萝卜硫素对结肠癌HT-29细胞增殖、血管形成及miR-34a/Notch1信号通路的影响

Effects of Sulforaphane on the Proliferation,Angiogenesis and miR-34a/Notch1 Signal Pathway of Colon Cancer HT-29 Cells

目的:探讨萝卜硫素对结肠癌HT-29细胞增殖、血管形成及miR-34a/Notch1信号通路的影响。方法:设结肠癌HT-29细胞组、氟尿嘧啶组(20.0μg·ml-1)、萝卜硫素低(100.0μg·ml-1)、高(1000.0μg·ml-1)剂量组,置于37℃、5%CO2的环境中培养72 h,培养结束后,细胞计数试剂盒-8测定法测定细胞增殖能力,Giemsa溶液染色细胞计算集落总数,BD Matrigel基质培养测定结肠癌HT-29细胞相对总血管长度、相对总血管数目,流式细胞仪测定凋亡率,RT-PCR法及Western blot测定miR-34a、Notch1 mRNA及Notch1蛋白水平。结果:与结肠癌HT-29细胞组比较,氟尿嘧啶组、萝卜硫素低、高剂量组存活率、单克隆形成数目、相对总血管长度、相对总血管数目、miR-34a mRNA、Notch1 mRNA及蛋白显著降低(P<0.05),凋亡率显著升高(P<0.05);随着萝卜硫素剂量的增加,萝卜硫素各剂量组存活率、单克隆形成数目、相对总血管长度、相对总血管数目、miR-34a mRNA、Notch1 mRNA及蛋白显著降低(P<0.05),凋亡率显著升高(P<0.05);与氟尿嘧啶组比较,萝卜硫素各剂量组存活率、单克隆形成数目、相对总血管长度、相对总血管数目、miR-34a mRNA、Notch1 mRNA及蛋白显著升高(P<0.05),凋亡率显著降低(P<0.05)。结论:萝卜硫素能抑制结肠癌HT-29细胞增殖、血管形成,并能促进HT-29细胞凋亡,其机制与萝卜硫素抑制miR-34a、Notch1基因和蛋白的表达有关。

Objective:To investigate the effects of sulforaphane on the proliferation,angiogenesis and miR-34a/Notch1 signal pathway of colon cancer HT-29 cells.Methods:Colon cancer HT-29 cell group,HT-29 cell with 5-fluorouracil treatment group(20.0μg·ml-1),HT-29 cell with low-dose of sulforaphane(100.0μg·ml-1)treatment group and HT-29 cell with high-dose of sulforaphane(1000.0μg·ml-1)treatment group were incubated at 37℃in 5%CO2 for 72 hours.After the culture,the cell proliferation was measured by cell counting kit-8 assay.The cells were stained with Giemsa solution and the total number of colony was calculated by BD Matrigel matrix culture.The relative total length and the number of vessels generated by colon cancer HT-29 cells were measured.The apoptotic rate of HT-29 was determined by flow cytometry.The protein levels of miR-34a,Notch1 mRNA and Notch1 were measured by RT-PCR and Western blot.Results:Compared with those of HT-29 cell group,the survival rate,number of monoclonal formation,relative total blood vessel length,relative total blood vessel number,miR-34a mRNA,Notch1 mRNA and protein in 5-fluorouracil group,low-and high-dose of sulforaphane groups decreased significantly(P<0.05),while the apoptotic rate increased significantly(P<0.05);with the dose increase of sulforaphane,the survival rate,number of monoclonal formation,relative total blood vessel length,relative total blood vessel number,miR-34a mRNA,Notch1 mRNA and protein were gradually decreased(P<0.05),and the apoptotic rate was gradually increased(P<0.05).Compared with those of 5-fluorouracil group,the survival rate,number of monoclonal formation,relative total blood vessel length,relative total blood vessel number,miR-34a mRNA,Notch1 mRNA and protein increased significantly(P<0.05),and the apoptotic rate decreased significantly(P<0.05).Conclusion:Sulforaphane can inhibit the proliferation and angiogenesis of HT-29 cells,and promote the apoptosis of HT-29 cells.Its mechanism is related to the inhibition of miR-34a,Notch1 gene and protein by sulforaphane.