RNA干扰抑制HMGB1基因表达对宫颈癌HeLa细胞生物学行为的影响

Effects of silencing HMGB1 gene expression by RNA interference on biological behavior in cervical cancer cell line HeLa

背景与目的:高迁移率族蛋白1(high mobility group box-l,HMGB1)是一种非组蛋白染色体蛋白质,在多种高转移性肿瘤中高表达。前期研究结果证实,HMGB1在宫颈鳞癌组织及宫颈癌HeLa细胞中高表达,并与肿瘤临床分期、侵袭、转移密切相关。本研究通过RNA干扰(RNA interference,RNAi)抑制HMGB1基因表达,观察其对HeLa细胞增殖、侵袭和迁移的影响。方法:将HMGB1 siRNA真核表达质粒PGCsi3.0-1在脂质体介导下转染HeLa细胞,G418筛选阳性克隆,以转染PGCsi3.0-Neg(无关序列siRNA)质粒的细胞和未转染HeLa细胞作为对照。采用RT-PCR和Western blot检测HMGB1 mRNA和蛋白表达,通过MTT实验、细胞划痕实验和Transwell实验分别检测HeLa细胞增殖、侵袭和迁移能力。结果:PGCsi3.0-1组HMGB1 mRNA和蛋白表达水平分别为0.38±0.12和0.10±0.44,PGCsi3.0-Neg组分别为1.19±0.25和1.90±0.35,未转染组分别为1.42±0.14和2.55±0.32,前组HMGB1 mRNA及蛋白表达水平与后两组比较,差异均有统计学意义(P<0.05)。MTT和细胞划痕实验显示,PGCsi3.0-1组细胞生长受到明显抑制,细胞迁移率降低(P<0.05)。Transwell实验显示PGCsi3.0-1组穿膜细胞数为18.0±4.2,低于PGCsi3.0-Neg组的62.0±3.5和未转染组的58.0±4.9(P<0.05)。结论:应用RNAi能有效抑制HMGB1基因表达,进而抑制HeLa细胞增殖、侵袭和迁移,为宫颈癌的基因治疗提供了新思路。

Background and purpose：High mobility group box-l（HMGB1）,a non-histone chromosomal protein,is over-expressed in a variety of tumors that have high metastasis.Preliminary studies have confirmed that HMGB1 was over-expressed in cervical squamous cell cancer tissues and cervical cancer HeLa cells,and its expression level was closely associated with the clinical stage,invasion and metastasis.This study evaluated the effects of HMGB1 expression suppressed by siRNA on proliferation,invasion and migration of human cervical cancer cell line HeLa.Methods：The eukaryotic expression vector PGCsi3.0-1/HMGB1 siRNA was transfected into HeLa cells by lipofectamine2000（PGCsi3.0-1 group）,and then the successfully transfected single cell was selected by G418 and amplified.Cells transfected with PGCsi3.0-Neg and non-transfected cells were used as control.HMGB1 expression was detected by RT-PCR and Western blot.The MTT method was used for observing cell proliferation and drawing a growth curve.The ability of invasion and speed of cell migration were evaluated through transwell chamber invasion models and the cell scratch method.Results：The eukaryotic expression vector PGCsi3.0-1/HMGB1 siRNA was transfected into HeLa cells successfully,and the highest transfection efficiency was 75%.The expression of HMGB1 mRNA and protein in PGCsi3.0-1 group were 0.38±0.12 and 0.10±0.44,respectively.Compared with the PGCsi3.0-Neg group（1.19±0.25,1.90±0.35） and non-transfected group（1.42±0.14,2.55±0.32）,the difference was significant（P0.05）.The curves of cell growth indicated that cell proliferation in the PGCsi3.0-1 group was inhibited significantly compared with those in the PGCsi3.0-Neg group and non-transfected group（P0.05）.The scrape wound healed more slowly in PGCsi3.0-1 group than the other two groups（P0.05）.The numbers of trans-membrane cells in PGCsi3.0-Neg group and non-transfected group were 62.0±3.5 and 58.0±4.9.In comparison,PGCsi3.0-1group had a small number 18.0±4.2 （P0.05）.Conclusion：HMGB1 expression could be successfully inhibited by RNAi induced through eukaryotic expression vector PGCsi3.0-1/HMGB1 siRNA,and consequently inhibits the proliferation,migration and invasion of human cervical cancer cell line HeLa.This study provided preliminary results in the searching of RNAi therapy for cervical cancer.