AHR小干扰RNA对肝癌HCCLM3细胞体外增殖和体内肿瘤生长的影响

The effect of AHR small interference on hepatoma HCCLM3 cell proliferation in vitro and tumor growth in vivo

目的 :探讨RNA干扰(RNA interference,RNAi)芳香烃受体(aryl hydrocarbon receptor,AHR)基因表达对肝癌HCCLM3细胞增殖和迁移的影响及其可能的作用机制。方法 :将AHR基因特异性小干扰RNA(small interference RNA,siRNA)转染人肝癌HCCLM3细胞后,应用实时荧光定量-PCR法检测AHR mRNA的表达水平,蛋白质印迹法检测AHR和应激激活的蛋白激酶(stress-activated protein kinase,SAPK)/c-Jun N-末端激酶(c-Jun N-terminal kinase,JNK)、细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK 1/2)和c-Jun蛋白及其磷酸化水平,CCK-8(cell counting kit-8)法和Transwell法检测AHR-siRNA转染后对HCCLM3细胞增殖和迁移能力的影响。合成AHR基因特异性小发夹RNA(small hairpin RNA,shRNA),构建稳定干扰AHR基因表达的重组病毒载体pMKO.1/puro-shAHR,并将其感染HCCLM3细胞,感染后的细胞接种于裸鼠皮下建立移植瘤模型,观察移植瘤的生长情况;蛋白质印迹法检测pMKO.1/puro-shAHR感染后HCCLM3细胞及裸鼠皮下移植瘤组织中AHR蛋白的表达水平。结果 :AHR-siRNA转染组HCCLM3细胞中AHR mRNA和蛋白的表达水平明显下降(P<0.01),细胞的增殖和迁移能力受到抑制(P<0.01);SAPK/JNK、ERK 1/2和c-Jun的磷酸化水平降低(P<0.01)。pMKO.1/puro-shAHR感染后的HCCLM3细胞裸鼠皮下移植瘤体积和质量明显低于对照组(pMKO.1/puro-shNC感染HCCLM3细胞)(P<0.01),pMKO.1/puro-shAHR感染后的HCCLM3细胞及裸鼠皮下移植瘤组织中AHR蛋白的表达水平降低(P<0.01)。结论 :AHR可能通过上调SAPK/JNK、ERK 1/2和c-Jun的磷酸化水平而促进HCCLM3细胞的体外增殖和迁移。

Objective： To investigate the effect of aryl hydrocarbon receptor （AHR） RNA interference on hepatoma HCCLM3 cell proliferation and migration and to explore its possible mechanism. Methods： After HCCLM3 cells transfection with specific AHR-small interference RNA （siRNA）, the expression level of AHR mRNA was detected by real-time fluorescence quantitative-PCR, and the expression levels of AHR, stress- activated protein kinases （SAPK）/c-Jun N-terminal kinase ~JNK）, extracellular signal-regulated kinase 1/2 （ERK 1/2） and c-Jun phosphorylation were detected by Western blotting. Then the proliferation and migration of HCCLM3 cells after transfection with AHR-siRNA were detected by cell counting kit-8 （CCK-8） and Transwell assay, respectively. Synthesize AHR gene-specific small hairpin RNA （shRNA） to construct recombinant virus vector pMKO. 1/puro-shAHR which stable knockdown AHR, and the recombinant vector was infected into HCCLM3 cells and then implanted into nude mice to construct subcutaneous tumor model. The growth of xenografted tumor in nude mice was examined. The expression levels of AHR protein in I-ICCLM3 cells and xenografted tumor tissues after infection with pMKO.1/puro- shAHR were detected by Western blotting. Results： The expression levels of AHR mRNA and protein were significantly reduced in HCCLM3 cells transfected with AHR-siRNA （P 〈 0.01）, and the proliferation and migration of HCCLM3 cells were inhibited （P 〈 0.01）. The expression levels of SAPK/JNK, ERK 1/2 andc-Jun phosphorylation were depressed （P 〈 0.01）. The volume and weight of HCCLM3 cells xenografted tumor in nude mice after infection with pMKO.1/puro-shAHR were lower than those of the control group （HCCLM3 cells were infected with pMKO.1/puro-shNC） （P 〈 0.01）. The expression levels of AHR protein in HCCLM3 cells and xenografted tissues after infection with pMKO.1/puro.-shAHR were decreased （P 〈 0.01）. Conclusion： AHR may promote HCCLM3 cell proliferation and migration in vitro by up-regulating the phosphorylation levels of ERK1/2, SAPK/JNK and c-Jun.