下调HOTAIR通过提高PTEN表达逆转HCC827细胞吉非替尼耐药

Down-regulation of HOTAIR Reverses the Resistance of Gefitinib in HCC827 Cells by Increasing the Expression of PTEN

背景与目的肺癌是发病率和死亡率最高的恶性肿瘤,其中80%以上为非小细胞肺癌。HOX转录反义RNA(HOX transcript antisense RNA, HOTAIR)异常表达于多种肿瘤组织,并参与调控肺癌的发生与发展,本研究旨在探讨下调HOTAIR对肺腺癌HCC827细胞对吉非替尼药物耐药的影响及其机制。方法应用实时荧光定量PCR(quantitative real-time PCR, RT-qPCR)法检测HCC827细胞及HCC827吉非替尼耐药细胞(HCC827GR)中HOTAIR的表达情况,利用生物信息学分析预测HOTAIR靶基因,应用脂质体转染法将体外合成的针对HOTAIR的siRNA转染入HCC827GR细胞中,采用RT-qPCR及Western blot方法检测其HOTAIR及PTEN、PI3K、AKT的表达水平,同时采用MTT法检测各组细胞的吉非替尼半数抑制浓度(50%inhibitory concentration, IC_(50)),运用流式细胞术分析各组细胞凋亡率的变化。结果 RT-qPCR结果显示HOTAIR在HCC827GR耐药细胞及吉非替尼耐药患者血清中表达增高,利用生物信息学分析预测到PTEN的为HOTAIR潜在靶基因。RT-qPCR及Western blot结果显示HCC827GR细胞经转染HOTAIR siRNA后,HOTAIR表达降低(P<0.05),而PTEN表达升高,PI3K及AKT表达下降(P<0.05);与对照组比较,下调HOTAIR的HCC827GR细胞对吉非替尼的IC_(50)值下降(P<0.05),细胞增殖能力下降、凋亡率升高(P<0.05)。结论下调HOTAIR表达可抑制HCC827GR细胞增殖,促进其凋亡,并可降低HCC827GR细胞的吉非替尼IC_(50),下调HOTAIR后PTEN表达升高,而PI3K及AKT表达下降提示下调HOTAIR可通过靶向调控PTEN/PI3K/AKT通路逆转HCC827GR细胞对吉非替尼的耐药。

Background and Objective Lung cancer is the most common cancer worldwide with the highest morbidity and mortality,in which the non-small cell lung cancer accounts for 80%of all cases.The expression of(HOX transcript antisense RNA)HOTAIR were abnormal in a variety of tumor tissues and is involved in the regulation of the occurrence and development of lung cancer.The purpose of this study is to investigate the effect and mechanism of downregulation of HOTAIR on gefitinib resistance of lung adenocarcinoma HCC827 cells by targeting PTEN.Methods The HOTAIR downstream target gene was predicted by bioinformatics database.The small interfering RNAs(siRNA)which is corresponding to HOTAIR was transfected using LipofectamineTM 2000.Quantitative real-time PCR(RT-qPCR)and Western blot were used to detect the expression of HOTAIR,PTEN,PI3K and AKT in HCC827 and HCC827GR cells.MTT assay was used to detect the changes in drug resistance of HCC827GR cells.Flow cytometry analysis were used to test the cell proliferation and the rate of apoptosis.Results The expression of HOTAIR increased in HCC827GR and the serum of NSCLC patients with gefitinib resistance(P<0.05).Transfection of HOTAIR siRNA decreased the expression of HOTAIR(P<0.05),and increased the expressions of PTEN(P<0.05),while the expression of PI3K and AKT were decreased(P<0.05).Compared with the blank control group,down-regulation of HOTAIR increased the sensitivity of HCC827GR cells to gefitinib.The cell proliferation ability was decreased and the apoptosis was promoted apparently(P<0.05).Conclusion Down-regulation of HOTAIR can suppress the cell growth and promote the apoptosis,and it can reverse the resistance of HCC827GR cells to gefitinib.Its potential mechanism may be related with the targeting of PTEN/PI3K/AKT pathway.