Objective To study the effect of knockdown of signal transducer and activator of transcription 3 (STAT3) expression by short hairpin RNA (shRNA) on proliferation, apoptosis and invasion of human gastric cancer cell line MKN-45 in vitro . Methods Specific shRNA plasmids to STAT3 were constructed, and then transfected into MKN-45 cells by lipofectamine methods. Cells were divided into three groups: control group, psiRNA-H1 transfected group as negative group and psiRNA-H1/STAT3 transfected group. Semi-quantitative RT-PCR and Western blotting were used to detect the expression of STAT3 mRNA and protein, respectively. Proliferation and apoptosis of the transfected cells were observed by methyl thiazolyl tetrazolium (MTT) method and flow cytometry (FCM), respectively. The invasion of the transfected MKN-45 cells was measured by Boyden chamber. Results Compared with the negative control cells, semi-quantitative RT-PCR and Western blotting showed that the expressions of STAT3 mRNA and protein were down-regulated in the psiRNA-H1/STAT3 transfected group ( P ＜ 0.05) . The subcloned recombinant plasmid expressing shRNA effectively inhibited MKN-45 cell growth and proliferation while empty plasmid had no such specific effect. Cell apoptosis rate increased significantly in psiRNA-H1/STAT3 transfected group （ P ＜ 0.01）, and the invasion of MKN-45 cells was efficiently inhabited in psiRNA-H1/STAT3 transfected group as compared with control group and psiRNA-H1 transfected group( P ＜ 0.01).Conclusion Recombinant plasmid psiRNA-H1/STAT3 shRNA significantly inhibits the proliferation and invasion of MKN-45 cells and promotes their apoptosis.
Objective To design, construct and select the optimal repl ication-defective recombinant adenovirus mediated short hairpin RNA (shRNA) which is transduced into human osteosarcoma cells to silence c-myc gene expression, and to construct the recombinant adenovirus vector expressing c-myc-shRNA and determine its viral titer. Methods Three pairs of complementary single-stranded ol igonucleotides (ss ol igos) were designed and synthesized, and then they were annealed to create a double-stranded ol igonucleotide (ds ol igos).The ds ol igos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into osteosarcoma cells by l iposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cells to produce repl ication-incompetent recombinant adenovirus mediated shRNA against c-myc whose cytopathic effect was observed and viral titer was determined by the viral particle (VP) method and 50% tissue culture infective dose (TCID50). Results Ds ol igos, which was verified by electrophoresis, was cloned into pENTR/U6 vector to produce pENTR/U6-shRNA shuttle plasmid, which was confirmed to be corrected by sequencing. The optimal plasmid with good silence effect was chosen by RT-PCR from the three pairs of double-stranded ol igonucleotide. By Pac I enzyme, the l inearrization repl ication-defective recombinant adenovirus mediated shRNA was constructed to perform the LR recombination reaction to the adenovirus backbone plasmid. The cytopathic effect and vacuole phenomenon of adenovirus mediated shRNA appeared at 3 days and became obvious at 6 days. The adenovirus virus titer in the first generation was 5.23 × 109 VP/mL, and reached 2.26 × 1012 VP/mL via 3-4 generations’ ampl ification. The viral titer was 10-3.8/0.1 mL determined by VP method and TCID50. Conclusion The recombinant adenovirus mediated shRNA c-myc is constructed in vitro through RNA interference technology.
Objective To construct vectors that express phosphatidylinositol-3-kinase, catalytic, beta polypeptide （PIK3cb） shRNA in eukaryon plasmid catalyzed by PI3K in rat, then test their effects on intimal hyperplasia in transplanted vein graft. Methods One hundred and fifty SD rats were randomly divided into six groups （n=25, in each group）: blank (25% Pluronic F-127), shRNA-1, shRNA-2, 1/2 （shRNA-1+shRNA-2）, negative control (pGenesil-1 scramble shRNA) and positive control （wortmannin） group. The jugular vein in rats were interpositioned autologously into the common carotid artery. shRNA and 25% Pluronic F-127 were mixed and coated around the transplanted vein in three PIK3cb shRNA groups. Every 5 samples were removed according to the time point (1, 3, 7, 14 and 28 days after operation), respectively. The thickness of intima and neointima area were calculated and analyzed by computer system. The PCNA expression was detected by Western blot and SP immunohistochemistry. Results The intimal thickness of three PIK3cb shRNA groups were lower than those in the blank group and negative control group on day 3, 7, 14, 28 after operation (P＜0.05); The neointima area in three PIK3cb shRNA groups (except shRNA-2 group on day 3, 7) began to decrease significantly from day one (P＜0.05). The protein expression of PCNA in three PIK3cb shRNA groups on day 3 after operation were decreased compared with blank group and negative group (P＜0.05). The percentage of the PCNA positive cells area in three PIK3cb shRNA groups were significantly lower than those in blank group and negative control group in each time point (Plt;0.05). There were no significant differences between blank and negative control group in different time points (Pgt;0.05). Conclusion The PIK3cb shRNA can effectively inhibit the proliferation of vascular smooth muscle cell, which may provide a new gene therapy for the prevention of vein graft restenosis after bypass grafting.