Tao Shaolin,Tan Qunyou,Deng Bo,Kang Boming,XuYi.Construction of siRNA vector for inhibiting expression of human positive co-activator 4[J].J Amry Med Univ (J Third Mil Med Univ),2011,33(18):1912-1916.
Tao Shaolin,Tan Qunyou,Deng Bo,Kang Boming,XuYi.Construction of siRNA vector for inhibiting expression of human positive co-activator 4[J].J Amry Med Univ (J Third Mil Med Univ),2011,33(18):1912-1916.DOI:
Construction of siRNA vector for inhibiting expression of human positive co-activator 4
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.