Rational SiRNA Targeting A549 Cell PLK1 MRNA And Inhibiting Gene Expression

Backgrounds and Objective:Lung cancer ranks the most common malignant disease in male patients, it takes about 16% of all malignant disease, and 28% of all death of malignancy. It' s the first cause of cancer in most countries in the world.In recent years, molecular targeting therapy of lung cancer has been developed rapidly, such as IRESSA and TARCEVA. They can selectively combine to EGFR and inhibit protein tyrosine kinase(PTK) activities, thus inhibit cancer growth. Compared with traditional drugs, they are more effective and have lower toxicity. But in the phase II clinical trail, the efficacy ratio was only 11.8%-18. 4% and 12. 5%. Therefore more researches are badly needed.Polo and Polo-like kinases (Plks) are a family of conservedregulators of multiple events during cell division. The founding member of this family, Polo, was originally identified in the fruit fly(Drosophila melanogaster) and shown to be a serine - threonine kinase that is required for mitosis. Protein kinases play a pivotal role in execution of cell division. Polo and Polo-like kinases have emerged as major regulators for various cell cycle checkpoints. Plks localize primarily to the centrosome during interphase and the mitotic apparatus during mitosis. Many key cell cycle regulators such as p53, Cdc25C,cyclin B, components of the anaphase-promoting complex(APC), and mitotic motor proteins are directly targeted by Plks. Plks are important mediators for various cell cycle checkpoints that monitor centrosome duplication, DNA replication, formation of bipolar mitotic spindle, segregation of chromosomes, and mitotic exit, thus protecting cells against genetic instability during cell division. Many researches have proven that when PLK1 gene expression is inhibited, malignant tumors stop growing to apoptosis, while normal cells growth rarely affected. In conclusion, inhibiting PLK1 gene expression may be an important method of malignancy cure. SiRNA(short or small interfering RNA) is believed to be the most important discovery in biotechnological progresses of recent years, and has now been adopted as a standard methodology for silencing the expression of specific genes in mammalian cells. The potential of siRNA based cancer therapy, that is, inhibiting cancer proliferation via targeting and silencing "key genes" of malignancy, is the most exciting and interesting one. Many studies have used siRNAs as an experimental tool to dissect the cellular pathways that lead to inhibit cell proliferation and RNAi has been proposed as a potential treatment for cancer.We aim at studying the knockdown effect of small interfering RNA(siRNA) targeting PLK1 (Polo-like kinase1) mRNA in lung cancercell line A549, and identifying the hyperactive siRNA targeting PLK1 and noval transfection ways for future in vivo RNAi experiments targeting PLK1 mRNA and inhibiting lung cancer growth in animal.Methods:Special siRNA molecules were designed targeting PLK1 mRNA sequence and chemically synthesized, that were transfected into A549 via lipofectamine2000. PLK1 and house keeping GAPDH mRNA levels were assayed by Real-Time PCR 24hours, 48hours and 72hours after transfection, and the expression levels of PLK1 mRNA were quantified based on the GAPDH mRNA levels. The changes of PLK1 and GAPDH protein levels, were checked by Western-blot.Statistical methods:Two-way ANOVA was adopted for comparation between two groups. All data were subjected to stata 8.0 for windows.Results:The siRNA molecules, on concerntrations of 50nmol/L and 25nmol/L, knocked PLK1 mRNA down at different levels, remaining 25-23. 1%, 17. 5-19. 5% and 13. 4%-8. 4%, 24hous, 48hours and 72hours after transfection respectively. 48hours after transfection, Western-blot detected dominantly decreased levels of PLK1 protein, but the GAPDH protein levels remained intact. Nonsense siRNA , as a negative control, didn' t affect PLK1 mRNA levels.Conclusions:1) SiRNA down regulated PLK1 mRNA apparently, but not GAPDH mRNA, suggesting siRNA' s targeting affects.2) SiRNA at 25nmol/L knocked down PLK1 mRNA level by 91.6% 72 hours after transfection, proving it' s hyperactivity and durability; Lipofectamine2000, as a transfection vehicle forA549 cell RNAi, has high efficacy.3) The study got the highest knocking down RNAi efficacy targeting A549 cell PLK1 mRNA, while compared with published literature(83%),which probably is due to the differences of targets selection and transfection vehicles.4) There was no knocking down efficacy difference between 25nmol/L and 50nmol/L groups,which calling for further experiments on RNAi at lower concentrations.5) We observed down regulation of PLK1 mRNA after RNAi,but the PLK1 protein level was undetectable, which probably due to the translation gene silencing(TGS).6) This siRNA can be a candidate, after methylation modifications to increase its serum stability, for in vivo RNAi research against tumour growth of A549 cell bearing nude mouse.