p21活化酶5预测骨肉瘤化疗敏感性的实验研究

Experimental study of prediction of p21-actived kinase 5 on chemosensitivity of osteosarcoma

目的探讨骨肉瘤组织p21活化酶5(PAK5)表达与新辅助化疗敏感性的关系,从细胞水平验证PAK5是否参与调节骨肉瘤细胞化疗敏感性。方法采用免疫组化法检测74例骨肉瘤患者活检组织中PAK5表达,分析PAK5表达与骨肉瘤临床病理特征的关系。设计合成siRNA-PAK5,分别转染进入Saos-2和MG63细胞,QPCR和Westtem blotting分别检测PAK5 mRNA和蛋白的表达情况。以不同浓度梯度的阿霉素(ADM 50、25、5、0.5μmol/L)、甲氨蝶呤(MTX 50、5、1、0.5、0.05μmol/L)分别处理Saos-2和MG63细胞,CCK-8法检测细胞增殖抑制率,计算转染siRNA-PAK5后骨肉瘤细胞半数抑制浓度(IC_(50))的差异。结果 74例骨肉瘤组织中PAK5阳性57例,阴性17例,PAK5表达与新辅助化疗后肿瘤坏死率及肺转移有关(P<0.05)。QPCR检测转染组PAK5 mRNA分别水平下降为未转染组29%和31%;Western blotting结果显示转染组PAK5蛋白表达较未转染组下调49%和42%。转染siRNA-PAK5后,ADM作用Saos-2和MG63细胞的IC_(50)分别由(8.35±0.07)μmol/L、(7.35±0.07)μmol/L降至(0.54±0.004)μmol/L、(0.50±0.002)μmol/L(P<0.05);MTX作用Saos-2和MG63细胞的IC_(50)分别由(1.255±0.021)μmol/L、(1.05±0.014)μmol/L降至(0.606±0.01)μmol/L、(0.72±0.014)μmol/L(P<0.05)。结论骨肉瘤PAK5表达与患者新辅助化疗敏感性相关,抑制骨肉瘤细胞PAK5表达可提高化疗敏感性。

Objective To investigate the relationship between expression of p21-activated kinase 5 （PAK5）in osteosarcoma tissue and sensitivity of neoadjuvant chemotherapy, and whether the activation of PAK5 is involved in regulating the sensitivity of osteo- sarcoma ceils to chemotherapy at cellular level. Methods PAK5 expression in biopsy tissues of 74 osteosarcoma patients were detected by immunohistochemistry.The relationship between PAK5 expression and clinicopathological features of osteosarcoma was analyzed, siR- NA-PAK5 was design and synthezed, and it was transferred into Saos-2 and MG63 cells. PAK5 mRNA and protein was detected by QPCR and Western blotting. Saos-2 and MG63 cell were treated with different concentrations of adriamycin （ ADM 50, 25, 5, 0. 5μmol/L） and methotrexate（ MTX 50, 5, 1, 0. 5, 0. 05 μmol/L） respectively. Cell proliferation inhibtion rate was detected by CCK-8 assay. The difference of half inhibiton concentration （ IC50 ） of osteosarcoma cells after transfection of siRNA-PAK5 was analyzed. Re- suits In total of 74 os , PAK5 was positive in 57 case.The expression of PAK5 was related to the tumor necrosis rate after neoadjuvant chemotherapy and lung metastasis.The levels of PAK5 mRNA in the transfected group were decreased to 29% and 31% in the non transfection group, respectively ; Western blotting showed that the expression of PAK5 protein in transfected group was lower than that in non transfected group by 49% and 42%. After transfeced with siRNA-PAK5, the IC50 of ADM in Saos-2 and MG63 were decreased from（ 8.35±0. 07） μmol/L, （7. 35±0. 07） μmol/L to（0. 54±0. 004） μmol/L, （0. 50±0. 002） μmol/L, respectively;the ICs0 of MTX in Saos-2 and MG63 were decreased from（ 1. 255±0. 021 ）μmol/L, （ 1.05±0. 014） μmol/L to（ 0. 606±0. 01 ） μmol/L, （0.72±0. 014） μmol/L, respectively. Conclusion The expression of PAK5 in osteosareoma is related to the sensitivity of neoadjuvant chemotherapy, inhibiting the expression of PAK5 in osteosarcoma ceils, which can improve the sensitivity of chemotherapy.