调控胎盘生长因子表达对骨肉瘤耐药逆转作用研究

目的胎盘生长因子(placenta growth factor,PlGF)是一种属于血管内皮生成因子家族的分泌性同二聚体糖蛋白,对肿瘤等病理情况下的血管生成有重要作用,其在影响骨肉瘤耐药中的作用研究较少。本研究应用RNA干扰下调PlGF基因表达在逆转骨肉瘤耐药中的作用。方法将骨肉瘤耐药MG-63细胞分为阴性对照组、空白对照组和转染组,转染组转染PlGFshRNA真核表达载体,空白对照组只添加空白转染剂,阴性转染组转染阴性对照RNA干扰试剂,实时荧光定量PCR和蛋白质印迹法分别检测转染前后PlGF基因和蛋白的表达变化,流式细胞仪检测细胞周期和细胞凋亡,细胞计数盒8法检测各组细胞对化疗药物的敏感性。结果PlGFshRNA可有效抑制MG-63骨肉瘤耐药细胞中PlGF基因和蛋白表达,PlGF基因下调71.3%(F=17.302,P=0.031),蛋白表达下调67.2%,F=27.112,P=0.023。转染组细胞凋亡高于空白对照组和阴性对照组,差异有统计学意义,F=22.34,P=0.037。3组细胞在加入多柔比星后增殖均具有不同程度的抑制,且呈明显的时间-效应关系,但转染组细胞的存活率均低于其他2组,差异有统计学意义,F=15.22,P=0.007。结论应用RNA干扰技术下调耐药MG-63骨肉瘤细胞PlGF基因表达后,可有效促进耐药细胞的凋亡,从而增加其对化疗药物的敏感性,PlGF基因有可能成为骨肉瘤耐药治疗的新靶点。

Silencing the PlGFgene expression by RNA interference enhanced the chemotherapeutic sensitivity of drug resistant MG-63 osteosarcoma cells

OBJECTIVE Placenta growth factor(PlGF)is a dimeric glycoprotein,and is structurally and functionally associated with VEGF.PlGFis not produced by the majority of types of normal human tissue and serves a function in pathological angiogenesis.To date,the effect of PlGFon drug resistance of osteosarcoma remains unknown.This study aimed to investigate the inhibiting effect of silencing of PlGFgene expression by RNA interference on drug-resistance of osteosarcoma cell.METHODS The osteosarcoma drug resistance cells were divided into siRNA group,negative control group and blank control group.The technology of gene recombination was used to construct the eukaryotic expression vector pSilencer3.1-H1 neo-PlGF.Then the pSilencer3.1-H1 neo-PlGF was transfected into human osteosarcoma MG-63 drug-resistant cells using liposome transfection reagents.The expressions of PlGF mRNA and protein were determined by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot respectively.The apoptosis of cell was determined using flow cytometry.The chemosensitivity of MG-63 drug-resistant cell to doxorubicin was performed by CCK8.RESULTS The recombinant eukaryotic expression vector pSilencer3.1-H1 neo-PlGF was successfully constructed.Compared with control groups,the expression of PlGF mRNA and protein were both significantly decreased in the transfected MG-63 drug-resistant cell(71.3%and 67.2%respectively,P=0.031 and P=0.023).The flow cytometry analysis showed there was higher percentage of apoptosis in transfected MG-63 drug-resistant cellsiRNA group(8.46±3.2);blank control group(4.72±1.1);negative control group(4.72±1.1),F=22.34,P=0.037].We also observed that suppression of PlGFexpression in osteosarcoma drug-resistant cells increased their chemosensitivity to doxorubicinsiRNA group(62.11±12.53)%;blank control group(91.32±13.44)%;negative control group(90.32±12.43)%,F=15.22,P=0.007].CONCLUSIONS This study showed that PlGFshRNA increased the apoptosis and increased the sensitivity of MG-63 drug-resistant cell to chemotherapy.Our data suggest that PlGFis involved in drug resistance of human osteosarcoma and may serve as a promising therapeutic target for osteosarcoma.