15脱氧前列腺素J2对人肝癌细胞增殖和凋亡的影响及其机制

Effects of 15-Deoxy-Δ12,14-prostaglandin J_2 on the Proliferation and Apoptosis of Human HepG2 Cells

目的研究15脱氧前列腺素J2(15-deoxy-Δ12,14-prostaglandin J2,15d-PGJ2)对体外培养的人肝癌HepG2细胞增殖和凋亡的影响,并探讨其作用机制。方法不同浓度的15d-PGJ2干预体外培养的HepG2细胞,采用MTT比色法检测细胞增殖能力,3H-TdR掺入实验检测细胞DNA合成速率,RT-PCR和Western blot检测PPARγmRNA和蛋白表达情况,流式细胞术检测细胞凋亡率和细胞周期;使用GW9662和/或转染pSG5-PPARγ真核表达质粒来观察PPARγ通路在15d-PGJ2抑制HepG2细胞增殖中的作用,并使用pGCsi-PPARγ转染观察PPARγ沉默后15d-PGJ2对HepG2细胞增殖作用的影响;最后采用凝胶迁移电泳实验(EMSA)检测NF-κB的DNA结合活性。结果 15d-PGJ2作用于HepG2细胞后能够导致使细胞增殖受到抑制、DNA合成速率减慢,并诱导了细胞凋亡,此作用呈一定的剂量依赖关系;在上述过程中,G0/G1期细胞比例明显增加,S期细胞比例明显减少,但PPARγmRNA和蛋白的表达并没有显著变化。GW9662可以部分拮抗15d-PGJ2的增殖抑制作用,但转染pSG5-PPARγ可以逆转GW9662的作用;15d-PGJ2在20μmol/L时对pGCsi-PPARγ转染的HepG2细胞仍可以表现出增殖抑制效应,EMSA结果表明其在50μmol/L时明显抑制了NF-κB的DNA结合活性。结论 15d-PGJ2能够抑制HepG2细胞的增殖、诱导其凋亡并干扰细胞周期,表现出明显的抑瘤作用,此过程涉及PPARγ依赖途径和非依赖途径,而其中PPARγ非依赖途径可能与其抑制NF-κB的作用有关。

Objective To investigate the effects of 15-deoxy-Δ12,14-prostaglandin J_2（15d-PGJ_2）on the proliferation and apoptosis of human HepG2 cells and explore the possible mechanisms.Methods Effects of 15d-PGJ_2 on the growth and DNA synthetic rate of human HepG2 cells were measured by MTT assay and 3H-TdR uptake,respectively.Cell apoptosis and cell cycle were detected by flow cytometry（FCM）.The expression of PPARγmRNA and protein was measured by reverse transcription-polymerase chain reaction（RT-PCR）and Western blotting,respectively.The role of PPARγin 15d-PGJ_2-induced inhibition of HepG2 cells was examined by use of GW9662,a PPARγantagonist and/or the eukaryotic expression plasmid pSG5-PPARγ.The effect of 15d-PGJ_2 on the proliferation of HepG2 was also examined after silence of PPARγ with pGCsiPPARγ.Additionally,NF-κB DNA binding activity was detected by electrophoretic mobility shift assay.Results 15d-PGJ_2 inhibited cell proliferation,decreased DNA synthetic rate and induced cell apoptosis in a dose-dependent manner.Cell cycle analysis revealed a decreased proportion of cells in S phase,with arrest at G0/G1 in this process.But the expression levels of PPARγmRNA and protein were not significantly changed.GW9662 patially reversed the cell grow inhibition effect of 15d-PGJ_2.Transfection with pSG5-PPARγcould suppress the effect of 15d-PGJ_2 on cell growth in the presence of GW9662.15d-PGJ_2 at 20μmol/L was found to inhibit the proliferation of HepG2 cells transfected with pGCsi-PPARγ.Furthermore,15d-PGJ_2 at 50μmol/L inhibited the DNA binding activity of NF-κB.Conclusion 15d-PGJ_2 inhibits cell growth,induces cell apoptosis and interferes with cell cycle in HepG2 cells through PPARγ-dependent and-independent pathways.The PPARγ-independent pathway is related to the activity of NF-κB signal.