特异性抑制PC4表达的siRNA载体,并发现PC4能影响上皮来源293T细胞的增殖和迁徙能力。

Objective To construct the specific siRNA vector with a high performance for inhibiting the expression of human positive co-activator 4（PC4）.Methods Four pairs of PC4 specific siRNA interfere targets were designed and synthesized.They were then cloned into the PSES-HUS adenovirus shuttle plasmid to construct recombinant pSES-HUS-PCi1,pSES-HUS-PCi2,pSES-HUS-PCi3,and pSES-HUS-PCi4 plasmids which were packaged with lipid bodies and transiently transfected into 293T cells.Effect of the 4 plasmids on expression of PC4 mRNA and protein was detected by real time quantitative PCR（qRT-PCR）and Western blotting,respectively.Optimal recombinant plasmids and interfere targets were screened,and their effect on proliferation and migration of 293T cells was studied by cell scratch test.Results The 4 constructed pairs of recombinant plasmid vectors were identified by enzyme digestion and analyzed by gene sequencing.Their size and sequences were consistent as expected.The 4 constructed pairs of recombinant plasmids and pSES-HUS-PCi1 empty plasmids were used to transfect 293T cells.The expression level of PC4 mRNA and protein was lower in recombinant pSES-HUS-PCi1 plasmids than in pSES-HUS-PCi1 empty plasmids and normal 293T cells（P0.01）,indicating that the PC4 target sequences of recombinant pSES-HUS-PCi1 plasmids（sense： 5′-AACAGAGCAGCAGCAGCAGATTTT-3′;antisense： 5′-ATCTGCTGCTGCTGCTCTGTTTT-3′）can inhibit the expression of PC4 mRNA and synthesis of proteins（P0.01）,with an inhibitory rate of 85.30% at mRNA level and 80.57% at protein level,respectively.The in vitro proliferation and migration of 293T cells were decreased,especially in recombinant pSES-HUS-PCi1 plasmids,when the expression of PC4 was interfered（P0.05）.Conclusion The siRNA vector for specific inhibition of PC4 expression with a high performance has been successfully constructed and PC4 can influence the proliferation and migration of 293T cells in epithelium.