Abstract: Background　Current studies indicate that PAIP1 is highly expressed in various tumors, yet research on its role in breast cancer remains scarce, and the underlying mechanisms are not yet fully understood. Objective　To investigate the expression of PAIP1 protein in breast cancer and its relationship with clinicopathological characteristics, and to explore the role of PAIP1 in the biological functions of breast cancer cell proliferation, invasion and glycolysis. Methods　Collect data from the GEO database (841 cases of breast cancer tissue and 631 cases of adjacent tissue) and the TCGA database (1 060 cases of breast cancer tissue and 111 cases of adjacent tissue, including different clinical stages of breast cancer: stage Ⅰ , 351 cases; stage Ⅱ , 379 cases; stage Ⅲ , 364 cases; stage Ⅳ , 77 cases) to analyze PAIP1 mRNA expression. Enroll 159 patients with invasive breast cancer who were treated in the Breast Surgery Department of Yanbian University Hospital from February 2012 to March 2013 as the study subjects. Collect surgical specimens of cancerous and adjacent tissues, and detect PAIP1 expression using immunohistochemistry. Transfect small hairpin RNA (sh-RNA) to silence PAIP1 expression in two breast cancer cell lines, SK-BR-3 and MCF-7, and divide the cells into three groups: the blank control group, the PAIP1-shRNA group, and the PI3K activator (740Y-P)+PAIP1-shRNA group. Use the Cell Counting Kit-8 (CCK8) method and plate cloning experiments to assess the proliferative capacity of the two breast cancer cell lines. Conduct scratch assays and cell migration experiments to evaluate the migration ability of the two cell lines. Employ Western blotting to examine the expression of proteins related to the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, as well as E-cadherin and vimentin. Measure ATP, lactate, and glucose levels in the two breast cancer cell lines using colorimetric methods. Results　According to the immune histochemical analysis results from the GEO and TCGA databases and clinical collected case specimens, the PAIP1 mRNA expression levels in adjacent tissues were consistently lower than those in breast cancer tissues (P<0.05). Additionally, TCGA data analysis revealed that PAIP1 mRNA expression levels in clinical stage II were lower than those in stages III-IV (P<0.05). CCK8 and colony formation assays demonstrated that in both breast cancer cell lines, the PAIP1-shRNA group exhibited lower proliferation capacity and colony formation counts compared to the Control group (P<0.05), while the 740Y-P+PAIP-shRNA group showed higher values than the PAIP1-shRNA group (P<0.05). Scratch and cell migration assays indicated that in both breast cancer cell lines, the PAIP1-shRNA and 740Y-P+PAIP-shRNA groups displayed reduced lateral and longitudinal migration capabilities compared to the Control group (P<0.05), with the 740Y-P+PAIP-shRNA group showing higher migration than the PAIP1-shRNA group (P<0.05). Western blot results revealed that in both breast cancer cell lines, the PAIP-shRNA group exhibited lower protein expression levels of phosphorylated PI3K (p-PI3K), phosphorylated AKT (p-AKT), phosphorylated mTOR (p-mTOR), Vimentin, hexokinase 2 (HK2), glucose transporter 2 (GLUT2), glucose transporter 3 (GLUT3), and lactate dehydrogenase (LDH) compared to the Control group (P<0.05), while E-cadherin levels were higher (P<0.05). The 740Y-P+PAIP-shRNA group showed higher protein expression levels of p-PI3K, p-AKT, p-mTOR, Vimentin, HK2, GLUT2, GLUT3, and LDH than the PAIP1-shRNA group (P<0.05), but lower E-cadherin levels (P<0.01). Additionally, HK2, GLUT2, GLUT3, and LDH expression levels were lower than those in the Control group (P<0.05). Glucose, lactate, and ATP detection results showed that in both breast cancer cell lines, the PAIP1-shRNA and 740Y-P+PAIP-shRNA groups exhibited lower ATP, glucose, and lactate levels than the Control group (P<0.05), with the 740Y-P+PAIP-shRNA group showing higher levels than the PAIP1-shRNA group (P<0.01). Conclusion　PAIP1 protein is highly expressed in breast cancer tissue. Silencing PAIP1 inhibits the proliferation, migration and glycolysis of breast cancer cells.

Key words: Breast cancer, PAIP1, Proliferation, Glycolysis, Analysis of variance

摘要： 背景　多聚腺苷酸结合相互作用蛋白1(PAIP1)是哺乳动物特有的蛋白质，在多种肿瘤中高表达。研究显示，PAIP1在肿瘤细胞的增殖和周期进程中发挥重要调控作用。但PAIP1在乳腺癌组织中的表达及其对乳腺癌细胞的影响尚不明确。目的　探讨PAIP1与乳腺癌临床病理特征的关系以及PAIP1在乳腺癌细胞增殖、侵袭和糖酵解等生物学功能中的作用。方法　收集GEO数据库（841例乳腺癌组织和631例癌旁组织）和TCGA数据库（乳腺癌组织1060例和癌旁组织111例，不同乳腺癌临床分期包括Ⅰ期351例，Ⅱ期379例，Ⅲ期364例，Ⅳ期77例）数据资料，分析PAIP1 mRNA表达情况。纳入2012年2月—2013年3月就诊于延边大学附属延边医院乳腺外科的159例浸润性乳腺癌患者为研究对象，收集患者手术切除癌组织及癌旁组织标本，使用免疫组化法检测PAIP1的表达情况。通过转染小发夹RNA（sh-RNA）沉默2株乳腺癌细胞SK-BR-3和MCF-7中PAIP1的表达，并将细胞分为空白组（Control）、PAIP1-shRNA组和PI3K激活剂（740Y-P）+PAIPsh-RNA组。采用细胞计数试剂盒-8（CCK8）法和平板克隆实验检测2株乳腺癌细胞中增殖能力，通过划痕实验和细胞迁移实验检测2株乳腺癌细胞迁移能力；采用蛋白印迹法检测2株乳腺癌细胞中磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（AKT）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路相关蛋白和及上皮钙黏蛋白（E-Cadherin）、波形蛋白（Vimentin）表达情况，通过比色法检测2株乳腺癌细胞ATP、乳酸以及葡萄糖水平。结果　GEO和TCGA数据库及临床收集病例标本免疫组化分析结果显示，癌旁组织PAIP1 mRNA表达量均低于乳腺癌组织（P<0.05），且TCGA数据分析结果显示，临床Ⅱ期PAIP1 mRNA表达量低于Ⅲ~Ⅳ期（P<0.05）。CCK8和平板克隆实验结果显示，2株乳腺癌细胞中，PAIP1-shRNA组增殖能力和集落形成数低于Control组（P<0.05），740Y-P+PAIP-shRNA组高于PAIP1-shRNA组（P<0.05）。划痕实验和细胞迁移实验结果显示，2株乳腺癌细胞中，PAIP1-shRNA组和740Y-P+PAIP-shRNA组横向及纵向迁移能力均低于Control组（P<0.05），740Y-P+PAIP-shRNA组高于PAIP1-shRNA组（P<0.05）。蛋白印迹结果显示，2株乳腺癌细胞中，PAIP-shRNA组磷酸化PI3K（p-PI3K）、磷酸化AKT（p-AKT）、磷酸化mTOR（p-mTOR）、Vimentin以及己糖激酶2（HK2）、葡萄糖转运蛋白2（GLUT2）、葡萄糖转运蛋白3（GLUT3）、乳酸脱氢酶（LDH）蛋白表达水平均低于Control组（P<0.05），E-cadherin的蛋白水平高于Control组（P<0.05）。740Y-P+PAIP-shRNA组p-PI3K、p-AKT、p-mTOR、Vimentin、HK2、GLUT2、GLUT3、LDH蛋白表达水平高于PAIP1-shRNA组（P<0.05），E-cadherin的蛋白水平低于PAIP1-shRNA组（P<0.01），且HK2、GLUT2、GLUT3、LDH蛋白表达水平低于Control组（P<0.05）。葡萄糖、乳酸以及ATP检测结果显示，2株乳腺癌细胞中，PAIP1-shRNA组和740Y-P+PAIP-shRNA组ATP、葡萄糖和乳酸水平水平均低于Control组(P<0.05)，740Y-P+PAIP-shRNA组高于PAIP1-shRNA组（P<0.01）。结论　PAIP1蛋白在乳腺癌组织中高表达，沉默PAIP1通过抑制PI3K/AKT/mTOR信号通路，抑制乳腺癌细胞增殖、迁移以及糖酵解。

关键词: 乳腺癌, PAIP1, 增殖, 糖酵解, 方差分析