【作者】 姜炜；

【导师】 王志明；

【作者基本信息】 中南大学 ， 外科学， 2012， 博士

【摘要】 目的：肝细胞癌(hepatocellular carcinoma, HCC),是我国最常见的恶性肿瘤之一,在各种恶性肿瘤的死亡率中位居第二。高迁移率族蛋白1(high-mobility group box1, HMGB1)作为一种非组蛋白核蛋白被发现在多种肿瘤中高表达,但它在肝癌中所扮演的角色目前并不清楚。在本次研究中,我们检测肝癌组织中HMGB1的表达情况,并分析HMGB1mRNA和肝癌临床病理因素之间的关系；探讨RNA干扰对人肝癌细胞株HCCLM3生物学活性的影响,并初步研究其可能的下游作用机制。方法：(1)收集11例正常肝脏组织、32例肝癌组织及其配对的癌旁组织,逆转录PCR(reverse transcription-polymerase chain reaction, RT-PCR)和Western-blot分析HMGB1的表达水平,免疫组织化学法检测HMGB1的定位,并分析HMGB1mRNA表达水平和肝癌临床病理因素之间的关系。(2)设计并合成了3对特异性针对HMGB1的小干扰RNA序列(HMGB1-siRNA-1、HMGB1-siRNA-2和HMGB1-siRNA-3),通过脂质体LipofectamineTM2000转染HCCLM3细胞,同时设置阴性对照组和空白对照组。实时荧光定量PCR(Real-time PCR)和Western-blot检测HMGB1干扰序列对HMGB1表达的影响；MTT法检测细胞的增殖并绘制生长曲线；流式细胞术检测细胞的凋亡；Transwell法检测细胞迁移和侵袭的能力。(3)Real-time PCR和Western-blot检测HMGB1干扰序列转染进入HCCLM3细胞后NF-KB/p65和VEGF-C的表达水平,间接免疫荧光法(indirect immunofluorescence)检测NF-κB/p65的定位,酶联免疫吸附测定法(enzyme-linked immunosorbent assay, ELISA)检测NF-KB/p65的活性。结果：(1)RT-PCR结果表明HMGB1mRNA的相对表达量在肝癌组织中最高,为0.859±0.159,明显高于癌旁组织的0.506±0.131和正常肝脏组织的0.413±0.086(P<0.001)。HMGB1mRNA的过表达与肝癌的TNM分期、Edmondson分级、血管侵犯和包膜侵犯密切相关。Western-blot结果显示HMGB1蛋白在肝癌组织中的表达也是三组中最高的。相关性分析表明,组织中的HMGB1mRNA与HMGB1蛋白表达呈明显正相关(R=0.833,P<0.01)。此外,免疫组化结果显示HMGB1在肝癌中过表达,免疫染色颗粒主要位于细胞核,正常肝脏组织中未见染色,在癌旁组织中有少量染色位于细胞浆。(2) Real-timePCR和Western-blot结果表明,3条特异性针对HMGB1的小干扰RNA序列都可以抑制HMGB1的表达,其中以HMGB1-siRNA-1抑制效率最高。MTT结果显示HMGB1-siRNA-1组细胞的生长速度明显低于阴性对照组和空白对照组(P<0.01)；流式细胞术显示HMGB1-siRNA-1组细胞凋亡显著增加(P<0.01)；Transwell法结果表明在HMGB1表达抑制的HMGB1-siRNA-1组细胞中,细胞迁移和侵袭的能力较阴性对照组和空白对照组明显下降(P<0.01)。(3)Real-time PCR和Western-blot结果表明,HCCLM3细胞转染HMGB1-siRNA-1后NF-KB/p65和VEGF-C的表达较阴性对照组和空白对照组明显下降(P<0.01)；间接免疫荧光法结果显示HMGB1-siRNA-1组的NF-κB/P65核转位减少；ELISA结果显示HMGB1-siRNA-1组的NF-κB/P65活性降低。结论：(1)肝癌组织中HMGB1的表达明显升高；肝癌组织中的HMGB1表达水平与肝癌的TNM分期、Edmondson分级、血管侵犯、包膜侵犯正相关,其过表达可能与肝癌的发生、发展密切相关。(2)特异性针对HMGB1的siRNA序列可以有效地抑制HCCLM3细胞的增殖,促进细胞凋亡,同时抑制HCCLM3细胞迁移和侵袭的能力,HMGB1可能作为肝癌治疗的一个潜在靶点。(3)HMGB1表达抑制后,HCCLM3细胞中NF-KB/p65和VEGF-C的表达均降低,同时可以抑制HCCLM3细胞中NF-KB/p65的核转位并降低其活性,HMGB1可能通过NF-κB信号通路调节VEGF-C的表达。更多还原

【Abstract】 Objectives:Hepatocellular carcinoma (HCC), one of the most common malignant tumors, is the second cause of cancer mortality in China. High-mobility group box1(HMGB1), as a non-histone nuclear protein, has been associated with many human cancers, but the role of HMGB1in HCC remains unclear. In this study, we investigated the expression of HMGB1in human HCC and analyzed the relationship between HMGB1mRNA and HCC clinicopathologic significance. We also examined the effects of RNA interference (RNAi) HMGB1on the bioactivity of HCC cell line HCCLM3and explored possible downstream mechanism.Methods:(1)11cases of normal liver tissues,32cases of HCC and the corresponding liver tissue just around the tumor (LAT) were collected, Then, all the samples were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western-blot to analysis the expression of HMGB1. At the same time, immunohistochemisty was used to detect the location of HMGB1. The relationships between HMGB1mRNA expression and clinicopathologic parameters were analyzed.(2) We designed and synthesized three specific small interfering RNA of HMGB1(HMGB1-siRNA-1、HMGB1-siRNA-2and HMGB1-siRNA-3) and transfected these into HCCLM3cells by use of LipofectamineTM2000. Cells transfected with no significant homology with human gene sequences (negative group) and without transfection treatment (blank group) were treated in parallel. Real time-PCR and Western blot were performed to determine the effects of HMGB1-siRNAs on HMGB1expression. In vitro proliferation was assessed by MTT assay. Apoptosis was demonstrated by flow cytometry. Migration and invasive ability were determined by use of the Transwell assay.(3) RT-PCR and Western blot were performed to detect NF-κB/p65and VEGF-C expression after transfection of HMGB1-siRNA-1into HCCLM3. Indirect immunofluorescence assay was used to detect the location of NF-κB/p65. Then, we used enzyme-linked immunosorbent assay (ELISA) to detect NF-KB/p65activity.Results:(1) RT-PCR demonstrated that the expression of relative HMGB1mRNA was0.859±0.159; the highest in the tissue of HCC, significantly up-regulated compared with that of0.506±0.131in LAT and of0.413±0.086in normal liver tissues (P<0.001). HMGB1mRNA overexpression was significantly associated with Edmondson stage, TNM stage, vascular invasion and capsule invasion. Western-blot showed the expression of HMGB1protein in HCC also as the highest among all the groups. Correlation analysis between HMGB1mRNA and protein indicated that the expression of HMGB1mRNA had a positive correlation with HMGB1protein (Person correlation analysis, R=0.833, P<0.01). Furthermore this overexpression revealed by immunostaining was predominantly localized in the nuclei of HCC; whereas, none of the stains were seen in normal liver tissues and only a trace of it was detected in the cytoplasm of LAT cells.(2) Real-time PCR and Western-blot showed that all three specific HMGB1-siRNAs significantly inhibited HMGB1expression, with inhibition by HMGB1-siRNA-1being highest. MTT assay revealed that the HMGB1-siRNA-1group significantly reduced cell proliferation compared with the negative group or blank group cells (P<0.01). FCM revealed that apoptosis was significantly increased in HMGB1-siRNA-1-transfected cells compared with negative group or blank group cells (P<0.01). The Transwell assay revealed that HCCLM3cells transfected with HMGB1-siRNA-1had much lower migratory ability and invasive activity than the negative group or blank group cells (P<0.01).(3) Real-Time PCR and Western-blot analysis showed that the mRNA and protein levels of NF-κB/p65and VEGF-C of HCCLM3cells transfected with HMGB1-siRNA-1were significantly decreased compared with the negative group or blank group cells (P<0.01). Indirect immunofluorescence assay revealed that NF-κB/p65nuclear translocation was decreased after HCCLM3cells transfected with HMGB1-siRNA-1. At the same time, ELISA showed that NF-κB/p65activity was also decreased. Conclusions:(1) HMGB1levels in HCC were significantly elevated. HMGB1overexpression was significantly associated with Edmondson stage, TNM stage, vascular invasion and capsule invasion. Overexpression of HMGB1might associated with HCC development and progress.(2) Downregulation of HMGB1by specific small interfering RNA could obviously inhibit the growth and of promotes the apoptosis of HCCLM3cells. It also obviously inhibits their migration and invasion ability. HMGB1may serve as a potential target for treatment of HCC.(3) Downregulation of HMGB1can inhibit the expression of NF-κB/p65and VEGF-C in HCCLM3cells. Meanwhile, NF-κB/p65nuclear translocation and activity were also decreased. HMGB1possibly via the NF-κB signaling pathway that regulates VEGF-C expression.更多还原