肺癌患者外周血miR-214表达及对肺癌细胞凋亡和多药耐药的影响

Expression of miR-214 in peripheral blood in patients with lung cancer and its influence on cell apoptosis and multidrug resistance

目的探讨miR-214在肺癌患者外周血中的表达及其对肺癌细胞存活、凋亡、多药耐药的影响。方法小细胞肺癌(small cell lung cancer,SCLC)患者30例为SCLC组,非小细胞肺癌(non-small cell lung cancer,NSCLC)患者30例为NSCLC组,30例体检健康者为对照组,采用实时荧光定量PCR法检测3组外周血miR-214相对表达量。取对数生长期人SCLC H446细胞、人NSCLC A549细胞,人正常肺上皮BEAs-2B细胞,采用实时荧光定量PCR法检测3组细胞miR-214相对表达量;H446、A549细胞转染miR-214mimics质粒,BEAs-2B细胞不转染miR-214mimics质粒,于转染24h采用CCK-8法检测3组细胞存活率,TUNEL法检测细胞凋亡率,实时荧光定量PCR法检测P-糖蛋白(P-glycoprotein,P-gp)、肺耐药相关蛋白(lung resistance-related protein,LRP)、多药耐药相关蛋白(multidrug resistance-associated protein,MRP)mRNA相对表达量,Western blot法检测P-gp、LRP、MRP蛋白表达,MTT比色法检测阿霉素、长春新碱、依托泊苷、顺铂、吉西他滨处理48h的半数抑制剂量(the half maximal inhibitory concentration,IC50)。结果SCLC组、NSCLC组、对照组外周血miR-214相对表达量(0.572±0.024、0.711±0.048、1.001±0.001)依次增高(P<0.05);H446细胞、A549细胞、BEAs-2B细胞miR-214相对表达量(0.476±0.078、0.710±0.042、1.000±0.001)依次增高(P<0.05);转染24h,H446细胞、A549细胞、BEAs-2B细胞存活率[(43.5±5.2)%、(73.3±4.5)%、(100.0±0.1)%]依次增高(P<0.05),细胞凋亡率[(18.221±1.721)%、(13.523±1.825)%、(5.296±0.331)%]依次降低(P<0.05);转染24h,A549、H446、BEAs-2B细胞P-gp mRNA相对表达量(0.683±0.047、0.817±0.021、1.002±0.005)、LRP mRNA相对表达量(0.415±0.062、0.678±0.077、1.001±0.002)和MRP mRNA相对表达量(0.502±0.072、0.708±0.049、1.000±0.003)依次增高(P<0.05),P-gp蛋白相对表达量(1.225±0.022、1.763±0.021、2.401±0.032)、MRP蛋白相对表达量(1.325±0.072、1.662±0.068、1.872±0.074)依次增高(P<0.05);LRP蛋白相对表达量在A549、H446细胞(1.214±0.121、1.342±0.054)低于BEAs-2B细胞(1.825±0.0354)(P<0.05),A549细胞与H446细胞比较差异无统计学意义(P>0.05);阿霉素、长春新碱、依托泊苷、顺铂、吉西他滨作用48h,H446、A549细胞IC50值低于BEAs-2B细胞(P<0.05)。结论肺癌患者miR-214表达增高,miR-214可抑制肺癌细胞的增殖和多药耐药性,促进肺癌细胞凋亡。

Objective To investigate the expression of miR-214 in peripheral blood of patients with lung cancer patients and its influence on the proliferation,apoptosis and multidrug resistance of lung cancer cells.Methods The expression of miR-214 in peripheral blood was detected by real-time fluorescent quantitative PCR(RTq-PCR)in 30 patients with small cell lung cancer(SCLC group),30 patients with non-small cell lung cancer(NSCLC group),and 30 healthy volunteers(control group).The miR-214 expressions of SCLC H446 cells,NSCLC A549 cells and human normal lung epithelial BEAs-2 B cells in logarithmic growth phase were detected by RTq-PCR.H446 and A549 cells were transfected with miR-214 mimics plasmid,and BEAs-2 B cells were not transfected with miR-214 mimics plasmid.The cell survival rate was detected by CCK-8 method,the apoptotic rate was detected by TUNEL flow cytometry,the mRNA expressions of P-glycoprotein(P-gp),lung resistance-related protein(LRP)and multidrug resistance-associated protein(MRP)were detected by RTq-PCR,the expressions of P-gp,LRP and MRP proteins were detected by Western blot,the half maximal inhibitory concentration(IC50)values were detected by MTT colorimetric assay after treating with doxorubicin,vincristine,etoposide,cisplatin and gemcitabine for 48 h.Results The relative expressions of miR-214 in peripheral blood increased in turn in SCLC group,NSCLC group and control group(0.572±0.024,0.711±0.048,1.001±0.001)(P<0.05),as well as in H446 cells,A549 cells and BEAs-2 Bcells(0.476±0.078,0.710±0.042,1.000±0.001)(P<0.05).After transfection for 24 h,the survival rate increased((43.5±5.2)%,(73.3±4.5)%,(100.0±0.1)%)and the apoptotic rate decreased((18.221±1.721)%,(13.523±1.825)%,(5.296±0.331)%)gradually in turn in H446 cells,A549 cells and BEAs-2 Bcells(P<0.05);the relative expressions of P-gp mRNA(0.683±0.047,0.817±0.021,1.002±0.005),LRP mRNA(0.415±0.062,0.678±0.077,1.001±0.002)and MRP mRNA(0.502±0.072,0.708±0.049,1.000±0.003),P-gp protein(1.225±0.022,1.763±0.021,2.401±0.032)and MRP protein(1.325±0.072,1.662±0.068,1.872±0.074)increased gradually in turn in A549 cells,H446 cells and BEAs-2 Bcells(P<0.05).The relative expression of LRP protein was significantly lower in A549 cells(1.214±0.121)and H446 cells(1.342±0.054)than that in BEAs-2 Bcells(1.825±0.0354)(P<0.05),and showed no significant difference between A549 cells and H446 cells(P>0.05).After treating with doxorubicin,vincristine,etoposide,cisplatin and gemcitabine for 48 h,the IC50 values were significantly lower in H446 cells and A549 cells than those in BEAs-2 Bcells(P<0.05).Conclusion miR-214 increases in lung cancer patients.It can inhibit the proliferation and multi-drug resistance of lung cancer cells and promote apoptosis.