shRNA干扰ASCT2基因表达对结肠癌细胞生物学行为的影响

Effect of ASCT2 gene knock-down by shRNA on biological behaviors of colorectal cancer cells

目的探讨shRNA下调谷氨酰胺转运体ASCT2对结肠癌细胞Lovo和SW480生物学行为的影响。方法利用Lipofectamine2000介导ASCT2-shRNA的表达载体转染结肠癌细胞Lovo和SW480，通过反转录PCR和western blot分别检测转染后ASCT2mRNA及蛋白的表达情况：分别应用MTY法和transwell法检测其结肠癌细胞增殖和侵袭能力；应用放射示踪法检测结肠癌细胞对谷氨酰胺的摄取情况。结果shASCT2转染后，Lovo和SW480细胞中ASCT2mRNA及蛋白水平均显著下调（P〈0．01）。shASCT2转染后，Lovo和SW480细胞增殖能力（A490）和细胞侵袭能力（透膜细胞数）均较对照组细胞显著减弱（均P〈0．01）；其中Lovo细胞中实验组和对照组的透膜细胞数分别为46．3±5．9和197，7±9．1，SW480细胞中实验组和对照组的透膜细胞数分别为29．7±3．8和139．0±9．5。放射示踪法检测显示，shASCT2转染后，Lovo和SW480细胞对谷氨酰胺的摄取受到显著抑制，其中，对Lzvo细胞的抑制率为79．15％，对SW480的抑制率为67．22％，差异均有统计学意义（均P〈0．01）。结论ASCT2在结肠癌中发挥癌基因的作用，其促进结肠癌进展的机制可能与谷氨酰胺代谢有关．有望成为肿瘤代谢治疗的重要靶点。

Objective To investigate the effect of ASCT2 gene （glutamine transporter） knock- down by shRNA on biological behaviors of eolorectal cancer cells. Methods shRNA was transfected into eolorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine. Results ASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells （P 〈 0.01 ）. MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells （A490） and decrease the number of invasive Lovo and SW480 cells from the membrane （both P〈 0.01）. The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells （both P〈0.01）. Conclusions ASCT2 plays an oncogenicrole in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.