Study Of The Influence Of SiRNA Targeting Plk1 Gene On The Biological Behaviors And Enhancement Of Vincristine Chemotherapeutic Sensitivity To Hepatocellular Carcinoma Cell Line BEL-7402

Operation is the main treatment to deal with primary hepatocellularcarcinoma(PHC)which is one of the most common malignant tumors inChina at present. Chemotherapy and radiotherapy play an important role inthe systemic treatment of PHC. However, the effects of chemotherapy areoverall dissatisfying because PHC is not sensitve to that. Therefore,how todegrade the toxicity of chemotherapeutics and strengthen the curative effectof chemotherapy deserve study and research thoroughly. With thedevelopment of molecular biology technique and gene research, genetictherapy has been applied on the liver cancer, which lead to a new treatmentfor PHC.Plk1(Polo-like kinase 1),a member of the polo-like kinase family whichis the mammalian serine/threonine protein kinase, plays pivotal roles in theregulation of cell cycle progression as a important DNA damage checkpointkinase in G₂/M phase of the mitosis. Plk1 activity is necessary for entry intomitosis, centrosome maturation, bipolar spindle formation and cytokinesis.Plk1contributes to the pathways which positively trigger entry into andprogression through mitosis and accelerate cellular proliferation. In multiplekind of tumors, Overexpression of Plk1 is observed and the level of Plk1expression has prognostic value for predicting outcomes in patients. PHCusually presented with simultaneouly underlying liver disease, eg, liver cirrhosis and chronic hepatitis which cause eontinueous liver damage andsubsequent hepatocyte regeneration result in a tendency for the control geneof cell cycle to mutate, and the intensified hepatocyte regeneration keep theDNA mutation from repair and make it multiplied in the regenerationprocess, and finally leading to the occurrence. Thus, We select Plk1 as thepotential gene therapy target to regulation the function of the DNA damagecheckpoint of G₂/M phase so as to develop a anticancer approach.RNA interference (RNAi) is a new powerful tool to inhibit the targetgene expression by post-transcriptional gene silencing, which mediated bydouble-stranded RNA (dsRNA) destructing the endogenous target mRNAssequence-specificly. The technical application has been utilized widely in theresearch of functional genome and tumor gene therapy. So silencing theexpression Plk1 in hepatocellular cells and strengthen the sensitive ofchemotherapeutics by RNA interference maybe bring a new pathway to treatPHC. Partâ… Expression and clinical significance of Polo-like kinase 1 inprimary hepatocellular carcinomaObjective To investigate the expressions of Polo-like kinase 1 mRNAand protein in primary hepatocellular carcinoma with regard to the clinicalsignificance.Methods Expressions of Pik1 mRNA and protein were detected bysemi-quantitative RT-PCR,Western blot technique in 21 cases ofPHC,7cases of hepatic cirrhosis and 7cases of normal liver tissues ascontrols. The relevance between the expressions of Plk1 mRNA and theclinical pathology of the patients of PHC were studied.Results Semi-quantitative RT-PCR demonstrated that the expressionratio of Plk1 mRNA were 0.572±0.144 in the tissues of PHC, whichsignificantly up-regulated compared with that in hepatic cirrhosis as0.250±0.057 and normal hepatic tissues as 0.218±0.073(P＜0.01). Theexpression of Plk1 mRNA was significantly positive correlated with the sizeof the tumor and the portal vein embolism. Western blot demonstrated thatthe expression of Plk1 protein also were higher than that in hepatic cirrhosisand normal hepatic tissues.Conclusion1. The expressions of Plk1 mRNA and protein were significantlyincreased in PHC tissues.2. The expressions of Plk1 mRNA was in high relevance with the tumorsize and the portal vein embolism of PHC. Partâ…¡Constuction and identificantion of Eukaryotic expressionvector of siRNA targeting Plk1 geneObjective To construct the eukaryotic expression vector of siRNAtargeting Plk1 gene.Methods One pair of short hairpin RNA targeting Plk1 and anegative control nonsense short hairpin RNA were designed and synthesizedand inserted into plasmid pGenesil-2 to generate siRNA eukaryoticexpression vector. Their correct construction were tested by PCR,releasedby Barn H and Hindâ…¢and recombinatant plasmid sequencing.Results The short hairpin RNA motifs of targeting Plk1 gene 251—269 site and 1396—1417 site and the negative control short hairpin RNAmotifs were synthesized and inserted into plasmid pGenesil-2.Therecombinatant siRNA plasmid were successfully constructed by directbacteria solution PCR,double digestion and nucleic acid sequencingidentification.Conclusion Construct the eukaryotic expression vector of siRNAtargeting Plk1 gene successfully Partâ…¢Effect of siRNA targeting Plk1 gene on the biological behaviorof hepatocellular carcinoma cell line BEL-7402Objective To study the influence of shRNA targeting Plk1 on thebiological behavior of hepatocellular carcinoma cell line BEL-7402.Methods The recombinant eukaryotic plasmids were transmittedinto BEL-7402 cells by electroporation. After gained stable expressedclones,the biological behabiors of the Plk1 siRNA transfected cells wereobserved by the experiments such as semi-quantitive RT-PCR,Westernblot,MTr and flow cytometry.Results The stable expressed Plk1 siRNA cell clone were gainedselected by G418 after electroporation transfection into BEL-7402successfully. We identified cell sublines stably transfected byPlk1siRNA-251, Plk1siRNA-1396, Plk1siRNA-hk. Semi-quanfitive RT-PCRdemonstrated that the expression of Plk1 and Cdc25C mRNA decreased andthe expression of p53 mRNA increased in Plk1siRNA-251,Plk1siRNA-1396groups significantly compared with that in Plk1siRNA-hk and controlgroups. Western blot demonstrated that the expression ratio of Plk1 proteinwere 0.032±0.007,0.040±0.008,0.272±0.041,0.278±0.053 in Plk1siRNA-251,Plk1siRNA-1396, Plk1siRNA-hk and control groups separately. Comparedwith other two groups, Plk1siRNA-251 and Plk1siRNA-1396 groups grewmore slowly and had a higher proportion of cells in G₂/M phase whereas lesscells in G₀/G₁ phase.Conclusion:1. Selected a pair of shRNA targeting Plk1 gene, which provide a new meas of the functional and mechanism to research Plk1 gene.2. The expressions of Plk1 mRNA and protein were inhibited which tocause the expression of p53 mRNA to elevate and Cdc25C mRNA todescend in stable expressed Plk1 siRNA cell done.3. The transfection of targeting Plk1 siRNA vector could inhabit theproliferation of BEL-7402 cells and change the cell cycle.. Partâ…£Study of the siRNA targeting Plk1 gene inducing sensitivity ofhepatocellular carcinoma cell line BEL-7402 to chemotherapyObjective To study the influence of chemotherapy sensitivity ofshRNA targeting Plk1 to hepatocellular carcinoma cell line BEL-7402.Methods Using the hepatocellular carcinoma cells constructed inprevious experiment, MTT assay was used to detect the cell growthinhibiting rate of transgene tumor cells affected by vincristine and IC₅₀ werecalculated.Flow cytometry and Hoechst dyeing was used to demonstrate theapoptosis index of different cells.Results Observed by MTT assay to hepatocellular carcinoma cellsaffected by vincristine, the growth inhibiting rate of Plk1siRNA-251 andPlk1siRNA-1396 group cells were higher significantly than that ofPlk1siRNA-hk and control group cells(P＜0.01).The IC₅₀ of Plk1siRNA-251group was 14.541 ug/ml and cut down 35.02%, 31.33% compared withPlk1siRNA-hk group and control group. The IC₅₀ of Plk1siRNA-1396 groupwas 13.413ug/ml and cut down 40.07 %,36.66 % compared withPlk1siRNA-hk group and control group. Afer given 5ug/ml vincristine 24hlater, the apoptosis index of Plk1siRNA-251 and Plk1siRNA-1396 groupwere 19.09%±3.97 %,19.80%±5.47% and increased significantlycompared with Plk1siRNA-hk group and control group(P＜0.01).Conclusion The siRNAs targeting Plk1 can not only suppress theexpression of Plk1 in hepatocellular cell, but can also enhance thechemotherapy sensitivity,which deserve to be studied further.