核纤层蛋白B2通过细胞分裂周期相关蛋白3调控内皮细胞血管新生

Lamin B2 mediates the angiogenesis of endothelial cells via regulating cell division cycle associated protein 3

目的探讨核纤层蛋白B2(LMNB2)在血管内皮细胞介导的血管新生中的作用及其调控方式。方法人脐静脉内皮细胞(HUVEC)购自美国典型菌种保藏中心(ATCC)。利用LMNB2的短发夹RNA(shRNA)质粒在HUVEC细胞中下调LMNB2的表达,检测对内皮细胞体外成管的影响,统计单视野管状结构长度及节点数目;通过噻唑蓝(MTT)、细胞计数试剂盒(CCK-8)及划痕实验检测LMNB2对内皮细胞增殖及迁移的影响;蛋白质印迹法(Western blot)检测细胞增殖及迁移相关标志物,包括细胞核增殖抗原(Ki-67)、增殖细胞核抗原(PCNA)、基质金属蛋白酶(MMP)-2及MMP-9的表达;实时定量PCR(qPCR)检测细胞分裂周期相关蛋白3(CDCA3)的mRNA表达;荧光素酶报告基因实验及CHIP实验检测LMNB2对CDCA3的调控作用。采用Student’st检验进行分析。结果在HUVEC细胞中,下调LMNB2会抑制内皮细胞血管新生,单视野平均管状结构总长由(6.45±0.63) mm下降至(2.61±0.52) mm,下降59.5%(t=4.709,P<0.01),节点数量下降由单视野的(14.75±1.25)个下降为(7.75±0.48)个,下降57.5%(t=5.230,P<0.01);LMNB2的敲低会抑制内皮细胞的增殖与迁移,伤口愈合率下降25%(t=4.541,P<0.05),且Ki-67、PCNA、MMP-2及MMP-9表达均显著降低;在HUVEC细胞中,LMNB2结合CDCA3启动子位点,促进CDCA3的转录。回复实验证实下调LMNB2后过表达CDCA3会回复由LMNB2缺失导致的增殖及血管新生缺陷。结论 LMNB2可调控内皮细胞增殖、迁移,并通过调控CDCA3的表达调控血管新生。

Objective:To investigate the role of lamin B2(LMNB2)in vascular endothelial cells-mediated angiogenesis and its regulatory pattern.Methods:Human umbilical vein endothelial cells(HUVECs)were purchased from American Center for the Collection of Typical Strains(ATCC).The expression of LMNB2 was down-regulated by short hairclip RNA(shRNA)plasmid in HUVECs,and the effect on tube formation of endothelial cells was detected.The length of single-field tube structure and the number of nodes were counted.The effects of LMNB2 on proliferation and migration of endothelial cells were detected by methyl thiazolyl tetrazolium(MTT),the cell counting kit-8(CCK-8)and wound closure assays.Western blotting was used to detect cell proliferation and migration related markers,including proliferation cell nuclear antigen(Ki-67),proliferating cell nuclear antigen(PCNA),matrix metalloproteinase(MMP)-2 and MMP-9.The expression of cell division cycle associated protein 3(CDCA3)mRNA was detected by quantitative PCR(qPCR).Luciferase reporter assay and CHIP assay were used to detect the regulatory effect of LMNB2 on CDCA3.GraphPad 7.0 was used for statistical analysis in this study.The data were shown by Mean±SD,and analyzed using Student’s t-test.Results:In HUVECs,down-regulation of LMNB2 inhibited angiogenesis of endothelial cells,the average length of tubular structure per field of vision decreased from(6.45±0.63)mm to(2.61±0.52)mm,a decrease of 59.5%(t=4.709,P<0.01),and the number of nodes decreased 14.75±1.25 to 7.75±0.48,a decrease of 57.5%(t=5.230,P<0.01).Knockdown of LMNB2 inhibited proliferation and migration of endothelial cells,wound healing rate decreased by 25%(t=4.541,P<0.05),and the expression of Ki-67,PCNA,MMP-2 and MMP-9 was significantly decreased.In HUVECs,LMNB2 bound to the CDCA3 promoter site and promoted CDCA3 transcription.Rescue experiments confirmed that overexpression of CDCA3 after downregulation of LMNB2 would restore proliferation and angiogenesis defects caused by LMNB2 deficiency.Conclusion:LMNB2 regulates endothelial cell proliferation and migration,and LMNB2 mediates the angiogenesis of endothelial cells via regulating CDCA3.