| The tumor is one kind disease of cell cycle regulation disorder, its basic reason lies in the expression of cell positive factor is strengthen or the expression of cell negative factor is weaken, causes the cell over growth and fission, presents the cell growth to lose control. The ubiquitin-proteasome pathway through the degeneration of cell cycle regulators involved in cell cycle regulation, is playing an important role in adjusting cell cycle regulators to balance, closely related to occurrence and development of tumor.S-phase kinase-associated protein 2 (Skp2) belongs to the ubiquitin-proteasome body system, it can recruite a variety of specific substrate proteins to the ubiquitination complex for ubiquitination and degradation involved in cell cycle regulation. Skp2, a kind of the oncogenes, correlates to cell cycle regulation closely by regulating the expression of p27kip1. p27kip1 is one of the members in CDK-interacting protein/kinase inhibition protein (Cip/Kip) family, inducing G1 cell cycle arrest. Skp2 and p27kip1 affect the proceeding and prognosis of leukemia by regulating the proliferation, apoptosis and differentiation of leukemia cells.RNA interference (RNAi) is the most effective gene silencing technology, which can specifically inhibit the transcription of target genes, and in turn reduce the expression level and function of the corresponding protein. Therefore, using RNAi technology, we choose Skp2 as the target gene, Here we constructed three recombinant plasmids expressing Skp2 short hairpin RNA (shRNA), which were transiently transfected into K562 cells mediated by lipofectamine reagent. Use RNA interference technology to down-regulation the expression of Skp2, observe change in expression of p27kip1, and investigate whether Skp2 down-regulation can effect the apoptosis, proliferation of K562 cells. The experiments contained two parts as below:Part 1: construction and screening of specific recombinant plasmids targeting Skp2Objective: To construct recombinant plasmids expressing Skp2 shRNA by pGenesil1.1 plasmid targeting Skp2, and to screen the highly efficient shRNA.Methods: Using online design software, three pairs of DNA sequences containing short hairpin structure and one pair of unspecific control sequence were designed and synthesized respectively. The complement forms were obtained by annealing and inserted into plasmid pGenesil1.1 with U6 promoter and enhanced green fluorecence protein (EGFP), and the recombinant plasmids were transformed into DH5α. Finally the plasmids identified by restriction enzyme used for sequence analysis. Four pairs of recombinant plasmids were transfected into K562 cells, and the expression of Skp2 mRNA and protein levels were determined by RT-PCR and flow cytometry respectively. The cell transfection efficiency was analyzed by fluorescent microscope and flow cytometry. The cells were divided into 7 groups:①blank control group;②transfection reagent group of lipofectamineTM 2000;③plasmid KB group;④pGenesil1.1-NC shRNA group;⑤pGenesil1.1-Skp2 shRNA1 group;⑥pGenesil1.1-Skp2 shRNA2 group;⑦pGenesil1.1-Skp2 shRNA3 group.Results:①Sequencing analysis showed that three recombinant pGenesil1.1-Skp2 shRNA plasmids and negative control pGenesil1.1-NC shRNA plasmid had been successfully constructed;②The transfection efficiency of Skp2 shRNA plasmids in K562 cells was about 40% at 48 hours;③Skp2 mRNA was down-regulated by Skp2 shRNA: Based on the experiment results, we select one time point to test the expression level of Skp2. The time point was 48h after transfection, which had the highest rate of inhibition. RT-PCR showed that there was no significant difference among CON group, NC group, LIP group and KB group. Skp2 mRNA in shRNA1 group, shRNA2 group and shRNA3 group was reduced by 40.00%, 76.36% and 56.36%, respectively.④Skp2 protein was down-regulated by Skp2 shRNA: Flow cytometry analysis showed that there was no significant difference among CON group, NC group, LIP group and KB group. Skp2 protein was significantly reduced by Skp2 shRNA, and the level of Skp2 protein expression was down-regulated by 36.77%, 67.25% and 45.30%, respectively. Therefore, we select pGenesil1.1 -Skp2 shRNA2 to carry on the following experiment.Conclusions: The recombinant plasmids targeting Skp2 can be successfully constructed, and can be successfully transfected into K562 cells, of which pGenesil1.1-Skp2 shRNA2 was more efficient.Part 2: The effects of Skp2 shRNA transfection on apoptosis and proliferation of K562 cellsObjective: Skp2 shRNA plasmid was transfected into K562 cells in order to observe the effects of Skp2 gene silencing on apoptosis and proliferation.Methods: Using in vitro cell culture technique, we detected the expression of Skp2 and p27kip1 in K562 cells using RT-PCR and flow cytomete, inorder to observe the change of Skp2 and p27kip1 expression after the Skp2 shRNA was transfeced. The growth status of K562 cells after the Skp2 shRNA trsanfected was detected by microscop. The inhibition ration of K562 cells was examined by methyl-thiazolyl- tetrazolium (MTT). Propidium iodide (PI) staining was used to evaluated the difference of cell cycle distribution and apoptosis rate in these different treatment groups.Results:①Skp2 shRNA inhibit Skp2 mRNA expression: RT-PCR results showed that Skp2 mRNA could be significantly reduced by Skp2 shRNA. The reduction appeared from 12h after interference, and the maximum reduction rate was 76.36%, which appeared at 48h. However, transient transfection of Skp2 shRNA could only present for a limited period, and the Skp2 mRNA had gone up at 72h after transfection;②Skp2 shRNA inhibited expression of Skp2 protein: Flow cytometry results showed that Skp2 protein expression could be significantly reduced by Skp2 shRNA, but the downward strend fell behind mRNA. The highest down–regulated rate was 67.25% at 48h, and the protein expression began to rebounded at 72h after transfection;③Skp2 shRNA influenced p27kip1 expression: there was no significant difference among CON group, LIP group, KB group and NC group. Flow cytometry analysis showed that p27kip1 protein expression of shRNA group was higher than NC group (591.03±2.60 vs 358.56±10.41), the difference was significant, the highest up-regulated rate was 64.83%, which appeared at 48h, and the protein expression began to back down at 72h after interference, the relationship between p27kip1 protein expression and Skp2 protein expression was a negative correlation (r=-0.969, p=0.000); but the expression of p27kip1 mRNA remained the same in the different treatment groups. This showed that p27kip1 expression was regulated in the level of post-transcriptional;④Skp2 shRNA affect cell cycle distribution of K562 cells: Based on the above results, we select one time point to test cell cycle distribution, cell inhibition rate and apoptosis rate. The time point was 48h after transfection, which had the highest rate of inhibition. The cell cycle distribution was basically the same among the CON group, LIP group, KB group and NC group. comparing to the NC group, the cell proportion of G0/G1 phase of Skp2 shRNA group significantly increased (64.57±2.36% vs 34.58±0.75%), the proportion of S phase cells and G2/M phase cells of Skp2 shRNA group significantly decreased (29.78±4.92% vs 47.65±1.85%, 5.65±3.03% vs 17.40±1.01%), the difference was significant. These showed that Skp2 shRNA prevented cells from G1 phase to enter S phase;⑤Skp2 shRNA inhibited K562 cells proliferation: Compared with the NC group, the K562 cell inhibition rate increased dramatically in Skp2 shRNA group (85.43±2.83% vs 1.23±6.51%), but there was no significant difference among the CON group, LIP group, KB group and NC group;⑥Skp2 shRNA promote apoptosis of K562 cells: The apoptosis rate of Skp2 shRNA group was higher than NC group (12.46±4.35% vs 0.34±0.08%), p=0.000, but there were no significant difference among the CON group, LIP group, KB group and NC group, and shRNA group appeared a hypodiploid peak before G0/G1 peak - apoptotic peak.Conclusions: After transient transfection of shRNA plasmid targeting Skp2 mediated by lipofectamine reagent, Skp2 expression was reduced both in the level of transcription and translation, the expression of Skp2 down-regulation could inhibited the ubiquitination and degradation of p27kip1, further more, a significant negative correlation was confirmed between the expression of Skp2 protein and the expression of p27kip1 protein. In the meantime, we could find that cell cycle of K562 cells arrested in the G1 phase , the growth status of K562 cells was significantly inhibited, and K562 cells were induced apoptosis. |
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