[微小RNA-145通过调节G蛋白偶联受体98抑制大肠癌细胞对奥沙利铂的耐药性]

[MiR-145 inhibits drug resistance to Oxaliplatin in colorectal cancer cells through regulating G protein coupled receptor 98].

作者信息

Fu Qiang, Cheng Jing, Zhang Jindai, Zhang Yonglei, Chen Xiaobing, Xie Jianguo, Luo Suxia

机构信息

Gastrointestinal Surgery Center, Henan Cancer Hospital (Affiliated Cancer Hospital of Zhengzhou University), Zhengzhou 450008, China.

Department of Medical Oncology, Zhengzhou Central Hospital (Zhengzhou Central Hospital Affiliated to Zhengzhou University), Zhengzhou 450002, China.

出版信息

Zhonghua Wei Chang Wai Ke Za Zhi. 2017 May 25;20(5):566-570.

PMID:28534337

Abstract

OBJECTIVE

To 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.

METHODS

L-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-OHP over-expressing miR-145 and HCT116/L-OHP. 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-OHP over-expressing GPR98 and HCT116/L-OHP. HCT116/L-OHP cells over-expressing both GPR98 and miR-145 (HCT116/L-OHP) 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.

RESULTS

HCT116/L-OHP cell line was successfully established with IC 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-OHP 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-OHP and 1.00±0.16 in HCT116/L-OHP (F=161.797, P=0.000). GPR98 was identified to be the target gene of miR-145. The relative mRNA and protein expressions of GPR98 in HCT116/L-OHP cells were 8.48±0.46 and 1.71±0.09, respectively, which were higher than those in HCT116/L-OHP (mRNA: 3.65±0.40, protein: 1.21±0.10) and HCT116/L-OHP (mRNA: 3.49±0.35, protein: 1.22±0.08; all P<0.05). The A value was 1.31±0.10, and the relative protein expressions of P-gp and MRP1 were 1.53±0.18 and 1.49±0.20 in HCT116/L-OHP cells, which were higher than those in HCT116/L-OHP (A value: 0.82±0.08, relative protein expression: 1.00±0.06 and 1.21±0.13, all P<0.05). The A value was 0.89±0.08, and the relative protein expressions of P-gp and MRP were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHP cells, which were higher than those in HCT116/L-OHP(A value: 0.20±0.05, relative protein expression: 0.20±0.07, 0.55±0.10, all P<0.05). The relative protein expression of PTEN in HCT116/L-OHP cells was 0.12±0.03, which was lower than 1.25±0.14 in HCT116/L-OHP cells(P<0.05). In addition, relative protein expressions of P-gp and MRP1 were 1.02±0.24 and 1.38±0.25 in HCT116/L-OHP cells, which were higher than those in HCT116/L-OHP cells (0.20±0.07 and 0.55±0.10), while PTEN expression in HCT116/L-OHP cells was lower as compared to HCT116/L-OHP cells (1.41±0.16 vs. 1.98±0.13, P<0.05).

CONCLUSION

MiR-145 inhibits drug resistance to L-OHP of HCT116 cells through suppressing the expression of target gene GPR98.

摘要

目的

预测并鉴定miR-145的靶基因，探讨miR-145抑制人结肠癌细胞对奥沙利铂（L-OHP）耐药的潜在机制。

方法

通过在细胞培养基中增加L-OHP浓度，体外建立L-OHP耐药人结肠癌细胞系（HCT116/L-OHP）。采用脂质体将miR-145模拟物及其阴性对照（NC-miRNA）转染至HCT116/L-OHP细胞，建立过表达miR-145的HCT116/L-OHP细胞和HCT116/L-OHP细胞。通过生物信息学分析预测miR-145的靶基因，并通过双荧光素酶活性测定进行验证。确定G蛋白偶联受体98（GPR98）为靶基因后，构建相应质粒并转染，建立过表达GPR98的HCT116/L-OHP细胞和HCT116/L-OHP细胞。通过修饰GPR98 cDNA与miR-145的结合位点，获得同时过表达GPR98和miR-145的HCT116/L-OHP细胞（HCT116/L-OHP）。采用CCK-8法评估HCT116/L-OHP细胞的增殖（A值）和对L-OHP的敏感性（IC50越低，敏感性越强）。采用实时定量PCR检测miR-145和GPR98的mRNA表达。采用蛋白质免疫印迹法检测GPR98和耐药相关蛋白的表达，如P-糖蛋白（gp）、多药耐药蛋白1（MRP1）、抑癌基因PTEN。

结果

成功建立HCT116/L-OHP细胞系，其IC为（42.34±1.05）mg/L，miR-145 mRNA表达为0.27±0.04，高于HCT116细胞中的（9.81±0.95）mg/L（t=39.784，P=0.000），低于1.00±0.09（t=13.021，P=0.000）。基于HCT116/L-OHP细胞，成功建立HCT116/L-OHP细胞，其miR-145相对表达为10.01±1.05，高于HCT116/L-OHP中的1.06±0.14和HCT116/L-OHP中的1.00±0.16（F=161.797，P=0.000）。鉴定GPR98为miR-145的靶基因。HCT116/L-OHP细胞中GPR98的相对mRNA和蛋白表达分别为8.48±0.46和1.71±0.09，高于HCT116/L-OHP（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；均P<0.05）。HCT116/L-OHP细胞的A值为1.31±0.10，P-gp和MRP1的相对蛋白表达分别为1.53±0.18和1.49±0.20高于HCT116/L-OHP（A值：0.82±0.08，相对蛋白表达：1.00±0.06和1.21±0.13，均P<0.05）。HCT116/L-OHP细胞的A值为0.89±0.08，P-gp和MRP的相对蛋白表达分别为1.02±0.24和1.38±0.25，高于HCT116/L-OHP（A值：0.20±0.05，相对蛋白表达：0.20±0.07，0.55±0.10，均P<0.05）。HCT116/L-OHP细胞中PTEN的相对蛋白表达为0.12±0.03，低于HCT116/L-OHP细胞中的1.25±0.14（P<0.05）。此外，HCT116/L-OHP细胞中P-gp和MRP1的相对蛋白表达分别为1.02±0.24和1.38±0.25，高于HCT116/L-OHP细胞（0.20±0.07和0.55±0.10），而HCT116/L-OHP细胞中PTEN的表达低于HCT116/L-OHP细胞（1.41±0.16 vs. 1.98±0.13，P<0.05）。