CAJ | 학술논문

目的研究甲胎蛋白(AFP)对肝癌细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)受体-2(DR5)表达及其在肝癌细胞耐受TRAIL中的作用.方法用Western blot法分析全反式维甲酸(ATRA)处理人肝癌细胞株Bel 7402细胞24 h后DR5表达的变化;免疫共沉淀(Co-IP)技术研究AFP与维甲酸受体(RAR)-β相互作用;激光共聚焦显微镜观察AFP与RAR-β的细胞共定位; RNA干扰技术抑制Bel 7402细胞AFP表达,再用ATRA处理24h后检测细胞内DR5表达的变化;用pcDNA3.1质粒和人AFP基因连接构建表达AFP的载体(称为pcDNA3.1-afp),然后转染到不表达AFP的人肝癌细胞株HLE细胞;细胞生长状况用四甲基偶氮唑盐法检测.组间比较用f检验进行统计学分析.结果 Bel 7402和HLE细胞低表达DR5,ATRA(160μmol/L)处理24h后能促进肝癌细胞DR5表达; Co-IP技术研究显示AFP能与RAR-β结合;共聚焦显微镜观察发现AFP与RAR-β共定位于细胞质;干扰AFP表达后,Bel 7402细胞的DR5表达明显提高,抑制AFP表达后,ATRA能显著促进Bel 7402细胞内DR5的表达,并增加Bel 7402细胞对TRAIL的敏感性;转染pcDNA3.1-afp载体后,HLE细胞内的AFP能与RAR-β结合,并发现pcDNA3.1-afp载体能对抗TRAIL诱导HLE细胞凋亡.结论 Bel 7402细胞内表达的AFP具有抑制DR5表达的生物学功能;AFP可能通过抑制RAR-β入核调节DR5的表达;细胞内高表达的AFP是导致Bel 7402细胞耐受TRAIL诱导细胞凋亡的重要原因.
목적 연구갑태단백(AFP)대간암세포종류배사인자상관조망유도배체(TRAIL)수체-2(DR5)표체급기재간암세포내수TRAIL중적작용.방법 용Western blot법분석전반식유갑산(ATRA)처리인간암세포주Bel 7402세포24 h후DR5표체적변화;면역공침정(Co-IP)기술연구AFP여유갑산수체(RAR)-β상호작용;격광공취초현미경관찰AFP여RAR-β적세포공정위; RNA간우기술억제Bel 7402세포AFP표체,재용ATRA처리24h후검측세포내DR5표체적변화;용pcDNA3.1질립화인AFP기인련접구건표체AFP적재체(칭위pcDNA3.1-afp),연후전염도불표체AFP적인간암세포주HLE세포;세포생장상황용사갑기우담서염법검측.조간비교용f검험진행통계학분석.결과 Bel 7402화HLE세포저표체DR5,ATRA(160μmol/L)처리24h후능촉진간암세포DR5표체; Co-IP기술연구현시AFP능여RAR-β결합;공취초현미경관찰발현AFP여RAR-β공정위우세포질;간우AFP표체후,Bel 7402세포적DR5표체명현제고,억제AFP표체후,ATRA능현저촉진Bel 7402세포내DR5적표체,병증가Bel 7402세포대TRAIL적민감성;전염pcDNA3.1-afp재체후,HLE세포내적AFP능여RAR-β결합,병발현pcDNA3.1-afp재체능대항TRAIL유도HLE세포조망.결론 Bel 7402세포내표체적AFP구유억제DR5표체적생물학공능;AFP가능통과억제RAR-β입핵조절DR5적표체;세포내고표체적AFP시도치Bel 7402세포내수TRAIL유도세포조망적중요원인.
Objective To explore the mechanism ofAlpha-fetoprotein (AFP) effects on hepetocellular cancinoma cells (HCC) resistances apoptosis induced by tumor necrosis factor-related apoptosis inducingligand (TRAIL). Methods The expressed alteration of TRAIL recepator-2 (DR5) after the human hepatoma cells line Bel 7402 (AFP-producing) and HLE cells (non-AFP producing) were treated with all trans retinoic acid (ATRA) were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation (Co-IP); Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering (RNAi) was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP (named pcDNA3, 1-afp); The growth of hepatoma cells was analyzed by MTT. Results Bel 7402 and HLE cells expressed DR5, lowed dosage of ATRA (40 μmol/L) had no influence on the expression of DR5 in Bel 7402 cells, but ATRA (160 μ mol/L) could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with PAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced. Conclusions These data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistanceinduced apoptosis by TRAIL.