Effect Of Small Interfering RNA Targeting Polo-like Kinase 1 On Apoptosis In Hepatocellular Cell

Effect of Small Interfering RNA Targeting Polo-like Kinase 1 on Apoptosis in Hepatocellular CellObjectiveTo investigate the expression and significance of Plk1 gene protein in hepatocellular carcinoma (HCC) tissues and the expression of Plk1 gene protein in hepatoma cell lines. Using small interfering RNA (siRNA) targetting Plk1 gene transfect hepatoma cell line BCL-7402 cells to observe the effects of Plk1 gene silence on Plk1 and p53 gene expression and apoptosis in BCL-7402 cells, and explore the feasibility and effect of the therapy targeted against human Plk1 gene in the treatments of hepatoma.MethodsImmunohistochemical technique(SP)was used to detect the expression of the Plk1 gene protein in 67 cases of HCC tissues, 27 cases of corresponding paracancerous cirrhosis tissues and 5cases normal tissues of liver. And the relationship between clinical pathology characters and expression of the Plk1 gene protein were analysed too. Western Blot was used to detect the expression of the Plk1 gene protein in hepatoma cell lines. Two siRNA sequences targeted against human Plkl gene were designed and synthesized, named siRNA1 and siRNA 2 respectively. The blank control, negetive control, siRNA1 and siRNA2 were transfected into BCL-7402 cells respectively via lipofection. Reverse transcription-polymerase chain reaction (RT- PCR) was used to examine the expression of Plk1 and p53 mRNA and Western Blot analyses was used to examine the expression of Plk1 and p53 protein in transfected BCL-7402 cells. The cell cycle distribution and apoptosis of cells transfected with siRNAs were also monitored by FCM. The changes of ultrastructure in transfected BCL-7402 cells were observed via transmission electron microscope(TEM). The expression of bcl-2,bax,Caspase-8,Caspase-9 and Caspase-3 mRNA was tested by RT-PCR 48 hours after transfected.ResultsThe positive rates of Plkl in HCC tissues was 76.1% and 44.8% of all carcinomas showed strong expression of Plk1. 27 cases of corresponding paracancerous cirrhosis tissues all showed positive expression. No significant Plk1 expression was observed in normal tissues of liver. The expression of Plk1 protein in well differentiation HCC tissues was markedly higher than in poor differentiation (P＜0.01). In addition, HCC tissues with peplos encroachment showed lower expression rates than others without peplos encroachment (P＜0.05). There was no other significant correlations of Plk1 expression with either age, tumor diameter and number, lymphoid node metastasis, extrahepatic metastasis, or portal vein embolus could be established. Plk1 protein was found to be overexpressed in both hepatoma cell lines. BCL-7402 cells transfected with low doses of siRNAs targeted against Plk1 had greatly decreased levels of Plk1 mRNA and protein, siRNA1 reduced Plk1 mRNA by 51%, 62% and Plk1 protein by 65%, 81% 24 hours and 48h after transfection, respectively, siRNA2 reduced Plk1 mRNA by 42%, 56% and Plk1 protein by 51%, 65% 24 hours and 48h after transfected, respectively. The ulteration status of Plk1 protein levels were similar to that of Plk1 mRNA levels. The p53 protein levels increased obviously to accompany with decrease of Plk1 protein levels. Cell cycle was changed and cell fraction at G₂/M phase was increased obviously 24 hours after transfected. Apoptosis rate was increased remarkably and the phenotypes of apoptosis could be seen in transfected cells 48 hours after transfected via TEM. The expression of bcl-2 mRNA decreased obviously, meanwhile the expression of bax mRNA increased remarkablely 48h after transfected. The expression of Caspase-8, -3 mRNA were up-regulated and Caspase-9 mRNA levels were unchanged 48h after transfected. Conclusiona high rate of Plk1-positivity in HCC which suggests involvement of Plk1 in tumorigenesis in this tumor entity and maybe a important early event of this process.siRNAs targetting against human Plk1 gene can remarkably inhibit Plk1 expression, increase p53 gene expression, lead to mitotic arrest at G₂/M phase and promote apoptosis.The pathway which promote apoptosis of hepatoma cells by siRNAs targetting against human Plk1 gene transfected is relationship with both the intrinsic apoptotic pathway---mitochondria and endoplasmic reticulum pathway and extrinsic apototic pathway---death ligands and receptors pathway.