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| Polo样激酶1在肝细胞肝癌中的作用及临床意义 |
| Polo-like kinase 1 Expression in hepatocellular carcinoma and its effect on cell proliferation |
| 投稿时间：2022-04-18 |
| DOI：10.3969/j.issn.1000-0399.2023.10.006 |
| 中文关键词: Polo样激酶1 小分子抑制剂 肝细胞肝癌 增殖抑制 诊疗靶点 |
| 英文关键词: Polo-like kinase 1 Small molecule inhibitor Hepatocellular carcinoma Proliferation inhibition Clinical target |
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| 中文摘要: |
| 目的 探讨PLK1在肝细胞肝癌中的表达、对肝癌细胞生物学行为的影响及临床转化意义。方法 使用TCGA数据库分析PLK1 mRNA在369例肝细胞癌肿瘤患者组织及160例患者癌旁组织中的表达及对无病生存期的影响。提取川北医学院附属医院肝胆外一科2019年1~6月收治的30例肝细胞癌患者的肿瘤组织及癌旁组织，应用实时荧光定量聚合酶链反应（qRT-PCR）检测PLK1的mRNA表达水平进行验证。选取HepG2细胞系进行功能实验，分别使用小干扰及GSK461364处理，Western blot实验检测PLK1小干扰RNA敲低效率，检测不同处理肝癌细胞生物学行为变化；流式细胞术检测不同处理细胞周期改变。使用HepG2细胞构建裸鼠皮下成瘤模型，分别给予50 mg/kg GSK461364腹膜注射和等体积生理盐水腹膜注射，20 d后收获皮下肿瘤，进行后续的拍照及免疫组化染色，比较移植瘤体积及Ki-67染色阳性率。提取数据库中患者两年随访资料，同时以PLK1表达中位值为界，将患者分为PLK1高表达组及低表达组，并采用log-rank检验进行生存分析。结果 TCGA数据库分析结果显示，PLK1在肝癌组织中的表达明显高于癌旁组织log2（TPM+1）值分别为（2.3±7.5）、（0.8±2.7），差异有统计学意义（P<0.05）。生存分析显示，PLK1高表达组无病生存期低于PLK1低表达者，差异有统计学意义（P<0.05）。肝癌肿瘤组织标本分析PLK1 mRNA表达结果显示，PLK1在肿瘤组织中的表达为（2.4±1.0）高于癌旁组织（1.3±0.8），差异有统计学意义（P<0.001）。使用两条siRNA敲低HepG2细胞中PLK1的表达，敲低效率分别为（71.3±4.8）%、（65.3±5.3）%，细胞增殖、平板克隆形成及细胞周期实验结果显示，与siCtrl组相比，siPLK1-1、siPLK1-2组肝癌细胞HpG2的细胞相对活力明显降低，细胞克隆数减少，HepG2细胞周期阻滞在G2期，差异均有统计学意义（P<0.001）。使用PLK1特异性小分子抑制剂GSK461364处理HepG2细胞，IC50=25.0 nM，CCK8、平板克隆形成及细胞周期实验结果显示，与DMSO组相比，GSK461364组HpG2的细胞相对活力明显降低，细胞克隆数明显减少，HepG2细胞周期阻滞在G2期，与小干扰结果一致，差异均有统计学意义（P<0.05）。裸鼠体内成瘤实验结果显示，相对于生理盐水注射组，GSK461364注射组小鼠的移植瘤体积明显减小[（328.3±28.4） mm3比（527.7±26.5） mm3]；Ki-67阳性率明显减少，[（52.5±1.9）%比（15.2±1.9）%]，差异均有统计学意义（P<0.05）。而体质量无明显变化，差异无统计学意义（P>0.05）。结论 PLK1在肝细胞肝癌中高表达，与患者的总体生存率呈负相关，抑制其表达可显著降低肝癌细胞的增殖能力，减慢肝细胞肝癌进展，可作为肝细胞肝癌的潜在治疗靶点。 |
| 英文摘要: |
| Objective To investigate the expression of PLK1 in hepatocellular carcinoma(HCC), its effect on the biological behavior of HCC cells and its clinical transformation significance. Methods TCGA database was used to analyze the expression of PLK1 mRNA in 369patients with hepatocellular carcinoma and 160 patients with paracancerous tissues and its effect on disease-free survival. The tumor tissue and adjacent tissues of 30 patients with hepatocellular carcinoma admitted to our hospital from January 2019 to June 2019 were extracted, and the mRNA expression level of PLK1 was detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR) for validation.Subsequently, HepG2 cell lines were selected for functional experiments, and treated with small interference and GSK461364 respectively.Western blot was used to detect the knockdown efficiency of PLK1 small interference RNA, and to detect the biological behavior changes of liver cancer cells treated with different treatments; Cell cycle changes were detected by flow cytometry. HepG2 cells were used to construct the subcutaneous tumorigenic model in nude mice, and 50 mg/kg GSK461364 and equal volume of normal saline were injected into the peritoneum respectively. Subcutaneous tumor was harvested 20 days later, the subcutaneous tumor was harvested, and the follow-up photos and immunohistochemical staining were performed to compare the volume of transplanted tumor and the positive rate of Ki-67 staining. Two years of followup data of patients in the database were extracted. At the same time, patients were divided into PLK1 high expression group and low expression group by taking the median value of PLK1 expression as the boundary, and survival analysis was performed by log-rank test. Results The results of TCGA database analysis showed that PLK1 expression in liver cancer tissue was significantly higher than that in adjacent tissues[the value of log2(TPM+1) was(2.3 ±7.5) and(0.8 ±2.7), respectively, P<0.05], and the difference was statistically significant. The survival analysis showed that the disease-free survival of patients with high expression of PLK1 was significantly lower than those with low expression of PLK1(P<0.05). The analysis of PLK1 mRNA expression in liver cancer tissue samples showed that the expression of PLK1 in tumor tissue was(2.4 ±1.0), significantly higher than that in paracancerous tissue(1.3 ±0.8), and the difference was statistically significant(P<0.001). Using two siRNAs to knock down the expression of PLK1 in HepG2 cells, the knockdown efficiency was(71.3 ±4.8)% and(65.3 ±5.3)%, respectively. The results of cell proliferation, plate cloning and cell cycle experiments showed that compared with the siCtrl group, the relative viability of HepG2 cells in the siPLK1-1 and siPLK1-2 groups was significantly reduced, the number of cell clones was significantly reduced, and the cell cycle of HepG2 cells was blocked in G2 phase, all with statistically significant differences(P<0.001). HepG2 cells were treated with PLK1-specific small molecule inhibitor GSK461364. The results of IC50=25.0 nM, CCK8, plate cloning and cell cycle experiment showed that the relative activity of HpG2 cells in GSK461364 group was significantly lower than that in DMSO group, the number of cell clones was significantly reduced, and the cell cycle of HepG2 was blocked in G2 phase, which were consistent with the results of small interference, and all the difference was statistically significant. The results of tumorigenesis experiment in nude mice showed that compared with the saline injection group, the transplanted tumor volume of GSK461364 injection group was significantly reduced[(328.3 ±28.4) mm3 vs(527.7 ±26.5) mm3, P<0.001], the positive rate of Ki-67 was significantly reduced,[(52.5 ±1.9)% vs(15.2 ±1.9)%, P<0.001], and the difference was statistically significant. There was no significant difference in body mass(P>0.05). Conclusion The high expression of PLK1 in hepatocellular carcinoma is negatively correlated with the overall survival rate of patients. Inhibition of PLK1 expression can significantly reduce the proliferative capacity of hepatocellular carcinoma cells, slow down the progression of hepatocellular carcinoma, and can be a potential therapeutic target for hepatocellular carcinoma. |
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