趋化因子CCL18调控LDHA、LDHB促进前列腺癌细胞糖酵解的机制研究

The glycolysis of tumor has been a hotspot in the field of tumor development and treatment.Lactate dehydrogenase (LDH) is the tetramer emzyme, which composed of two major subunits called LDHA and LDHB, is the key enzyme of glycolytic pathway. Our previous study proved that CCL18 is closely related to the occurrence, development and prognosis of prostate cancer (Bio Med Int, 2014), and in the preliminary experiments found that CCL18 up-regulated LDHA but down-regulated expression, and raised the lactate level. Our TMA data showed that LDHA and LDHB were differential expression between prostate cancer and non-cancerous prostatic tissues which were related to prognosis of prostate cancer. Whether CCL18 involved in prostate cancer cells glycolysis? On this basis, we hypothesized that CCL18 regulating the LDHs expression pattern which may involved in prostate cancer cells glycolysis. CRISPR/Cas9 technology was utilized to establish LDHA ko and LDHB ko cell lines and mice models to elucidate the molecular mechanism of how CCL18 involved in regulating the glycolysis pathway. A further analysis of clinical data correlation which LDHs is needed to assess clinical significance of prostate cancer diagnosis and treatment. And we hope to provide a reliable evidence for a new targeted therapy with metabolism in prostate cancer.

肿瘤糖代谢一直是肿瘤发展及治疗领域的研究热点，乳酸脱氢酶（LDH）是糖代谢的关键酶。我们已证实CCL18促进前列腺癌发生、发展，与前列腺癌预后密切相关（Bio Med Int, 2014）：预实验发现CCL18上调LDHA、下调LDHB表达，促进乳酸形成；应用组织芯片发现LDHA、LDHB在前列腺癌与非癌中存在差异性表达并与疾病预后相关，同时LDHA\LDHB比值在良性前列腺疾病及前列腺癌的前列腺液、组织中均存在表达差异。但CCL18调控LDHA、LDHB影响前列腺癌糖酵解的机制是什么？据此，我们提出CCL18信号通路调控LDHs参与前列腺癌细胞糖酵解的假设。拟通过CRISPR/Cas9 技术构建实验细胞株，建立小鼠模型，揭示CCL18通路调控糖酵解的机制，通过临床数据相关性分析评估LDHs对前列腺癌诊疗的临床意义，为前列腺癌的糖代谢靶向治疗提供可靠依据。

课题组利用人重组CCL18蛋白处理前列腺癌雄激素非依赖型细胞株PC3和雄激素依赖型细胞株LNCaP，检测发现前列腺癌细胞受到CCL18刺激后p-LDHA、LDHA蛋白表达水平升高，而LDHB蛋白表达水平下降；同时检测细胞乳酸产量的改变发现CCL18能够使前列腺癌细胞PC3、LNCaP的乳酸产量明显升高（p＜0.01）。证明了CCL18能够影响前列腺细胞的糖酵解；课题组筛选出一系列与细胞代谢、增殖相关转录因子（如：HIF-1、ARNT、CEBPB、TP53、JUN、FOX等），并构建了上述基因的混合CRISPR CAS9载体系统，发现转录因子ARNT基因表达抑制后，能够降低LDHA蛋白表达水平同时使LDHB蛋白表达升高。同时，我们发现作为CCL18的受体CCR8被表达抑制后，能够使ARNT得表达下降。通过TCGA数据库分析，我们发现CCR8与ARNT表达具有正相关性（R=0.403，p＜0.01）。因此我们提出潜在CCL18调控前列腺癌细胞糖酵解的通路：CCL18-CCR8-ARNT-LDHA/LDHB；我们构建了CRISPR Cas9 △ARNT的ARNT表达抑制细胞株，western blot证实：当ARNT表达抑制后，使LDHA/LDHB表达比例降低。说明ARNT可调控糖酵解基因表达。另一方面，为了验证CCR8能够调控ARNT，我们构建了CRISPR Cas9 △CCR8的CCR8表达抑制细胞株，western blot证实：当CCR8表达抑制后，ARNT的表达也随之受到抑制，同时，CCR8的表达降低，使LDHA/LDHB表达比例降低。LDHA在前列腺良性组织与癌组织中表达差异不显著（PCa:3.78±1.74 VS Non-cancerous:3.65±1.29, p=0.65），而LDHB在前列腺良性组织与癌组织中具有差异性表达（PCa: 3.26±0.14 VS Non-cancerous: 5.91±0.37, p＜0.01）。我们已初步统计组织芯片中LDHA、LDHB不同表达水平（即LDHA/LDHB比例）与生化复发时间相关。COX风险回归模型单因素分析中，LDHA是预测前列腺癌生化复发的风险因素，而LDHB则是前列腺癌生化复发的保护因素。