HMGB1及其受体RAGE分子信号通路参与调节肝细胞肝癌增殖和侵袭迁移机制的研究

Study on HMGB1 and its receptors RAGE molecular signaling pathway involved in regulating the mechanism of proliferation and infiltrative migration of HCC

目的观察高迁移率族蛋白B1(HMGB1)及其受体晚期糖基化终产物(RAGE)在人体肝细胞癌(HCC)组织中的表达情况,了解HMGB1及其受体RAGE对HCC细胞增殖和侵袭转移的影响,研究HMGB1及其受体分子信号通路参与调节HCC细胞增殖和侵袭转移的相关机制。方法选取10例人体HCC组织标本和HCC细胞培养标本,以正常的肝脏组织细胞作为对照。通过免疫荧光染色、Western blot、RT-PCR检查方法分析HMGB1对HCC细胞内丝裂原活化蛋白激酶(MAPKs)、蛋白激酶B(AKT)、NT-κB等信号通路的调节,检测HCC细胞中HMGB1及其受体分子RAGE蛋白的表达量,对HMGB1及其受体RAGE的表达量与其临床症状作相关性分析。结果 1在HMGB1抑制剂甘草皂甙、重组HMGB1(r HMGB1)、r HMGB1+sRAGE的作用下,能够有效干扰HMGB1在HCC细胞中的表达。干扰HMGB1可以抑制HCC细胞的增殖、促使细胞凋亡、引起细胞周期阻滞,同时降低其体外迀移和侵袭能力。这些结果提示HMGB1在HCC中具有促增殖、促迁移、抗凋亡和侵袭的作用。2HMGB1能增强MAPKs和NF-κB信号通路活性。稳定干扰HMGB1的HCCLM3细胞内MAPKs的磷酸化水平降低,包括p38和JNK,同时它们的上游激酶(MEK1/2、SEK1)和下游底物(c-Jun、-Myc)的磷酸化也被抑制;NF-κB的表达量及Ser536位点的磷酸化水平也下调。结论 1HMGB1与RAGE结合通过激活MAPK信号通路最终导致核转录因子NF-κB激活并促进RAGE基因转录,增加RAGE的mRNA含量,使RAGE表达上调,正反馈的扩大HMGB1引起的反应;2HMGB1通过作用于RAGE激活下游RAC1分子,并最终引起VEGF表达上调,进而促进HCC的增殖和侵袭转移。

Objective To observe the expression of HMGB1 and its receptor RAGE in human liver tissue,and to understand the influence of HMGB1 and its receptor RAGE on proliferation,invasion and metastasis of human hepatic carcinoma cells（ HCC）,and to study the mechanism of HMGB1 and its receptor molecular signaling pathway involved in regulating the mechanism of invasion and metastasis of HCC cells and its related mechanism.Through the study of its signaling pathway,it is expected to clarify the HMGB1 in HCC and its receptor molecules related mechanism of signaling pathways,in order to provide new target for prevention and treatment of HCC.Methods Ten samples of human HCC tissue specimens and the specimens of cultured HCC cells were applied for this study,and normal liver tissue cells were used as controls.The immunofluorescence staining method,Western blot and RT-PCR were applied to analyze HMGB1 in HCC cells for the regulation of signaling pathways of MAPKs,AKT,NT-κB etc,and to detect HMGB1 and its receptor molecules RAGE protein expression in HCC cells,and the expression of HMGB1 and its receptor RAGE in quantity and its clinical symptoms for correlation analysis.Results 1Under the action of liquorice saponin,restructuring HMGB1,HMGB1 inhibitors（ r HMGB1） plus sRAGE,effective jamming the expression of HMGB1 in HCC cells.The interference of HMGB1 can inhibit the proliferation of HCC cells,inducing cell apoptosis,and arrest the cell cycle,at the same time reduce the movement in vitro and the ability of attack.These results indicated that HMGB1 could promote the proliferation and migration of HCC,the action of anti-apoptosis and anti-attack.2HMGB1 can enhance the activity of sinaling passways of MAPKs and NF-κB.It can stablely interfere the levels of cell phosphalation of HMGB1 and MAPKs in HCCLM3 cells,including p38 and JNK,at the same time their upstream kinase（ MEK1 /2,SEK1） and suppressed the phospholation of downstream substrates（ c-Jun,-Myc）.The quantity of expression of NF-κB and the phosphorylation level of Ser536 loci was also cut down.Conclusion 1HMGB1 combined with RAGE by activating MAPK signaling pathways leading to the activition of nuclear transcription factor NF-κB and promote RAGE gene transcription,increasing levels of mRNA in the RAGE,the up-regulation of RAGE expression caused by the expansion of positive feedback repons of HMGB1.2 By acting on RAGE and activationg the downstream molecule of RAC1,HMGB1 eventually cause the expression of VEGF,and promote the proliferation,invasion and metastasis of HCC.