Background In most colorectal carcinomas, the level of phospholipase C （PLC）-gamma 1 expression is greatly elevated. Increased expression of PLC-gamma 1 may play an important role in colon carcinogenesis, but the mechanism is not well known. The aim of this study was to evaluate the role of PLC-gamma 1 in colon carcinogenesis by using recombinant lentivirus that stably suppressed the PLC-gamma 1 expression in human colorectal carcinoma LoVo cells. Methods Recombinant lentivirus producing PLC-gamma 1 siRNA were prepared. After LoVo cells were transduced by each lentivirus, stably transduced cells were selected by Blasticidin. The protein and mRNA expression of PLC-gamma 1 were examined by Western-blot and reverse transcription-polymerase chain reaction （RT-PCR） analysis, and the effects of the lentivirus on the cell adhesion, migration and apoptosis were analyzed. Results Stable LoVo cell line deficient in PLC-gamma 1, was established. Notably, PLC-gamma 1 was silenced without affecting the levels of other subtypes of PLC so that the role of PLC-gamma 1 in colon carcinogenesis could be examined Silencing of endogenous PLC-gamma 1 resulted in efficient inhibition of the adhesion and migration of LoVo cells in vitro and a great increase of 5-fluorouracil induced apoptosis （30%-40%） of LoVo cells.Conclusions PLC-gamma 1 may play an important role in metastasis and anti-apoptosis in human colorectal carcinomas.
TAN LiXIAO Bing-xiangZENG Wei-senLIN JunZOU Zhi-pengXU Ai-minLUO Shen-qiu
背景与目的:磷脂酶C-γ1(phospholipase C gamma1,PLC-γ1)是跨膜信号转导中关键和重要的一个信号中介,是细胞增殖与细胞凋亡调控的一个重要分子,最近研究发现它在大肠癌等许多肿瘤组织中呈过表达状态,与肿瘤的发生、发展有密切关系。本研究主要探讨阻断PLC-γ1信号通路后对大肠癌LoVo细胞增殖、凋亡的影响,及其上述影响的信号机制。方法:以人大肠癌LoVo细胞作为研究模型,利用PLC-γ1特异的化学阻断剂U73122处理以阻断LoVo细胞中PLC-γ1信号通路,通过绘制细胞生长曲线、PI单染的流式细胞仪检测细胞周期而评估对其细胞增殖的影响,通过细胞形态观察及DNA片段琼脂糖凝胶电泳评估是否启动细胞凋亡,同时检测阻断PLC-γ1信号通路后HSP70、Caspase-3表达水平的变化来探讨可能的信号机制。结果:阻断PLC-γ1信号通路明显减缓大肠癌LoVo细胞的生长,其细胞增殖抑制率随药物作用时间和浓度的增加逐渐增高,10μmol/LU73122作用24、48h后其抑制效果可分别达到35%和45%,使LoVo细胞G1期细胞比例增加,而S期细胞比例降低,延缓细胞从G1期向S期的过渡,即抑制细胞周期的进行,阻断PLC-γ1信号通路后LoVo细胞未能出现凋亡特征性的形态学改变,DNA琼脂糖凝胶电泳未能检测到凋亡特征的梯状带的出现,不能引起Caspase-3的激活,同时PLC-γ1信号通路的阻断可上调HSP70的表达水平,HSP70的分子伴侣作用可能是其抑制大肠癌细胞周期进行的机制。结论:阻断磷脂酶C-γ1信号通路能够抑制大肠癌LoVo细胞的过度增殖、抑制其细胞周期的进行,其机制可能是通过上调热休克蛋白70的表达水平而实现,但不能启动LoVo细胞凋亡,磷脂酶C-γ1不是调控LoVo细胞凋亡的关键信号分子。
