摘要
目的
1.研究细胞周期调控蛋白p27kip1及其相关分子JAB1在肝细胞癌(human hepatocellular carcinoma, HCC)中的表达,并结合增殖指标Ki67和临床病理资料初步探讨p27及JAB1与HCC发生发展的关系。
2.克隆人类p27kip1基因,构建其真核表达载体。
3.研究化疗药物三氧化二砷(As2O3)对肝癌细胞活性、细胞周期及细胞凋亡的影响,初步探讨p27kip1及JAB1在此过程中的作用。
方法
1.首先在组织水平,选取76例HCC及癌旁组织,l0例肝血管瘤旁肝组织病例标本,10例HCC及癌旁肝新鲜组织,采用免疫组化,10例新鲜组织中有6例采用免疫印迹(Western blot)方法,检测p27kip1及其相关分子JAB1的表达情况,并结合增殖指标Ki67、PCNA和临床病理资料初步探讨p27kip1及JAB1与HCC发生发展的关系。
2.采用RT-PCR技术扩增人类p27kip1cDNA全长序列,通过DNA重组技术将其重组于pcDNA3.1及pEGFP-N2载体,构建p27kip1正、反义真核表达质粒及p27-EGFP融合蛋白表达质粒,通过酶切电泳分析及DNA测序的方法对重组表达质粒进行鉴定。
3.以WST-1法、流式细胞术及Hochest染色法检测三氧化二砷对人肝癌SMMC-7721细胞活性、细胞周期及细胞凋亡的影响, Western blot技术检测p27kip1分子网络中主要成员在此过程中的表达变化,运用细胞免疫荧光技术检测p27kip1相关分子在药物作用后亚细胞定位的变化。
结果
1.肝癌和癌旁组织免疫印迹结果显示,p27kip1主要在癌旁高表达,而JAB1蛋白在大部分HCC组织中的表达高于对应癌旁肝组织。免疫组化结果显示:JAB1在HCC中的表达高于癌旁组织和血管瘤旁肝组织。JAB1的表达与肝癌组织分化程度,血清AFP值及转移相关。p27kip1在正常肝组织和癌旁组织中的表达高于HCC组织,表达强度与肝癌组织分化程度,血清AFP值、坏死程度及转移相关。相关性分析表明HCC组织中JAB1蛋白表达与p27kip1蛋白表达呈负相关,与ki67表达正相关,p27的表达与ki67呈负相关。
2.构建p27kip1正、反义真核表达质粒及p27-EGFP融合蛋白表达质粒,通过酶切电泳分析及DNA测序的方法对重组表达质粒进行鉴定,证实构建成功。
3.三氧化二砷能够抑制肝癌SMMC-7721细胞生长活性,小剂量三氧化二砷可诱导肝癌细胞发生G2/M期生长阻滞,大剂量可诱发细胞凋亡;随As2O3作用时间的延长,SMMC-7721中p27kip1表达增加,CDK2、CRM1、JAB1等蛋白表达下降。免疫荧光结果显示,加药刺激后,p27kip1发生从胞浆向胞核的易位,伴有JAB1胞浆向胞核的易位,CRM1的胞浆定位变化不明显。。
结论
1. p27kip1蛋白表达的降低、JAB1表达的增高与HCC的发生及肿瘤恶性程度密切相关。JAB1蛋白的表达与p27kip1负相关,与ki67正相关,提示JAB1在HCC组织中表达上调,可能是通过作用于细胞周期调控蛋白p27kip1,加速了p27kip1的出核降解,使其表达及代谢发生异常。导致细胞周期失控并促进细胞异常增殖,从而参与了HCC的发生和发展。
2.成功构建了人类p27kip1基因的正、反义真核表达质粒和p27-EGFP融合蛋白表达质粒,为进一步探讨p27kip1基因在肝癌发生中的作用奠定了实验基础。
3.三氧化二砷能够抑制肝癌细胞活性,引起细胞周期阻滞或凋亡,上调p27kip1蛋白表达,下调CRM1、JAB1等蛋白表达,同时伴有p27kip1相关分子亚细胞定位的改变。提示,CRM1、JAB1可能通过加速p27kip1的降解、改变p27kip1的亚细胞定位来影响p27kip1的功能,参与As2O3对SMMC-7721细胞生长抑制的分子调控机制。
Objective
Investigate the expression of JAB1 and p27 protein in human hepatocellular carcinoma (HCC) and its clinicopathologic significance. The expression of JAB1 and p27 were analyzed and compared with that of proliferative cell nuclear antigen (ki67) in these specimens. Clone human p27kip1 gene, construct its eukaryotic expressive vectors, Evaluate the effect of arsenic trioxide(As2O3) (a chemotherapeutic drug) on activity, cell cycle and apoptosis of SMMC-7721 cells, and preliminarily investigate the role of p27kip1 linked protein network during the process.
Methods
1. Immunohistochemical study for JAB1, p27 was performed on 76 cases of hepatocellular carcinoma tissue and adjacent nontumorous tissues, 10 cases of native liver tissue adjacent to hepatic hemangioma and 10cases of fresh HCC tissue and the adjacent liver tissue .Fresh specimens from 6 cases of HCC and the adjacent liver tissue were also collected for Western blot analysis.
2. Human p27kip1 complete cDNA was amplified by RT-PCR method, and was reconstructed into the eukaryotic expressive vector pcDNA3.1 and pEGFP-N2 to form the p27kip1 sense or antisense expressive plasmid and p27-EGFP fusing protein expressive plasmid. The reconstructed DNA was identified by enzyme digestion and DNA sequencing.
3. The influence of As2O3 on the activity, cell cycle or apoptosis of hepatomacellular carcinoma SMMC-7721 cell, cells was detected by MTT colorimetry, Flow cytometry and Hochest staining. The expression of p27kip1 and p27kip1-related molecule was investigated by Western blot. The subcellular localization of p27kip1 and p27kip1-related molecule were detected by immunoflurescence.
Results
1. Western blot analysis showed that in most HCC, JAB1 expression was higher than that in adjacent liver tissue. Statistically significant difference in expressions of JAB1 and p27kip1 were noted in the three groups studied (P<0.001).The expression of JAB1 in HCC was significantly higher than that in other two groups.The expression of JAB1 was correlated with histological differentiation, serum alpha-fetal protein levels and metastasis (P<0.05).The levels of p27kip1 is lower in cancer tissues than in nontumorous tissues. There was a inverse correlation between JAB1 and p27 in HCC. Positive correlation was found between JAB1 and ki67 in HCC (P<0.001).
2. The cloned DNA was confirmed to be human p27kip1 cDNA.
3. As2O3 significantly inhibited the growth of SMMC-7721 cell, compared with untreated control cell. A striking increase in p27kip1 expression and a reciprocal decrease in p27kip1-related protein (JAB1, CRM1) expression were found in As2O3-treated SMMC-7721 cell. Meanwhile, As2O3 decreased the protein levels of Cdk2 and cyclinE .The location of p27kip1 and JAB1 were transferred from cytoplasm to nucleus which detected by immunoflurescence.CRM1 was mainly located in cytoplasm and no significant changing of subcellular localization has been detected during this time.
Conculusions
1.Decreased expression of p27kip1 and increased expression of JAB1 were closely correlated with the aggressiveness of HCC. There was a inverse correlation between JAB1 and p27kip1 in HCC, and positive correlation was found between JAB1 and ki67. overexpression of JAB1 may reduce the protein level of p27kip1 by accelerating p27kip1 degradation via nuclear export mechanism and causes disruption of cell cycle control and enhancement of the proliferative activity, indicating JAB1 playes an important role in oncogenesis and development of HCC.
2. We successfully cloned human p27kip1 gene and constructed its eukaryotic expressive plasmids, which was an ideal model for further study.
3. Arsenic trioxide attenuated JAB1, CRM1 expression, thereby disturbing the location and expression of p27kip1, and then may participate in regulating the growth of human hepatoma cell through interfering with the function of p27kip1.
引文
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