靶向沉默CXCR4表达逆转肺癌细胞吉西他滨耐药性的作用研究

摘要

Objective To explore the reversion effect of Gemcitabine-resistance A549 cell (A549/Gem) by silencing CXCR4.Methods A549 cell was induced by continuous stepwise exposure to Gemcitabine in order to obtain Gemcitabine-resistance A549 cell ( A549/Gem) in vitro.The CXCR4 expressions level of A549 and A549/Gem were detected by Quantitative RT-PCR ( RT-qPCR) and Western blot analyses.The CXCR4 shRNA vector was transfected into the A549/Gem cell by targeting silence CXCR4.Furthermore, MTT assay was used to explore the IC50 and RI in A549, A549/Gem and A549/Gem-CXCR4 cells.Moreover, Western blot analysis was performed to detect the expressions of phospho-JNK, phospho-p38 and phospho-ERK 1/2 in A549, A549/Gem and A549/Gem-CXCR4 cells.Results Gemcitabine-resistance A549 cell ( A549/Gem) was successful constructed by using continuous stepwise exposure to Gemcitabine in vitro.The expression level of CXCR4 was up-regulated in A549/Gem cell than in A549 cell.The CXCR4 shRNA vector could significantly decrease CXCR4 expression in A549/Gem cell.The IC50 values of Gemcitabine in A549, A549/Gem and A549/Gem-CXCR4 cell were (0.08 ±0.01)μmol/L, (14.01 ±0.21)μmol/L and (1.84 ±0.61)μmol/L, respectively.The RI value of Gemcitabine was (127.12 ±12.28) in A549/Gem cells, while the value of RI was (27.3 ±0.98) in A549/Gem-CXCR4 cells.The expression level of phospho-JNK, phospho-p38 and phospho-ERK 1/2 were also markedly inhibited in A549/Gem-CXCR4 cell than in A549/Gem cell.Conclusion CXCR4 is up-regulated in A549/Gem cell.Targeting silence CXCR4 can successfully reverse drug-resistance of Gemcitabine in A549/Gem cells, which hints CXCR4 is associated with lung cancer radiation therapy as an effective molecular target.%目的：观察靶向沉默CXCR4基因表达对非小细胞肺癌吉西他滨耐药株（ A549／Gem）逆转作用的影响。方法采用体外浓度梯度递增的诱导方法建立非小细胞肺癌吉西他滨耐（Gemcitabine）获得性耐药细胞株（A549／Gem）。通过Quantitative RT－PCR （RT－qPCR）检测A549与A549／Gem细胞中CXCR4的表达量。通过体外转染CXCR4 shRNA干扰载体到A549／Gem细胞，RT－qPCR及蛋白质印迹法（ Western blot ）验证shRNA对CXCR4基因的靶向沉默效率。进而采用MTT法检测A549、A549／Gem和CXCR4沉默表达细胞株（A549／Gem－CXCR4）对Gemcitabine的半数抑制浓度（50％inhibitory concentration，IC50）和耐药指数（resistance index，RI）；最后采用Western blot法检测A549、A549／Gem和A549／Gem－CXCR4细胞中JNK、p38和ERK1／2磷酸化蛋白的表达水平的变化。结果通过体外浓度梯度递增的诱导方法成功建立A549／Gem细胞株（ P＜0．05）。 A549／Gem细胞株中CXCR4的表达量明显高于A549细胞株。体外转染CXCR4 shRNA可明显下调A549／Gem细胞株中CXCR4表达。 MTT法检测A549，A549／Gem和A549／Gem－CXCR4对Gemcitabine的IC50分别为（0．08±0．01）μmol／L，（14．01±0．21）μmol／L和（1．84±0．61）μmol／L。 A549／Gem细胞株对Gemcitabine的耐药指数（RI）是（127．12±12．28），远高于A549／Gem－CXCR4对Gemcitabine 的耐药指数（RI）（0．27±0．08）；Western blot结果显示A549／Gem－CXCR4细胞株中JNK、p38和ERK1／2磷酸化蛋白的表达量较A549／Gem细胞株明显下调。结论 A549／Gem细胞株中CXCR4的表达明显上调。靶向沉默CXCR4表达能够逆转肺癌细胞吉西他滨耐药性，提示CXCR4是肺癌临床放射治疗相关的有效分子靶点。