高精度腹腔热灌注化疗上调miR-218逆转胃癌多药耐药的作用及分子机制

Multidrug resistance (MDR) in gastric cancer remains a major challenge to chemotherapy treatment.Thermal therapy plays an important role in the reversal of MDR.Studies have also shown that the activation of Hh/SMO signal pathway or the overexpression of CyclinD1 protein can lead to the MDR of tumors. But until now, the mechanisms of Hh/SMO and CyclinD1 are unclear in gastric cancer. Applicant's earlier studies showed that high-precision hyperthermic intraperitoneal chemotherapy (HHIPC)could upregulate the expression of miR-218 and inhibit the expression of MDR-related genes to enhance the sensitivity of chemotherapy in gastric cancer. So, we propose that HHIPC can upregulate the expression of miR-218 and inhibit Hh/SMO signal pathway or the expression of CyclinD1 and reverse the MDR of gastric cancer. Therefore, we designed the research project as follows:first,HHIPC can reverse the MDR of gastric cancer effectively; second,to clarify the detailed mechanism of Hh/SMO signal pathway and the expression of CyclinD1 during the process of HHIPC. This project will establish the new target of miR-218 in high-precision hyperthermic intraperitoneal chemotherapy and provide the scientific basis for it.

多药耐药（MDR）是胃癌患者化疗失败的关键原因,热疗在逆转胃癌MDR的过程中起重要作用。研究表明，Hedgehog（Hh）/SMO信号通路的激活或CyclinD1蛋白的高表达均可引起肿瘤细胞的MDR，但二者在胃癌MDR中的作用机制尚不清楚。申请者前期研究表明高精度腹腔热灌注化疗(HHIPC)可上调miR-218的表达并通过抑制MDR相关基因的表达来增强胃癌化疗的敏感性。因此，我们提出"高精度腹腔热灌注化疗上调miR-218并通过抑制Hh/SMO信号通路和/或CyclinD1的表达来逆转胃癌MDR"这一科学假说。本课题拟设计下列内容：①HHIPC上调miR-218能有效逆转胃癌MDR；②阐明Hh/SMO信号通路和CyclinD1在其中的分子机制，并对上述结果进行临床验证。本项目将为确立miR-218在HHIPC中作为抑制胃癌MDR的新靶点提供科学依据。

多药耐药（MDR）是导致胃癌化疗失败的重要原因。前期我们对5例胃癌患者经腹腔热灌注化疗（HIPEC）前后的血清采用miRNA表达谱筛选，发现miR-218在HIPEC后胃癌患者血清中的表达明显上调且高达8倍以上，同时miR-218还是胃癌MDR的候选分子，进一步分析发现HIPEC后MDR相关基因的表达显著下降，这一现象提示miR-218可能具有增强胃癌化疗疗效甚至逆转胃癌MDR的作用。为了明确miR-218在胃癌及其胃癌化疗耐药中发挥的作用及其机制，首先我们检测了miR-218在胃癌及相应癌旁组织中的表达，发现其在胃癌组织中低表达且与胃癌进展相关，在胃癌细胞株中过表达miR-218可以显著抑制胃癌细胞增殖、集落形成及裸鼠皮下成瘤能力，我们还发现miR-218通过靶向mTOR更增强了胃癌细胞对化疗药物的敏感性。接着我们通过RT-PCR检测发现miR-218在胃癌耐药细胞株中低表达及MDR相关基因高表达，在胃癌耐药细胞株中过表达miR-218可以显著提高耐药的胃癌细胞对化疗药的敏感性，并且显著促进了化疗药诱导的胃癌耐药细胞凋亡。为了阐明miR-218在胃癌中发挥的抑癌作用及逆转胃癌MDR的分子机制，我们经网站预测可见miR-218靶向SMO、cyclinD1 及Gli2，且荧光素酶报告基因试验证实了SMO、cyclinD1 及Gli2是miR-218的下游靶基因，上调miR-218表达可以明显抑制SMO、cyclinD1 和Gli2的表达，且抑制MDR相关基因MDR-1和P-gp、抗凋亡基因Bcl-2的表达及增强了促凋亡基因BAX、cleaved caspase-3和转移相关基因E-cadherin的表达，进而发挥了miR-218逆转胃癌细胞MDR及抑制胃癌细胞增殖和侵袭，促进胃癌细胞凋亡的作用。我们研究发现热化疗可以上调miR-218的表达，抑制下游基因Bcl2和Gli2的表达和促进Bax、cleaved caspase-3和E-cadherin的表达，进而抑制胃癌细胞增殖、侵袭及增强胃癌细胞对顺铂的化疗敏感性。本项目探索了miR-218对胃癌MDR的影响及生物学机制，为以HIPEC后上调的miR-218为研究靶向，克服胃癌细胞的MDR、提高胃癌化疗效果提供理论基础和临床依据。