Silencing Of PLK1 Gene Inhibits Proliferation And Invasion Of Lymphoma Raji Cells

[Objective] Polo-like kinase 1(hereinafter referred to as PLK1) is a serine/threonine protein kinase which plays key roles in cell cycle regulation, maintenance of genome stability, repair of damaged DNA. Some studies have shown that PLK1 is overexpressed in most human tumors, and in our previous experiments, we found up-regulated expression of PLK1 in lymphoblastoid cells transformed by EBV. By virtue of in vitro cell culture experiment and animal experiment, this study is to observe the effects of silencing of PLK1 by RNA interference on biological behaviors such as proliferation, migration, invasion, cell cycle and apoptosis of lymphoma Raji cells and the formation of xenograft tumor in nude mice, to explore the role of PLK1 in the pathogenesis of lymphoma and to provide some experimental evidence for the study on the molecular mechanism and target therapy of lymphoma.[Methods] Through the design of sh RNA sequences targeting PLK1 genes, construction of RNA interference plasmids(p Len R-GPH-h PLK1-sh), recombination of lentivirus, stable transfection of Raji cell lines, the stable cell lines of Raji with down-regulation of PLK1 expression were established. The green fluorescent images in cells were observed by an inverted fluorescence microscope. By means of Western Blot and q RT-PCR, the expression level of PLK1 in these cells was detected; and by means of CCK-8 assay, flow cytometry, transwell migration and invasion assays, assay of formation of xenograft tumor in nude mice, the effects of silencing of PLK1 by RNA interference on biological behaviors were observed.[Results] 1. The construction of stable Raji cell lines with downregulation of PLK1 expression was successful.The results of enzyme digestion and electrophoresis of recombinant plasmid were in agreement with the expected ones. On the basis of comparison of the sequencing results and designed interference sequence for the target gene by using BLAST, it was shown that the coincidence rate between the sequence of interfering plasmid p Len R-GPH-h PLK1-sh and the designed interference sequence was 100%. This proved that the construction of the interfering plasmid p Len R-GPH-h PLK1-sh was successful. GPH-positive cells were evaluated by fluorescence microscopy. The number of cells transfected with green fluorescent reached 80%. Compared with cells transfected with p Len R-GPH(vector group, Raji/GPH) and ordinary Raji cells(control group), the level of m RNA and protein expressions of PLK1 in cells transfected with p Len R-GPHh PLK1-sh(interfered group, Raji/PLK1 sh RNA) was obviously lower. This proved that the construction of Raji cell lines with down-regulation of PLK1 expression was successful. And the interfered group PLK1-SH1 showed the best interference effect, in which the decrease of m RNA expression of PLK1 was greatest, so the group was selected for follow-up experiment.2. The interference on PLK1 expression could inhibit the proliferation, migration and invasion of Raji cells and the formation of xenograft tumor in nude mice, and induce cell apoptosis and arrest in G2 of Raji cells.The results of CCK-8 showed that the interfered group’s cell growth decreased more significantly when compared with the vector group and the control group, and the difference between the interfered group and the later two groups was a statistically significant one(P<0.01). This proved that the interference on PLK1 expression could inhibit the proliferation of Raji cells.The results of migration and invasion experiments showed that there were fewer migrating cells in the interfered group than those in the vector group and the control group, and the difference between the interfered group and the later two groups was a statistically significant difference(P<0.05); there were significantly fewer cells penetrating Matrigel in the interfered group than those in the vector group and the control group, and the difference between the interfered group and the later two groups was a statistically significant one(P<0.01). This proved that the interference on PLK1 expression could inhibit the migration and invasion of lymphoma Raji cells.Compared to the vector group and the control group, the results of flow cytometry showed that the percentage of G2 cells in the interfered group increased significantly while G1 cells decreased(P<0.01), and the apoptosis rate of cells also increased significantly(P<0.01). And both the differences of percentage and apoptosis rate of cells between the interfered group and the two groups were statistically significant. It suggested that the interference on PLK1 expression could lead to cell apoptosis and cell arrest in G2.The results of xenograft tumor growth assay showed that there were 80% immunodeficient mice with xenograft tumor in the vector group and the control group, while 0% in the interfered group. Compared with the two groups, the tumor formation rate in the interfered group decreased significantly. It suggested that the interference on PLK1 expression in Raji cells could inhibit xenograft tumor growth.[Conclusions] The interference on PLK1 expression can inhibit the proliferation, migration and invasion of Raji cells, xenograft tumor growth, and induce cell apoptosis and cycle arrest.