PTTG沉默通过下调糖酵解相关酶抑制卵巢癌细胞增殖

摘要

Objective To explore effect of pituitary tumor transforming gene ( PTTG ) on ovarian cancer cells proliferation and its mechanism.Methods From 2010 to 2013 totally 68 pathological samples were collected from cases of ovarian carcinoma with complete clinical data in Xi'an Fourth Hospital .Relationship between PTTG and aerobic glycolytic correlative enzymes protein including hexokinase (HK), phosphofructokinase (PFK), pyruvate phosphokinase 2 (PKM2), lactate dehydrogenase (LDHA) and Glucose transporter 1 ( GLUT-1) was detected by immunohistochemistry and Western blotting from the aspects of tissue and cell .Stable PTTG silenced cell lines were established by downregulating PTTG with lentivirus interference vector .Cellular proliferating ability was detected by MTT and soft agarose clone trial .Protein expression levels of correlative enzymes involving in aerobic glycolysis after PTTG knockdown and glucose transporter ( GLUT) were detected with Western blotting , and the mechanism was explored .Results PTTG expression levels showed positive correlation with enzymes involving in aerobic glycolysis including HK , PFK, PKM2, LDHA and GLUT-1 on ovarian cancerous tissue and cell levels (r value ranged from 0.839 to 0.987, P<0.01).The proliferating ability of ovarian cancer cells were inhibited correspondingly when suppressing PTTG by lentivirus interference vector , and the protein levels of correlative enzymes involving in aerobic glycolysis reduced significantly (gray level before and after interference was 2.212 ±0.36 and 0.782 ±0.18, respectively,t=2.36, P<0.01).The expression levels of PTTG, LDHA, PKM2 and GLUT-1 assayed by Western blotting were similar to immunohistochemistry results between normal tissue and ovarian cancer tissue .PTTG protein level showed subtle expression ( gray level was 0.705 ±0.13,n=13)but high expression level in ovarian cancer tissue (gray level was 2.616 ±0.33,n=58),and it presented positive correlation with malignant differentiation(r=0.908,P=0.012).Conclusion PTTG could inhibit metabolism of acrobic glycolysis and thus inhibit rapid proliferation ovarian cancer cells probably through downregualting protein expression levels of correlative enzymes involving in aerobic glycolysis.%目的：探讨原癌基因垂体肿瘤转化基因（ PTTG）对卵巢癌细胞增殖的影响及其机制。方法收集西安市第四医院医院2010至2013年有完整病例资料的卵巢癌手术患者病理标本68例。通过免疫组织化学和Western blotting方法，从组织和细胞水平检测PTTG与有氧糖酵解相关酶包括己糖激酶（ HK ）、磷酸果糖激酶（ PFK ）、丙酮酸激酶2（ PKM2）、乳酸脱氢酶 A （LDHA）和葡萄糖转运子1（GLUT-1）表达水平间的关系；利用慢病毒干扰载体下调PTTG表达，建立PTTG沉默的稳定卵巢癌细胞株，通过MTT、软琼脂糖克隆实验检测细胞的增殖能力改变；Western blotting检测PTTG沉默后有氧糖酵解相关酶和葡萄转运子的表达水平变化，并探讨其内在机制。结果通过组织和细胞水平检测发现，PTTG的表达水平与有氧糖酵解相关酶HK、PFK、PKM2、LDHA及GLUT-1表达水平呈正相关性（相关系数r＝0．839～0．987区间，均P＜0．01），慢病毒干扰载体沉默PTTG后，卵巢癌细胞的增殖能力显著受到抑制，有氧糖酵解相关酶的表达水平也显著下降（干扰前后灰度2．212±0．36 vs 0．782±0．18，t＝2．36，P＜0．01）。 Western blotting检测正常卵巢及卵巢癌组织PTTG、LDHA、PKM2和GLUT－1的表达情况，结果与免疫组化结果相一致，即在正常的卵巢组织中PTTG表达微弱（灰度为0．705±0．13，n＝13），而在卵巢癌组织中呈现高表达（灰度为2．616±0．33，n＝58），并与卵巢癌组织的恶性分化呈正相关性（Pearson检验r＝0．908，P＝0．012）。结论 PTTG可能通过降低有氧糖酵解反应过程中的相关调节酶的表达水平，而实现其抑制有氧糖酵解代谢的作用，从而抑制卵巢癌细胞的快速增殖。