CAJ | 학술논문

目的目的探讨靶向HBV X的siRNA(X-siRNA)和5-氮-2′-脱氧胞苷(5-aza-dC)对HBV相关肝细胞癌生长的影响及可能机制.方法设计合成X-siRNA及对照siRNA,用siRNA处理HepG2/GFP-HBx细胞,RT-PCR法测定处理细胞的HBV X基因表达;将HepG2/GFP、HepG2/GFP-HBx细胞接种于裸鼠皮下建立裸鼠肝癌皮下移植瘤,分别用X-siRNA、5-aza-dC单独或联合处理裸鼠并观察移植瘤生长;甲基化PCR测定移植瘤组织p16基因甲基化.结果 RT-PCR检测示X-siRNA处理的细胞HBV X mRNA水平明显降低;裸鼠体内实验显示HepG2/GFP-HBx组的皮下移植瘤体积明显大于HepG2/GFP组(P＜0.05);X-siRNA与5-aza-dC处理组移植瘤体积明显小于未处理组(P＜0.05);甲基化PCR检测示HepG2/GFP-HBx组移植瘤组织存在p16甲基化而HepG2/GFP组未检出p16甲基化;X-siRNA、5-aza-dC处理的移植瘤组织中p16基因甲基化减低.结论 X-siRNA及甲基化抑制剂可能通过逆转p16甲基化而能有效抑制肝细胞癌生长,具有潜在应用价值.
목적 목적 탐토파향HBV X적siRNA(X-siRNA)화5-담-2′-탈양포감(5-aza-dC)대HBV상관간세포암생장적영향급가능궤제.방법 설계합성X-siRNA급대조siRNA,용siRNA처리HepG2/GFP-HBx세포,RT-PCR법측정처리세포적HBV X기인표체;장HepG2/GFP、HepG2/GFP-HBx세포접충우라서피하건립라서간암피하이식류,분별용X-siRNA、5-aza-dC단독혹연합처리라서병관찰이식류생장;갑기화PCR측정이식류조직p16기인갑기화.결과 RT-PCR검측시X-siRNA처리적세포HBV X mRNA수평명현강저;라서체내실험현시HepG2/GFP-HBx조적피하이식류체적명현대우HepG2/GFP조(P＜0.05);X-siRNA여5-aza-dC처리조이식류체적명현소우미처리조(P＜0.05);갑기화PCR검측시HepG2/GFP-HBx조이식류조직존재p16갑기화이HepG2/GFP조미검출p16갑기화;X-siRNA、5-aza-dC처리적이식류조직중p16기인갑기화감저.결론 X-siRNA급갑기화억제제가능통과역전p16갑기화이능유효억제간세포암생장,구유잠재응용개치.
Objective To investigate the anti-tumor effect of small interfering RNA targeting to HBV X gene (X-siRNA) and 5-aza-2'-deoxycytidine (5-aza-dC) on HBV-related hepatocellular carcinoma.Methods X-siRNA and control siRNA were synthesized.HepG2/GFP-HBx cells were treated with X-siRNA,and the levels of HBV X mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR).Nude mice were inoculated with HepG2/GFP and HepG2/GFP-HBx cells subcutaneous respectively to establish implant models of hepatocellular carcinoma,and were treated with X-siRNA,5-aza-dC alone or in combination,and tumor growth was observed.The methylation of p16 gene promoter was detected by methylation specific polymerase chain reaction (MSP).Results RT-PCR showed the expression of HBV X mRNA in HepG2/GFP-HBx cells was inhibited markedly by X-siRNA.The nude mice experiment showed that the gross tumor volume was much bigger in HepG2/GFP-HBx group than that in HepG2/GFP group (P ＜ 0.05).The growth of palpable tumors in X-siRNA or 5-aza-dC treatment group notably decreased (P ＜ 0.05).MSP analysis showed that p16 gene methylation was observed in HepG2/GFP-HBx-caused palpable tumors,while no methylation was detected in HepG2/GFP group.However,after treatment with X-siRNA or 5-aza-dC,p16 gene methylation reduced.Conclusions HBV X-siRNA and methylation inhibitor can inhibit the growth of hepatoma cells via reversing p16 methylation.