MiR-145调节G蛋白偶联受体98抑制结直肠癌细胞的奥沙利铂耐药性  

MiR-145 inhibits drug resistance to Oxaliplatin in colorectal cancer cells through regulating Gprotein coupled receptor 98

作　　者：付强[1] 成静[3] 张金岱[1] 张永磊[1] 陈小兵[2] 谢建国[1] 罗素霞[2] 

机构地区：[1]河南省肿瘤医院(郑州大学附属肿瘤医院)胃肠外科中心,450008 [2]河南省肿瘤医院(郑州大学附属肿瘤医院)消化肿瘤内科,450008 [3]郑州市中心医院(郑州大学附属郑州中心医院)肿瘤内科,450002

出　　处：《中华胃肠外科杂志》2017年第5期566-573,共8页Chinese Journal of Gastrointestinal Surgery

基　　金：河南省科技厅科技创新人才计划（201301014）

摘　　要：目的预测鉴定miR-145靶基因，探讨miR-145抑制结直肠癌细胞株HCT116奥沙利铂（L-OHP）耐药性的机制。方法在人结直肠癌细胞株HCT116的基础上，建立人结直肠癌L-OHP耐药细胞株HCT116/L-OHP。脂质体转染法将miR-145的模拟体miRNA-mimics及阴性对照NC-miRNA转染入HCT116/L-OHP，得到过表达miR-145的细胞株HCT116/L-OHPmimics及其阴性对照HCT116/L-OHPNC。预测miR-145的靶基因并用荧光素酶方法验证。确定靶基因为G蛋白偶联受体98（GPR98）后，构建质粒并转染细胞，得到过表达GPR98的HCT116/L-OHPGPR98细胞及其对照HCT116/L-OHPcontrol；同时通过更改GPR98 cDNA中与miR-145结合的序列，得到同时过表达GPR98和miR-145的HCT116/L-OHPmimics＋GPR98细胞。本研究采用CCK-8法检测细胞的增殖能力[吸光值（A）]值和对L-OHP的敏感性（被测拮抗剂的半抑制浓度IC50越低，代表对药物的敏感性越强）。实时定量PCR检测miR-145和GPR98的mRNA表达水平；western blot法检测GPR98和耐药相关蛋白P-糖蛋白（P-gp）、多药耐药蛋白1（MRP1）以及抑癌基因PTEN的蛋白表达水平。结果成功建立耐药细胞株HCT116/L-OHP[IC50：（42.34 ± 1.05）μg/ml，高于HCT116细胞的（9.81 ± 0.95）μg/ml，t= 39.784，P= 0.000；miR-145：0.27±0.04，低于HCT116细胞的1.00 ± 0.09，t= 13.021，P= 0.000]。在HCT116/L-OHP基础上，成功构建HCT116/L-OHPmimics细胞[miR-145：10.01 ± 1.05，高于HCT116/L-OHPNC（1.06 ± 0.14）和HCT116/L-OHP（1.00 ± 0.16），F= 161.797，P= 0.000]。GPR98经鉴定是miR-145的靶基因。HCT116/L-OHPGPR98细胞中GPR98的mRNA和蛋白相对表达分别为8.48 ± 0.46和1.71 ± 0.09，与HCT116/L-OHPcontrol（mRNA：3.65 ± 0.40；蛋白：1.21 ± 0.10）和HCT116/L-OHP（mRNA：3.49 ± 0.35；蛋白：1.22 ± 0.08）比较，差异有统计学意义（均P 〈 0.05）。HCT116/L-OHPGPR98细胞A450值为1.31 ± 0.10，高于HCT116/L-OHP（0.82±0.08，t=6.251，P=0.000）；HCT116/L-OHPmimics＋GPR98ObjectiveTo predict and identify the target gene of miR-145, and to explore the underlying mechanism of the inhibition of miR-145 on drug resistance to Oxaliplatin （L-OHP） in human colorectal cancer cells.MethodsL-OHP-resistant human colorectal cancer cell line （HCT116/L-OHP） was established in vitro by exposing to increased concentrations of L-OHP in cell culture medium. MiR-145-mimics and its negative control （NC-miRNA） were transfected into HCT116/L-OHP cells using liposome to establish HCT116/L-OHPmimics over-expressing miR-145 and HCT116/L-OHPNC. The target genes of miR-145 were predicted by bioinformatic analysis, and validated by dual luciferase activity assay. After determination of G protein coupled receptor 98 （GPR98） as target gene, corresponding plasmids were constructed and transfected to establish HCT116/L-OHPGPR98 over-expressing GPR98 and HCT116/L-OHPcontrol. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 （HCT116/L-OHPmimics＋GPR98） were acquired through modification of the binding sites of GPR98 cDNA with miR-145. CCK-8 assay was used to assess the proliferation （A value） and sensitivity to L-OHP （the lower the IC50, the stronger the sensitivity） in HCT116/L-OHP cells. Real-time quantitative PCR was used to measure the mRNA expression of miR-145 and GPR98. Western blot was used to examine the protein expression of GPR98 and drug-resistant associated protein, such as P-glycoprotein （gp） , multiple drug-resistance protein 1 （MRP1） , cancer-inhibition gene PTEN.ResultsHCT116/L-OHP cell line was successfully established with IC50 of （42.34 ± 1.05） mg/L and miR-145 mRNA expression of 0.27±0.04, which was higher than （9.81 ± 0.95） mg/L （t= 39.784, P= 0.000） and lower than 1.00 ± 0.09 （t= 13.021, P= 0.000） in HCT116 cells. Based on HCT116/L-OHP cells, HCT116/L-OHPmimics cells were established successfully, with relative miR-145 expression of 10.01 ± 1.05, which was higher than 1.06 ± 0.14 in HCT116/L-OHPNC and 1.00 ± 0.16 in HCT116/L

关 键 词：结直肠肿瘤 G蛋白偶联受体98 MIR-145 HCT116细胞株:奥沙利铂 

分 类 号：R735.3[医药卫生—肿瘤]