长链非编码RNA AFAP1-AS1在肺腺癌吉非替尼耐药细胞中的作用及机制研究

Role and mechanism of long non-coding RNA AFAP1-AS1 in Gefitinib resistant lung adenocarcinoma cells

目的研究长链非编码RNA AFAP1-AS1对非小细胞肺癌耐吉非替尼细胞株PC9/GR耐药性的影响及其可能的作用机制。方法采用不同浓度的吉非替尼处理亲本PC9和耐药PC9/GR细胞,CCK-8法检测细胞增殖抑制率,计算半数致死量(IC_(50))。应用qRT-PCR检测AFAP1-AS1在吉非替尼耐药PC9/GR及亲本细胞株PC9、H1975中AFAP1-AS1的表达水平。将Scrambled和si-AFAP1-AS1转染PC9/GR细胞,qRT-PCR检测干扰效率,CCK-8法检测对照组(Scrambled组)和干扰组(si-AFAP1-AS1组)的IC_(50)。分别应用CCK-8法、克隆形成实验、流式细胞术检测PC9/GR对照组和干扰组细胞对吉非替尼药物敏感性、联合吉非替尼处理后细胞增殖能力、细胞凋亡率及细胞周期分布;Western blot检测对照组和干扰组PC9/GR细胞E-cadherin的蛋白表达水平。结果AFAP1-AS1在PC9/GR耐药细胞中的表达量高于PC9亲本细胞,差异有统计学意义(P<0.05);PC9/GR干扰组细胞的IC_(50)、增殖能力低于对照组,细胞凋亡率高于对照组,差异有统计学意义(P<0.05);干扰组细胞周期阻滞在G_(0)/G_(1)期比例高于对照组,差异有统计学意义(P<0.05)。Western blot结果显示,与对照组比较,干扰AFAP1-AS1表达的干扰组PC9/GR细胞E-cadherin表达水平明显升高。结论干扰AFAP1-AS1表达可能通过抑制细胞增殖,诱导细胞凋亡,上调E-cadherin蛋白表达而逆转PC9/GR细胞对吉非替尼的耐药性。

Objective To investigate the effect of long non-coding RNA AFAP1-AS1 on the resistance of PC9/GR in non-small cell lung cancer cell lines resistant to Gefitinib and its possible mechanism.Methods Parental PC9 and drug-resistant PC9/GR cells were treated with Gefitinib at different concentrations.The cell proliferation inhibition rate was detected by CCK-8 method,and the inhibitory concentration to produce 50%cell death(IC_(50))was calculated.The expression level of AFAP1-AS1 in Gefitinib-resistant PC9/GR and parent cell lines PC9 and H1975 was detected by qRT-PCR.PC9/GR cells were transfected with Scrambled and si-AFAP1-AS1,interference efficiency was detected by qRT-PCR,IC_(50)of control group(Scrambled)and interference group(si-AFAP1-AS1)was detected by CCK-8 method.CCK-8 assay,clonogenesis assay and flow cytometry were used to detect the sensitivity of PC9/GR control group and interference group to Gefitinib,cell proliferation ability,apoptosis rate and cell cycle distribution after combined treatment with Gefitinib.The expression of E-cadherin protein in PC9/GR cells of control group and interference group was detected by Western blot.Results The expression of AFAP1-AS1 in PC9/GR resistant cells was higher than that in PC9 parent cells,and the difference was statistically significant(P<0.05).The IC_(50)and proliferation capacity of cells in PC9/GR interference group were lower than those in control group,and the apoptosis rate was higher than that in control group,with statistical significances(P<0.05).The proportion of cell cycle arrest in G_(0)/G_(1)phase in interference group was higher than that in control group,and the difference was statistically significant(P<0.05).Western blot results showed that compared with the control group,the interference group interfered with AFAP1-AS1 expression of PC9/GR cells E-cadherin expression level increased significantly.Conclusion Knockdown AFAP1-AS1 can reverse the resistance of PC9/GR cells to Gefitinib by inhibiting cell proliferation,inducing apoptosis,and up-regulating the expression of E-cadherin protein.