Interaction between CD74 and RUNX3 in human pancreatic cancer cells
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摘要:
目的 探讨CD74和RUNX3在人胰腺癌Capan-2细胞内是否存在相互作用。 方法 构建CD74-siRNA的慢病毒表达载体和空白载体，酶切鉴定正确后，分别转染人胰腺癌Capan-2细胞，使用Western blot和RT-PCR等方法对CD74的表达进行检测验证；运用Western blot和RT-PCR等方法观察CD74低表达对RUNX3蛋白和mRNA表达的影响；利用免疫共沉淀法验证Capan-2细胞内CD74与RUNX3之间的相互作用。 结果 CD74在实验组Capan-2细胞中稳定低表达，实验组CD74蛋白（0.09±0.01 vs 2.78±0.04）、CD74 mRNA（0.39±0.001 vs 1.00±0.00）低于对照组（P<0.05）；抑制CD74可降低RUNX3蛋白和mRNA表达水平（P<0.05）；CD74抗体免疫共沉淀下来的蛋白复合物中检测到RUNX3，同时RUNX3抗体免疫共沉淀下来的蛋白复合物中也检测到CD74。 结论 CD74和RUNX3在人胰腺癌Capan-2细胞内存在相互作用。 Abstract:Objective To investigate the interaction between CD74 and RUNX3 in human pancreatic cancer capan-2 cells. Methods The lentiviral CD74-siRNA vector and blank vector were constructed. Restriction enzyme digestion was constructed correctly. Then Capan-2 cells were transfected by the lentivirus vector. The cell line stably low expression of CD74 was confirmed by Western blot and RT-PCR. The effect of low CD74 expression on RUNX3 protein and mRNA expression were detected by Western blot and RT-PCR. Co-immunoprecipitation assay was performed to verify the interaction between CD74 and RUNX3 in capan-2 cells. Results Protein and mRNA of CD74 were stably and lowly expressed in Capan-2 cells of the experimental group (P<0.05). Decreased CD74 level significantly inhibited RUNX3 protein and mRNA expression (P<0.05). RUNX3 was detected in the protein complex from the immunoprecipitation by using anti-CD74 antibody. CD74 in the protein complex from the immunoprecipitation was detected by using anti-RUNX3 antibody. Conclusion There is an interaction between CD74 and RUNX3 in human pancreatic cancer Capan-2 cells. -
Key words:
- CD74 /
- RUNX3 /
- immunoprecipitation /
- protein-protein interaction
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表 1 引物的序列
基因名称 引物方向 引物序列（5’to 3’） CD74 CD74-F CCAGAACCTGCAGCTGGAGAA CD74-R GACTCTGGAGCAGGTGCATCA RUNX3 RUNX3-F AGGCAATGACGAGAACTACTCC RUNX3-R CGAAGGTCGTTGAACCTGG β-actin Β-actin-F TGGCACCCAGCACAATGAA Β-actin-R CTAAGTCATAGTCCGCCTAGAAGCA 
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