RNA干扰沉默高迁移率族蛋白B1的表达对结直肠癌LoVo细胞增殖和侵袭的抑制作用  

Silencing HMGB1 expression by lentivirus-mediated small interfering RNA （siRNA） inhibits the proliferation and invasion of colorectal cancer LoVo cells in vitro and in vivo

作　　者：李增军[1] 王海鹏[3] 宋宝[2] 孙燕来[1] 徐忠法[1] 韩建军[1] 

机构地区：[1]山东省肿瘤医院山东省肿瘤防治研究院普外科,济南250117 [2]山东省肿瘤医院山东省肿瘤防治研究院基础研究中心,济南250117 [3]济南大学山东省医学科学院

出　　处：《中华肿瘤杂志》2015年第9期664-670,共7页Chinese Journal of Oncology

基　　金：山东省科技发展计划项目（2013GSF11834）;山东省医药卫生科技发展项目（2011HW069）;山东省自然科技发展项目（ZR2009CM138）

摘　　要：目的探讨小分子RNA（siRNA）干扰高迁移率族蛋白B1（HMGB1）基因的表达对结直肠癌细胞株kv0增殖和侵袭的抑制作用。方法采用慢病毒介导的RNA干扰技术沉默HMGB1的表达，以逆转录聚合酶链反应和Western blot法检测干扰后LoVo细胞中HMGB1 mRNA和蛋白的表达情况。采用四甲基偶氮唑蓝（MTr）法、流式细胞术、Transwell小室实验、细胞划痕实验及裸鼠成瘤实验，分析HMGB1基因沉默对LoVo细胞生长、增殖、侵袭及转移的影响。结果慢病毒介导siRNA可成功转染结直肠癌细胞株LoVo。HMGB1-siRNA转染组LoVo细胞中HMGB1 mRNA和蛋白的相对表达水平分别为0．24±0．04和0．21±0．03，阴性对照组分别为0．82±0．13和1．15±0．18，空白对照组分别为0．93±0．15和1．21±0．20；HMGB1-siRNA转染组HMGB1 mRNA和蛋白的表达水平均明显低于阴性对照组和空白对照组（P〈0．05）。MTT法检测结果显示，HMGB1-siRNA转染组LoVo细胞的生长速度较阴性对照组和空白对照组降低（P〈0．05）。流式细胞术检测结果显示，HMGBI—siRNA转染组LoVo细胞的增殖指数为38．27±1．32，明显低于阴性对照组（54．66±1．74）和空白对照组（57．43±1．29，P〈0．05）。Transwell小室实验显示，HMGB1-siRNA转染组穿膜细胞数为（14．0±3．5）个／高倍视野，明显低于阴性对照组[（51．0±6．7）个／高倍视野]和空白对照组[（68．0±5．3）个／高倍视野，P〈0．05]。细胞划痕实验显示，划痕后培养48h时，HMGB1-siRNA转染组LoVo细胞的迁移距离为（83．61±23．21）μm，明显低于阴性对照组[（202．86±46．46）μm]和空白对照组[（214．58±57．38）μm，P〈0．05]。裸鼠成瘤实验显示，21d后，肿瘤最终体积为（521±34）mm^3,明显小于阴性对照组[（763±46）mm^3]和空白对照组[（802±51）mm^3，P〈0．05]。结论慢病毒介导的RNA干扰技术可有效抑制结直肠癌LoVo细胞中HMGBObjective To inquire into the influence of silencing HMGB1 expression by small interfering RNA （siRNA） on cell growth, proliferation, invasion and metastasis of colorectal cancer LoVo cells both in vitro and in vivo. Methods Lentivirus-mediated HMGB1 siRNA was transfected into LoVo cells to silence the HMGB1 expression. The HMGB1 mRNA and protein expression after siRNA transfection was detected by RT-PCR and Western blot. MTT assay was used to observe the cell proliferation and to draw a growth curve. Cell cycle was measured by flow cytometry. The ability of invasion and speed of cell migration were evaluated by transwell chamber invasion and cell scratch assay. The influence of HMGB1 silencing on the proliferation of LoVo cells in vivo was observed in LoVo tumor-bearing nude mice. Results Lentivirusmediated siRNA was successfully transfected into colorectal cancer cell line LoVo. The expression of HMGB1 mRNA and protein in the HMGBI-siRNA group were 0.24±0.04 and 0.21±0.03, respectively. Compared with the HMGBI-siRNA-Neg group （0.82±0.13, 1.15±0.18） and control group （0.93±0.15, 1.21±0.20）, the difference was significant （P〈0.05）. MTT assay showed that the cell proliferation in the HMGBI-siRNA group was significantly inhibited when compared with that in the HMGBI-siRNA-Neg group and control group （P〈0.05）. Flow cytometry showed that the proliferation index （PI） of HMGBI-siRNA group was 38.27± 1.32, significantly lower than 54.66±1.74 in the HMGBI-siRNA-Neg group and 57.43±1.29 in the control group （P〈0.05）. The transwell assay showed that the number of penetrated cells in the HMGBI-siRNA group was 14.0±3.5, significantly lower than 51.0±6.7 in the HMGBI-siRNA-Neg group and 68.0±5.3 in the control group （P〈0.05）. Similarly, the scrape wound recovered significantly slower in the HMGB1- siRNA group （83.61±23.21） μm than that in the other two groups （202.86±46.46） bun and （214.58±57.38） txm （P〈0.05）. The nude mouse xenograft tumor experiment s

关 键 词：结直肠肿瘤 高迁移率族蛋白B1 LOVO细胞 RNA干扰 肿瘤侵袭 肿瘤 转移 裸鼠 

分 类 号：R735.34[医药卫生—肿瘤]