The Polymorphism Of PLK1 Gene In Lung Cancer And PLK1 SiRNA Interfering On A549 Cells

Lung cancer is now one of the most common malignant cancer in the world, and is the leading cause of all malignant diseases in China. Besides the traditional treatment, such as operation, radiotherapy and chemotherapeutics, molecular targeting therapy of lung cancer has been developed rapidly in recent years, but it is still the second-line treatment to lung cancer. With the development of molecular biology, more and more attentions have been paid to the gene therapy, especially the small interfering RNA (siRNA) technique.PLK1 is one of the serine/threonine kinases. It is an important regulator of many cell-cycle-related events, such as chromosome segregation, centrosome maturation and so on. Many extensive genetic and biochemical studies have revealed that PLK1 was positively correlated with the formation and development of certain cancers, including lung cancer, so the inhibiting of PLK1 gene expression by siRNA interference technique may be a new method to treat lung cancer. Many researchers have used the Lipofectamine 2000-based small interfering RNA technique to specifically down-regulate Plk1 gene in cancer cells, and then successfully inhibited cell proliferation, decreased viability, and induced apoptosis in cancer cells. So it is probably that we can successfully use siRNA targeting PLK1 to inhibit A549 Cells.To ensure the specific inhibiting effect of siRNA, it is very important to choose a target gene sequence. Single nucleotide mutations (SNP) and other mutations may affect the specific binding of siRNA to target gene. Some researches found that there were some SNP presenting in PLK3 gene, so we can not completely rule out some similar mutation presenting in PLK1 gene. So it is necessary to investigate the polymorphism of PLK1 gene before designing. And we need to figure out the best time and the best concentration in the process of transfection, because of that the efficiency of siRNA technique variates depending on the detecting time and the transfected siRNA concentration.We investigated the polymorphism of the open reading frame (ORF) of PLK1 gene in A549 cells (human lung adenocarcinoma), SK-MES-1 (human lung squamous cell carcinoma), NCI-H446 (human small cell lung cancer), BEAS-2B (human bronchic epithelial cells) and lung cancer tissues, then designed a siRNA targeting a stable conserved sequence of PLK1 gene, and transfected it into A549 cells with Lipofectsamine2000. Before we investigated the effect of siRNA on PLK1 gene expression and A549, we determined a best time of detection and a suitable concentration of transfection at first.Part one:Find out the possible polymorphism of ORF of PLK1 gene in small cell lung cancer cell line, non-small cell lung cancer cell line, human bronchic epithelial cell line, and 30 lung cancer tissues.Part two:Design a siRNA targeting PLK1 mRNA, evading the unstable region of PLK1, transfect it into A549 cells with Lipofectsamine 2000, and choose the best time and concentration at first. A549 cells are treated with siRNA, Lipofectamine 2000, missense nucleotide sequence, and GAPDH siRNA as the control groups. Use Real-Time PCR and Western-blot to investigate the the effect of siRNA on plkl gene expression, then use methyl thiazolyl tetrazolium(MTT) method and Flow cytometry analysis(FCA) to investigate the effect of siRNA-mediated PLK1 gene silencing on proliferation and apoptosis of human adenocarcinoma cell line A549.Statistical analysis method:One-way ANOVA was adopted for comparison between two groups, and one sample t-test was adopted for comparison between a single sample and a cluster sample. All data were analyzed by SPSS 11.0.The ORF of PLK1 gene of A549,NCI-H446,SK-MES-1 and BEAS-2B has no SNP site and other mutation, but the expression levels of PLK1 gene of the first three lung cancer cell lines are significantly higher than BEAS-2B. So we inferred that the high expression of PLK1 in lung cancer cells is not resulted from the plkl gene mutation, so it implied that the expression of PLK1 gene may be regulated on the levels of transcription or post-transcription. A SNP in PLK1 gene was found in one tissue of 30 lung cancer tissues, but the relationship between the SNP and the lung cancer is still unclear. And we found that this SNP is a heterozygous mutation, so it is probably located on the recessive alleles.We designed a siRNA targeting 606-627 site of PLK1 gene fragment, then used MTT method to compare the cell viability after transfected on different concentrations and at different times. As a result, we found that 120nM of siRNA is the best concentration, and 72h after the transfection is the best time to detect the efficiency and accuracy of transfection.The results of Real-Time PCR and Western-blot suggest that the siRNA which we designed to target PLK1 can effectively decrease the expression of PLK1 gene in A549 cells (P＜0.05).The inhibition ratio of PLK1 protein expression (62.0%) is lower than the inhibition of PLK1 mRNA (95.93%). The results of MTT and FCA methods suggest that the down-regulation of PLK1 gene mediated by siRNA can significantly inhibit the proliferation of A549 (inhibition ratio is 70.72%), and A549 death was also accelerated (death ratio is 14.94%). 1 Three lung cancer cells has no SNP site and other mutation, but the expression of PLK1 gene of them are significantly higher than BEAS-2B; 2 A SNP in PLK1 gene was found in one tissue of 30 lung cancer tissues, so PLK1 gene is conservative approximately; This SNP locates on 1114 site. It's a heterozygous mutation, and it dosen't affect the expression of PLK1 gene; 3 The depletion of PLK1 mediated by siRNA which we design can significantly decrease the expression of PLK1 gene, inhibit the proliferation of A549, and accelerate its death. It implies that PLK1 siRNA can be possibly used as a method to treat lung cancer.