摘要
目的:研究miRNA-200c对人胃癌耐药SGC7901/DDP细胞顺铂敏感性的影响及其机制。方法:Western blotting检测SGC7901/DDP及其亲代SGC7901细胞E-cadherin、PTEN、p-Akt及总Akt蛋白的表达;瞬时转染miRNA-200c前体(miR-NA-200c precursor,Pre-200c)提高SGC7901/DDP细胞miRNA-200c的表达,MTT法检测转染后SGC7901/DDP细胞对顺铂的敏感性,并分析p-Akt表达的改变;应用Akt通路抑制剂LY94002处理SGC7901/DDP细胞抑制Akt磷酸化,检测处理后细胞对顺铂的敏感性。结果:与SGC7901细胞相比,SGC7901/DDP细胞中p-Akt蛋白的表达量显著增高(1.02±0.09 vs 0.17±0.02,P<0.05),E-cadherin、PTEN蛋白的表达量显著降低(0.10±0.03 vs 0.47±0.06,0.18±0.06 vs 0.87±0.06;均P<0.05)。转染Pre-200c后,顺铂对SGC7901/DDP细胞的IC50显著低于对照组[(7.52±0.19)vs(12.18±0.29)mg/L,P<0.05],细胞中p-Akt蛋白的表达量也显著低于对照组(0.22±0.04 vs 0.69±0.09,P<0.05);LY94002处理后,SGC7901/DDP细胞p-Akt蛋白的表达显著抑制(0.18±0.06 vs 0.66±0.10,P<0.05),顺铂对细胞的IC50显著低于对照组[(6.80±0.28)vs(11.94±1.73)mg/L,P<0.05]。结论:SGC7901/DDP细胞的耐药可能与E-cadherin和PTEN蛋白的表达缺失及Akt通路的异常激活有关,而miRNA-200c提高该细胞对顺铂的敏感性可能是通过抑制Akt通路而发挥作用。
Objective： To explore the effect of miRNA-200c on chemosensitivity of drug-resistant human gastric cancer SGC7901/DDP cells to cisplatin （DDP） and its mechanism. Methods： The expressions of E-cadherin, PTEN, p-Akt and total Akt proteins in SGC7901/DDP cells and its parental SGC7901 cells were detected by Western blotting, miRNA-200c precursor （Pre-200c） was transiently transfected into SGC7901/DDP cells to increase the expression of miRNA-200c. The drug sensitivity of cells to cisplatin and the expression level of p-Akt were detected by MTT assay and Western blotting, respectively. Finally, SGC7901/DDP cells were treated with LY94002 to inactivate the phosphorylation of Akt, and the sensitivity of SGC7901/DDP cells to DDP was detected. Results： Compared with SGC7901 cells, the expression of p-Akt protein in SGC7901/DDP cells was significantly increased （[ 1.02 ±0.09] vs [0.17 ±0.02], P 〈0.05）, while the ex- pressions of E-cadherin and PTEN protein were significantly decreased （[0.10 ±0.03 ] vs [0.47 ± 0.06 ] ; [0.18 ± 0. 06 ] vs [ 0.87 ± 0.06 ] respectively, P 〈 0.05 ）. The ICs0 of cisplatin in the Pre-200c tansfected group was significantly lower than that in the negative control group （ [ 7.52 ± 0.19 ] mg/L vs [ 12.18 ± 0.29 ] mg/L, P 〈 0.05 ）. Furthermore.the expression of p-Akt protein in the Pre-200c transfected group was significantly lower than that in the control group （ [ 0.22± 0.04 ] vs [ 0.69± 0.09 ], P 〈 0.05 ） ; the level of p-Akt protein was significantly inhibited （ [ 0.18 ± 0.06 ] vs [0.66±0.10], P 〈0.05） in SGC7901/DDP cells treated with LY94002. Moreover, the ICs0 of DDP in the LY94002 treated group was significantly lower than that in the control group （ [ 6.80 ±0.28 ] mg/L vs [ 11.94 ± 1.73 ] mg/L, P 〈 0.05 ）. Conclusion： Drug resistance phenotype of SGC7901/DDP cells may be associated with the loss of E-cadherin and PTEN proteins and abnormally activation of Akt signaling pathway, and the chemotherapeutic sensitization of miRNA-200c may be through the inactivation of Akt signal pathway.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2012年第6期582-587,共6页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.81172333)~~
