胃癌耐药细胞株SGC7901/阿霉素中miR-185的表达及对细胞多药耐药性的影响

Expression of miR-185 in Gastric Cancer Cell Line SGC7901 / ADR and Its Influence on the Drug Resistance of the Cells

目的检测miR-185在胃癌组织及细胞株中的表达,探讨miR-185在胃癌多药耐药性(MDR)发生机制中的作用。方法选取2014年3—5月于河北医科大学第四医院行胃癌切除术的20例患者胃癌及癌旁组织标本,荧光定量PCR技术检测胃癌及癌旁组织和人胃腺癌细胞株SGC7901、正常胃上皮细胞株GES-1、耐阿霉素(ADR)的胃癌MDR细胞株SGC7901/ADR的miR-185表达水平。采用miR-185模拟物或无关对照序列转染细胞株SGC7901/ADR,检测其对化疗药物ADR、5-氟尿嘧啶(5-FU)、草酸铂(L-OHP)的敏感性,荧光定量PCR和Western blotting技术检测多药耐药基因MDR1/P-gp、MRP-1、GST-π、LRP的mRNA及蛋白表达水平。结果癌旁组织和胃癌组织miR-185相对表达水平分别为(0.910±0.142)、(0.243±0.045),差异有统计学意义(t=20.025,P<0.001)。细胞株GES-1、SGC7901、SGC7901/ADR miR-185相对表达水平分别为(0.903±0.117)、(0.630±0.101)和(0.191±0.030),差异有统计学意义(F=46.850,P<0.001)。其中,细胞株SGC7901、SGC7901/ADR miR-185相对表达水平低于GES-1,细胞株SGC7901/ADR miR-185相对表达水平低于SGC7901(P<0.05)。ADR、5-FU和L-OHP对不同转染处理后的细胞株SGC7901/ADR抑制率比较,差异均有统计学意义(P<0.05)。其中,ADR、5-FU和L-OHP对miR-185模拟物转染的细胞株SGC7901/ADR抑制率高于未转染和无关对照序列转染(P<0.05)。经不同转染处理后,细胞株SGC7901/ADR多药耐药基因MDR1/P-gp、MRP-1和GST-πmRNA和蛋白相对表达水平比较,差异有统计学意义(P<0.05)。其中,miR-185模拟物转染的细胞株SGC7901/ADR多药耐药基因MDR1/P-gp、MRP-1和GST-πmRNA和蛋白相对表达水平低于未转染和无关对照序列转染(P<0.05)。结论胃癌细胞miR-185表达下调,通过上调miR-185的表达可提高胃癌细胞对化疗药物的敏感性,其机制可能是miR-185调控部分多药耐药基因的表达。

Objective To test the expression of miR-185 in gastric cancer tissues and cell lines and to investigate the mechanism of miR-185 on the MDR of gastric cancer. Methods Samples of gastric cancer and para -cancer tissues were obtained from 20 patients who accepted gastric cancer resection operation in the Fourth Hospital of Hebei Medical University from March to May in 2014. The expressions of miR-185 in gastric cancer tissues,para-cancer tissues,SGC7901,ADR resistant cell line SGC7901/ADR,gastric epithelial cell line GES -1 were tested with fluorescent quantitation PCR. Then SGC7901/ADR was transfected by miR -185 mimics or irrelevant control sequence, and sensitivity of transfected SGC7901/ADR to chemotherapeutic drugs（ ADR,5-FU,L-OHP）was measured. The expressions of mRNA and their protein in MDR related genes MDR1/P-gp,MRP-1,GST-π,LRP were determined by PCR and Western blotting. Results Relative expressions of miR-185 was（0. 910 ± 0. 142） in para-cancer tissues,and（0. 243 ± 0. 045） in cancer tissue,which showed significant difference（t=20. 025,P〈0. 001）. Relative expressions of miR -185 in GES -1,SGC7901,SGC7901/ADR cells were （0. 903 ± 0. 117）,（0. 630 ± 0. 101）,（0. 191 ± 0. 030） respectively,which also showed significant difference（ F=46. 850, P〈0. 001）. Among these cell lines,relative expressions of miR-185 in SGC7901,SGC7901/ADR were lower than that in GES-1,and expressions of miR -185 in SGC7901/ADR was lower than that in SGC7901（P 〈0. 05）. Inhibition rates of ADR,5-FU and L -OHP to SGC7901/ADR cells obviously varied with different transfecting methods （ P 〈0. 05 ）. The inhibitions rate of ADR,5 -FU and L -OHP to SGC7901/ADR transfected by miR -185 mimic were higher than those to SGC7901/ADR transfected by irrelevant control sequence or without transfection. By different transfecting methods,the mRNA and protein expression levels of MDR1/P - gp, MRP -1 and GST - π in SGC7901/ADR were significantly different（ P 〈0. 05 ）. The mRNA and protein expression levels of MDR1/P-gp,MRP-1 and GST-πin SGC7901/ADR transfected by miR-185 mimic were lower than those in SGC7901/ADR transfected by irrelevant control sequence or without transfection. Conclusion miR-185 is down-regulated in gastric cancr cells,and upregulating miR-185 can enhance sensitivity of gastric cancer cells to chemotherapeutic drugs. The mechanism may be that miR -185 could regulate the expression of some MDR genes.