核纤层蛋白B1通过影响端粒酶活性调控肝癌细胞生长

Lamin B1 regulates the growth of hepatocellular carcinoma cells by influencing telomerase activity

摘要:

核纤层蛋白B1 (nuclear lamina protein B1, LMNB1)高表达于肝癌组织中，通过敲低LMNB1探讨其对肝癌细胞增殖的影响及其机制。利用siRNA在肝癌细胞中敲低LMNB1，Western blotting检测敲低效果，使用端粒重复序列扩增法(telomeric repeat amplification protocol assay, TRAP)检测其端粒酶活性变化。利用实时定量聚合酶链反应(quantitative real-time polymerase chain reaction, qPCR)检测其端粒长度变化。并通过CCK-8、克隆形成、Transwell、划痕实验检测其生长，侵袭和迁移能力变化。利用慢病毒系统构建稳定敲低LMNB1的HepG2细胞，检测其端粒长度及端粒酶活性变化，采用SA-β-gal衰老染色检测细胞衰老情况，通过裸鼠皮下成瘤实验及对肿瘤后续的组化染色，SA-β-gal衰老染色，端粒荧光原位杂交(fluorescencein situ hybridization, FISH)检测其对成瘤性的影响。最后利用生物信息分析的方法寻找LMNB1在临床肝癌组织中的表达情况，及其与临床分期、病人生存期的关系。HepG2和Hep3B中敲低LMNB1后端粒酶活性显著降低，细胞增殖、迁移和侵袭能力显著降低，细胞和裸鼠成瘤实验证明稳定敲低LMNB1后端粒酶活性降低的同时端粒长度缩短，细胞发生衰老，此外细胞成瘤性降低，Ki-67表达降低，生物信息分析结果显示，LMNB1高表达于肝癌组织，且与肿瘤分期和患者生存相关。LMNB1在肝癌细胞中过表达，其有望成为评估肝癌患者临床预后的指标和精准治疗的靶点。

Abstract:

Lamin B1 (LMNB1) is highly expressed in liver cancer tissues, and its influence and mechanism on the proliferation of hepatocellular carcinoma cells were explored by knocking down the expression of the protein. In liver cancer cells, siRNAs were used to knock down LMNB1. Knockdown effects were detected by Western blotting. Changes in telomerase activity were detected by telomeric repeat amplification protocol assay (TRAP) experiments. Telomere length changes were detected by quantitative real-time polymerase chain reaction (qPCR). CCK8, cloning formation, transwell and wound healing were performed to detect changes in its growth, invasion and migration capabilities. The lentiviral system was used to construct HepG2 cells that steadily knocked down LMNB1. Then the changes of telomere length and telomerase activity were detected, and the cell aging status was detected by SA-β-gal senescence staining. The effects of tumorigenesis were detected by nude mouse subcutaneous tumorigenesis experiments, subsequent histification staining of tumors, SA-β-gal senescence staining, fluorescence in situ hybridization (FISH) for telomere analysis and other experiments. Finally, the method of biogenesis analysis was used to find the expression of LMNB1 in clinical liver cancer tissues, and its relationship with clinical stages and patient survival. Knockdown of LMNB1 in HepG2 and Hep3B cells significantly reduced telomerase activity, cell proliferation, migration and invasion abilities. Experiments in cells and tumor formation in nude mice had demonstrated that stable knockdown of LMNB1 reduced telomerase activity, shortened telomere length, senesced cells, reduced cell tumorigenicity and KI-67 expression. Bioinformatics analysis showed that LMNB1 was highly expressed in liver cancer tissues and correlated with tumor stage and patient survival. In conclusion, LMNB1 is overexpressed in liver cancer cells, and it is expected to become an indicator for evaluating the clinical prognosis of liver cancer patients and a target for precise treatment.