HMGB1参与胃癌细胞端粒长度维持的生物学作用和分子机制

Telomere integrity is the crucial mechanism and marker of chromatin stability, and it is also the key factor in malignant mutation maintenance and overgrowth of gastric cancer cells. HMGB1 has recently emerged as a key factor in the pathogenesis of various diseases, placing it in the center of our modern understanding of cancer biology. The recent studies have showed that HMGB1 could affect the telomere length and telomerase activity, but the studies are rare, especially in cancer cells. In our work, HMGB1 overexpression was found in various cancerous tissues, including gastric adenocarcinoma. Furthermore, HMGB1 overexpression positively associated with cancer-free survival of resectable gastric adenocarcinomas. We also found that the constitutive telomere shortening andγ-radiation-induced telomerase activity in peripheral blood lymphacytes were associated with the elevated GC risk. The further preliminary study indicated HMGB1 expression was associated with the hTERT mRNA expression. Based on our preliminary work, we present the study to reveal the biological functions and molecular mechanisms of HMGB1 in the maintenance of telomere integrity. The main research contents include: 1) through detecting the HMGB1 expression, telomere length and telomerase activity in cancer tissues and cells, to assess the association of HMGB1 expression and telomere integrity. 2) Through influencing HMGB1 expression, to explicit the biological functions of HMGB1 on telomere integrity. 3) Through high-throughput screening techniques, just as gene chip, shRNA library screening, ChIP and phage-displaying polypeptides library, to investigate the hTERT expression dependent and non-dependent mechanism of HMGB1 regulation of telomere integrity. The associated study will reveal the biological functions of HMGB1 in the maintenance of telomere integrity, deepen the mechanism study associated with HMGB1 and telomere, and accelerate the therapeutic study targeting HMGB1 and telomere.

胃癌细胞快速增殖和永生化需要端粒长度维持，而端粒长度的维持又需要端粒酶活性保持或端粒延长替代机制激活。高迁移率族蛋白-1 (HMGB1）在肿瘤恶性演变进展的多个环节扮演着关键作用，我们前期研究证实胃癌等多种人类癌症存在高比例HMGB1过表达，并与胃癌预后相关。初步研究表明HMGB1与端粒长度正相关，可能通过调控端粒酶活性或激活端粒延长替代机制等途径，在肿瘤细胞端粒长度维持中发挥作用，但相关研究仅仅起步。基于此，本课题以胃癌为切入点，将研究内容设定为：①应用胃癌组织标本和细胞系，分析HMGB1表达与端粒长度相关性。②通过干预HMGB1作用，明确胃癌细胞中HMGB1参与端粒长度维持的生物学作用；③利用基因芯片等高通量筛选技术，探讨胃癌细胞中HMGB1参与维持端粒长度的分子机制。本课题的实施将促进以HMGB1和端粒为切入点的肿瘤分子机制研究，为以HMGB1和端粒为靶向的肿瘤治疗研究打下基础。

胃癌的发生与进展需要端粒长度维持。高迁移率族蛋白-1 (HMGB1）在肿瘤恶性演变进展的多个环节发挥重要作用。本课题意在明确HMGB1参与胃癌恶性生物学行为的作用及可能机制，特别是HMGB1参与胃癌细胞端粒功能维持可能作用机制。.（1）HMGB1的作用：胃癌组织芯片研究表明，HMGB1表达水平与胃癌恶性生物行为有关，但过高表达患者预后较好，并证实HMGB1等基因多态性与患者预后有关。构建RNAi慢病毒载体，转染胃癌细胞，抑制胃癌细胞HMGB1表达。HMGB1表达水平下降，胃癌细胞恶性生物学行为受到抑制，包括细胞增殖能力、迁移能力、克隆形成、凋亡发生、细胞分裂分期、体内成瘤能力等。Tet-on回复实验表明，HMGB1作用可能存在某种程度的“剂量依赖”效应。.（2）HMGB1作用通路：以SGC-7901RNAi和SGC-7901为对象，采用基因芯片技术，进行表达差异基因筛选。利用生物信息学技术，对HMGB1可能作用的信号通路进行分析，表明HMGB1表达改变可以导致不同作用机制、不同作用通路的分子出现表达改变。并以MAPK信号通路为例，进行初步的功能验证，表明HMGB1表达水平下降后，MAPK通路内多个分子表达出现改变。 .（3）HMGB1与端粒：HMGB1的表达水平影响胃癌细胞端粒长度，其机制可能为：1）利用组织芯片和免疫组化技术对HMGB1和端粒结合因子等表达相关性进行检测，发现在胃癌组织内，HMGB1的表达水平与TRF1水平呈现正相关，与TRF2表达水平呈现负相关。2）利用胃癌患者外周血单个核细胞，对HMGB1基因多态性和端粒长度进行检测，发现HMGB1的基因多态性和患者端粒长度相关。3）利用HMGB1RNAi胃癌细胞系（SGC-7901）研究发现，HMGB1表达抑制后，端粒长度缩短，端粒酶活性降低。4）分析基因芯片筛选结果，发现22个与端粒功能维持相关分子存在表达差异，其中POT1表达差异明显（FC值>2），初步验证提示HMGB1表达抑制可能影响POT1蛋白表达水平。同时，利用RT-PCR和Western Blot技术对HMGB1表达抑制后TRF1、TRF2等分子的表达水平进行检测，结果表明HMGB1表达水平影响端粒结合因子等相关分子的表达。