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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 investigate the effect of miRNA-200c on cisplatin sensitivity of human gastric cancer cell line SGC7901 / DDP and its mechanism. Methods: The expressions of E-cadherin, PTEN, p-Akt and total Akt protein in SGC7901 / DDP and its parental SGC7901 cells were detected by Western blotting. The transient transfection of miR-NA-200c precursor (Pre-200c) The expression of miRNA-200c in SGC7901 / DDP cells was detected by MTT assay. The sensitivity of transfected SGC7901 / DDP cells to cisplatin was detected and the expression of p-Akt was analyzed. The Akt phosphorylation was inhibited by Akt pathway inhibitor LY94002 The sensitivity of cells to cisplatin after the treatment was tested. 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), and the expression of E-cadherin and PTEN proteins was significantly decreased 0.03 vs 0.47 ± 0.06,0.18 ± 0.06 vs 0.87 ± 0.06; all P <0.05). After transfection with Pre-200c, the IC50 of cisplatin on SGC7901 / DDP cells was significantly lower than that of the control group [(7.52 ± 0.19) vs (12.18 ± 0.29) mg / L, P <0.05] (0.22 ± 0.04 vs 0.69 ± 0.09, P <0.05). After treatment with LY94002, the expression of p-Akt in SGC7901 / DDP cells was significantly inhibited (0.18 ± 0.06 vs 0.66 ± 0.10, P <0.05) , And the IC50 of cisplatin on cells was significantly lower than that of the control group [(6.80 ± 0.28) vs (11.94 ± 1.73) mg / L, P <0.05]. CONCLUSIONS: The drug resistance of SGC7901 / DDP cells may be related to the loss of expression of E-cadherin and PTEN protein and the abnormal activation of Akt pathway. However, the effect of miRNA-200c on cisplatin sensitivity may be mediated through inhibition of Akt pathway .