抑癌基因TIP30/CC3对肿瘤细胞生长的影响

Effects of tumor suppressing gene TIP30/CC3 on the growth of tumor cells

目的 将TIP30/CC3基因转染肝癌细胞PLC/PRF/5并筛选稳定表达的克隆,观察TIP30/CC3基因对其生长及细胞周期变化的影响。 方法 将正、反义TIP30/CC3基因导入PLC/PRF/5肝癌细胞并筛选稳定表达的克隆;绘制细胞生长曲线并检测细胞周期变化;将筛选的PLC/PRF/5细胞接种裸鼠皮下观察致瘤性及肿瘤生长。 结果 与转染正义TIP30/CC3基因的PLC/PRF/5细胞相比,转染反义的细胞生长明显加快,转染后第3天,PLC-anti-TIP30存活细胞数为14.0×104,与PLC-pcDNA3和PLC/PRF/5两对照组比较差异有统计学意义(P<0.05);PLC-TIP30组细胞数为4.9×104,与两对照组比较细胞数少,差异有统计学意义(P<0.05)。接种后第6天,PLC-anti-TIP30组细胞数为25.0×104,与两对照组比较差异有统计学意义(P<0.01);PLC-TIP30组细胞数为12.4×104,与两对照组比较差异有统计学意义(P<0.05)。细胞周期检测显示PLC-anti-TIP30组细胞增殖旺盛(G0/G1期及S期细胞分别为22.4％和58.6％)(P<0.05);PLC-TIP30细胞周期发生G1期阻滞(G0/G1期及S期细胞分别为56.9％和28.7％)(P<0.05)。裸鼠皮下致瘤实验也证实了TIP30基因可使PLC/PRF/5细胞的致瘤性降低,裸鼠皮下成瘤时间延迟,肿瘤生长相对延缓;而PLC-anti-TIP30细胞的致瘤性增强,裸鼠皮下成瘤时间缩短。

Objective To introduce the newly found gene TIP30/CC3 into a hepatoma cell line PLC/PRF/5 and select the stable expression clones. The growth and cell cycles were studied with the clones stably expressing TIP30/CC3 or anti-TIP30/CC3, and the effects of TIP30/CC3 gene on hepatoma cells were analyzed. Methods The internal expression of TIP30/CC3 protein was detected with Western blot, then TIP30/CC3 or anti-TIP30/CC3 cDNA was subcloned into a constitutive vector pcDNA3 followed by transfection into PLC/PRF/5. Stable expression clones were selected. The cell growth curve was made and cell cycles detected using flow cytometry. To confirm the results in vitro, stable-expressing cells were implanted subcutaneously into nude mice and time of tumor formation recorded and tumor volume measured. Results PLC-anti-TIP30 grew faster than the others. Three days after transfection, live cells of PLC-anti-TIP30 were 14.0 × 104, in comparison with the control PLC-DNA3 and PLC/PRF/5, the differences were statistically significant (P < 0.05). Live cells of PLC-TIP30 were 4.9 × 104, significantly less than the two control groups (P < 0.05). Six days after transfection, live cells of PLC-anti-TlP30 were 25.0 × 104, significantly more than the controls PLC-DNA3 and PLC/PRF/5 (P < 0.01). Live cells of PLC-TIP30 were 12.4 × 104, significantly less than the two control groups (P < 0.05). Cell cycle analysis showed that PLC-anti-TIP30 proliferated faster, 22.4% cells were in G0/G1 (gap) phases and 58.6% cells in S (DNA synthesis) phase. The growth of the PLC-anti-TIP30 cell was retarded and many cells were arrested from G1 to S phases. Cells in G0/G1 and S phase were 44.2% and 33.3% respectively. Furthermore,the average time of tumor formation was shorter in anti-TIP30 group and longer in TIP30/CC3 group, and times were 6.0 d (P < 0.01, with control groups) and 15.6 d (P < 0.05, with control groups) respectively. Tumors in the nude mice grew faster in PLC-anti-TIP30 group and slower in PLC-TIP30 group. Conclusion Tumor suppressor gene TIP30/CC3 can inhibit the proliferation of tumor cells and interfere in its cell cycles. It can be used as a valuable tool for hepatoma biotherapy including gene therapy.