微小RNA-17-5p在脑恶性胶质瘤患者血清和肿瘤组织的表达及其对U87MG和U251体外作用

MicroRNA-17-5p expression in tumor tissue and serum of glioblastomas patients and function analysis of microRNA-17-5p on U87MG and U251 in vitro

目的观察恶性胶质瘤患者肿瘤组织与血清微小RNA（miRNA，miR）-17-5p的表达，以及miR-17-5p对恶性胶质瘤细胞株U87MG和U251的体外作用。方法通过实时定量反转录聚合酶链反应（RT-qPCR）检测恶性胶质瘤患者肿瘤组织与血清miR-17-5p的表达水平；将U87MG和U251细胞用化疗药物20 μmol/L顺铂（DDP），50 μmol/L替莫唑胺或30 Gy伽马射线处理后检测miR-17-5p表达变化；将U87MG和U251细胞转染miR-17-5p mimic，并用无血清培养，检测miR-17-5p在无血清条件对细胞存活的作用；将U87MG和U251细胞转染miR-17-5p mimic，测定其细胞增殖、细胞迁移、集落形成以及肿瘤蛋白p53可诱导核蛋白1（TP53INP1）、Tripartite基序蛋白8（TRIM8）和含BTB结构域的锌指蛋白（ZBTB4）蛋白表达量的变化。结果通过RT-qPCR检测可见恶性胶质瘤患者肿瘤组织（7.40±1.55）和血清（2.70±0.55） miR-17-5p的表达较癌旁组织和健康人血清明显升高（P=0.019和P=0.028），同时采用化疗药物20 μmol/L DDP、50 μmol/L替莫唑胺或30 Gy伽马射线处理U87MG和U251细胞后，其miR-17-5p的表达量也明显上升（P=0.001）。将U87MG和U251细胞转染miR-17-5p mimic，并用无血清培养结果表明，miR-17-5p能够促进U87MG和U251在无血清条件下的细胞存活，同时检测显示miR-17-5P在低浓度（2.5%）血清下可以促进U87MG和U251细胞迁移[（20.50±2.33） mm和（14.50±1.35） mm]，促进U87MG和U251集落形成[（225±45）个和（312±58）个]，而在高浓度血清（10%）条件下，miR-17-5p并不能促进U87MG和U251细胞迁移，反而会抑制细胞增殖，表明miR-17-5p能够降低抑癌基因TP53INP1、TRIM8和ZBTB4蛋白表达。结论恶性胶质瘤患者血清和组织miR-17-5p表达增加，同时miR-17-5p对于恶性胶质瘤细胞株U87MG和U251在不良环境中的细胞生存具有促进作用。

Objective To study microRNA （miRNA, miR）-17-5p expression in the tumor tissue and serum of glioblastomas patients and the function of miR-17-5p on U87MG and U251 in vitro.Methods The miR-17-5p expression in the tumor tissue and serum of glioblastomas patients was detected by real-time quantitative reverse transcriptase-polymerase chain reaction （RT-qPCR）. U87MG and U251 cells were treated with 20 μmol/L Cisplatin （DDP）, 50 μmol/L Temozolomide or 30 Gy gamma ray irradiation and the expression level of miR-17-5p was evaluated. U87MG and U251 cells were transfected with miR-17-5p mimic and cultured in serum free medium to analyze the ability of miR-17-5p to promote cell survival. U87MG and U251 cells were transfected with miR-17-5p mimic, then the cell proliferation, cell migration, colony formation, and the protein expression levels of tumor protein p53 inducible nuclear protein 1 （TP53INP1）, tripartite motif protein 8 （TRIM8） and zinc finger and BTB domain-containing protein 4 （ZBTB4） were evaluated.Results The miR-17-5p expression level in the tumor tissue （7.40±1.55） and serum （2.70±0.55） in glioblastomas patients was significantly elevated compared with tumor adjacent tissue and health human serum （P=0.019, P=0.028）, and that in U87MG and U251 cells was increased after treatment with 20 μmol/L DDP, 50 μmol/L Temozolomide or 30 Gy gamma ray irradiation （P=0.001）. MiR-17-5p could promote the survival of U87MG and U251 cells when cultured in serum free medium, at the same time, under the low serum condition [2.5% fatal bovine serun （FBS）], miR-17-5p could promote the cell migration[（20.50±2.33） mm and （14.50±1.35） mm] and colony formation [（225±45） cells and （312±58） cells] of U87MG and U251 cells, but in normal serum level （10% FBS）, it could not promote the cell migration of U87MG and U251 cells, on the contrary, it could suppress the cell proliferation of U87MG and U251 cells. Western blotting results revealed that miR-17-5p could suppress the protein expression of tumor suppressor gene TP53INP1, TRIM8 and ZBTB4.Conclusion The expression level of miR-17-5p in the tumor tissue and serum of gliolbastomas patients was significantly upregulated. miR-17-5p could be used as a biomarker in glioblastomas detection. At the same time, miR-17-5p could promote the survival of U87MG and U251 cells under stress conditions.