MANF对结直肠癌Caco-2细胞增殖、迁移、侵袭及凋亡的影响

Effect of MANF on proliferation,migration,invasion and apoptosis of colorectal cancer Caco-2 cells

目的构建中脑星形胶质细胞源性神经营养因子(MANF)过表达的结直肠癌Caco-2细胞系,探讨MANF对结直肠癌Caco-2细胞增殖、迁移、侵袭和凋亡的影响以及潜在的机制。方法分别将重组质粒MANF-GFP与GFP空载体质粒转染Caco-2细胞,实时荧光定量PCR(RT-PCR)和蛋白印迹法(Western blot,WB)检测转染后Caco-2细胞中的MANF mRNA和蛋白产生量;Transwell检测MANF对Caco-2细胞迁移与侵袭能力的影响;用CCK8试验检测转染后各组Caco-2细胞增殖情况的不同;WB检测细胞中caspase-3的激活;使用flow cytometry检测Caco-2细胞凋亡情况;荧光素酶报告实验检测NF-κB的转录活性。结果MANF-GFP和GFP质粒均成功转入Caco-2细胞中。给予外源性MANF蛋白可有效降低Caco-2细胞迁移、侵袭能力(P<0.01),明显减弱细胞增殖能力(P<0.01);上调Caco-2细胞cleaved caspase-3蛋白表达水平,促进其凋亡;同时,与对照组相比,转染MANF质粒可降低NF-κB的转录活性(P<0.01)。结论MANF可能通过下调NF-κB的转录活性,抑制Caco-2细胞的增殖、迁移和侵袭能力,并促进其凋亡。

Aim To construct colorectal cancer Caco-2 cell line with overexpression of mesencephalic astrocyte-derived neurotrophic factor(MANF)and to investigate the effects of MANF on the proliferation,migration,invasion and apoptosis of human colorectal cancer Caco-2 cells.Methods Caco-2 cells were transfected with MANF-GFP plasmid and GFP empty vector plasmidrespectively.Real time polymerase chain reaction(RT-PCR)and Westernblot(WB)were used to detect the the expression level of MANF.Transwell was used to detect the effect of MANF on the migration and invasion ability of Caco-2 cells.CCK8 was used to dectet the influence of MANF on the proliferation of Caco-2 cells.WB and flow cytometrywere used to detect the activation ofcaspase-3andthe apoptosis rateof Caco-2 cells.Luciferase experiment was used to verify the effect of MANF on NF-κB pathway.Results MANF-GFP plasmid was successfully expressed in Caco-2 cells.The migration and invasion ability of Caco-2 cells were effectively reduced(P<0.01),and the ability of the cell proliferation was downregulated(P<0.01)in the MANF overexpression group;the expression of cleaved caspase-3 protein and apoptosis of Caco-2 cells were upregulated;compared with the control group,the transfection of MANF plasmid significantly reduced the transcription activity of NF-κB(P<0.01).Conclusions MANF can inhibit proliferation,migration,invasion of Caco-2 cells,while promote its apoptosis by negatively regulating NF-κB signal pathway.