miR-188-5p靶向PTEN对宫颈癌细胞SiHa顺铂化疗敏感性研究  

Effect of miR-188-5p on the sensitivity of cervical cancer cells SiHa tocisplatin chemotherapy by targeting PTEN

作　　者：钮红丽 牛琴 翟俊英 王韵一 马志宾 王莉[1] NIU Hong-li;NIU Qin;ZHAI Jun-ying;WANGYun-yi;MA Zhi-bin;WANG Li(Department of Reproductive Medicine,Nanyang First Peoples Hospital Affiliated to Henan University,Nanyang 473000,China)

机构地区：[1]河南大学附属南阳市第一人民医院生殖医学科,河南南阳473000

出　　处：《中华肿瘤防治杂志》2021年第15期1131-1137,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：河南省重点科技攻关项目(152102310328)。

摘　　要：目的探讨miR-188-5p对宫颈癌细胞SiHa顺铂(DDP)化疗敏感性的影响。方法 RT-PCR检测宫颈癌细胞株SiHa、耐药细胞株SiHa/顺铂(DDP)miR-188-5p、蛋白酪氨酸磷酸酶基因(PTEN)表达量,通过TargetScan/TargertScanS预测miR-188-5p靶基因PTEN并采用双色荧光素酶实验进行验证,构建miR-188-5p过表达(mimic组)、PTEN过表达(PTEN组)、miR-188-5p和PTEN双过表达(mimic+PTEN组)SiHa/DDP细胞并设置对照组(Control组),3-(4,5)-双甲基-2-噻唑-(2,5)-二苯基溴化四氮唑蓝(MTT)、5-溴脱氧尿嘧啶(BrdU)法、Transwell法、蛋白质印迹法检测DDP处理下细胞活力、增殖、侵袭能力及其相关蛋白表达。构建裸鼠移植瘤模型,比较30d存活率和肿瘤质量,免疫组化检测肿瘤组织Ki-67、P-糖蛋白(P-gp)蛋白表达。结果 Control组DDP的IC50值为49.80μmol/L、mimic组为24.96μmol/L、PTEN组为58.81μmol/L和mimic+PTEN组为40.66μmol/L。P-gp表达Control组(0.020±0.010)、mimic组(0.003±0.002)、PTEN组(0.100±0.030)和mimic+PTEN组(0.015±0.007)比较差异有统计学意义,Fmimic=30.952、P=0.001,FPTEN=24.836、P=0.001,Fmimic×PTEN=13.801、P=0.006;mimic组P-gp表达低于Control组,t=3.059,P=0.038;mimic+PTEN组P-gp表达低于PTEN组,t=4.867,P=0.008。mimic组与Control组比较,增殖阳性细胞数目、侵袭细胞数目、Ki-67、增殖细胞核抗原(PCNA)和N-钙黏蛋白(N-cadherin)蛋白表达均降低(P<0.05),E-钙黏蛋白(E-cadherin)表达升高,P<0.05;PTEN组与Control组比较,增殖阳性细胞数目、侵袭细胞数目、Ki-67、PCNA和N-cadherin蛋白表达均升高(P<0.05),E-cadherin蛋白表达降低,P<0.05;mimic+PTEN组与mimic组比较,增殖阳性细胞数目、侵袭细胞数目、Ki-67、PCNA和N-cadherin蛋白表达均升高(P<0.05),E-cadherin蛋白表达降低,P<0.05。Control组裸鼠存活率(46.67%)低于mimic组(86.67%),差异有统计学意义,χ2=5.136,P=0.023;mimic组Ki-67阳性率[(25.67±10.48)%]低于Control组[(76.17±15.84)%],差异有统计学意义,t=6.512,P<0.001;mimic�Objective To explore the effects of miR-188-5p on sensitivity of cervical cancer cells SiHa to cisplatin(DDP)chemotherapy.Methods The expression levels of cervical cancer cell line SiHa,drug-resistant cell line SiHa/DDPmiR-188-5p and protein tyrosine phosphatase gene(PTEN)were detected by RT-PCR.The miR-188-5p target gene PTENwas predicted by TargetScan/TargertScanS,and it was verified by dual-luciferase assay.SiHa/DDP cells with overexpres-sion of miR-188-5p(mimic group),PTEN(PTEN group),miR-188-5p and PTEN(mimic+PTEN group)were construc-ted.Control group was set up.Cells viability,proliferation and invasive abilities of cells,and expression of related proteinsunder DDP treatment were detected by 3-(4,5-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT),5-bromo-2-de-oxyuridine(BrdU),Transwell assay and Western blot.Xenograft models of nude mice were constructed.The 30 d survivalrate and tumors mass were compared.The expressions of Ki-67 and P-gp proteins in tumor tissues were detected by immunohistochemistry.Results IC_(50) values of DDP in Control group,mimic group,PTEN group and mimic+PTENgroup were 49.80,24.96,58.81 and 40.66 μmol/L,respectively.The differences in expression of P-glycoprotein(P-gp)were statistically significant among Control group,mimic group,PTEN group and mimic+PTEN group(0.020±0.010,0.003±0.002,0.100±0.030,0.015±0.007,F_(mimc)=30.952,P=0.001;F_(PTEN)=24.836,P=0.001 F_(mimic)×PTEN=13.801,P=0.006).P-gp expression in mimic group was lower than that in Control group(t=3.059,P=0.038).P-gp ex-pression in mimic+PTEN group was lower than that in PTEN group(t=4.867,P=0.008).Compared with Controlgroup,number of positive proliferation cells and invasion cells,expression of Ki-67,proliferating cell nuclear antigen(PC-NA)and N-cadherin were decreased in mimic group(P<0.05),while expression of E-cadherin was increased(P<0.05).Compared with Control group,number of positive proliferation cells and invasion cells,expression of Ki-67,PCNA andN-cadherin were increased in PTEN group(P<0.05),while

关 键 词：宫颈癌细胞 miR-188-5p 细胞增殖 细胞侵袭 耐药性 

分 类 号：R737.3[医药卫生—肿瘤]