摘要
目的探讨小分子干扰RNA（siRNA）抑制高迁移族蛋白B1（HMGBl）基因表达对胃癌细胞株MGC-803侵袭转移能力的影响及其作用机制。方法设计、合成靶向HMGBl基因的siRNA，以脂质体为载体转染胃癌细胞株MGC-803。Transwell小室模型和划痕实验测定细胞侵袭及迁移能力，四甲基偶氮唑蓝比色法检测细胞对Matrigel基质胶的黏附能力，电泳迁移实验检测化学合成核因子KB（NF—KB）的活性，逆转录聚合酶链反应和Westernblot方法检测HMGBl、基质金属蛋白酶9（MMP-9）mRNA和蛋白的表达。结果HMGBlsiRNA可明显抑制MGC-803胃癌细胞株HMGBImRNA和蛋白的表达。HMGBlsiRNA转染组细胞侵袭穿膜细胞的数量为（142．7±3．4）个／视野，与未转染组[（303．5±4．3）个／视野]比较，差异有统计学意义（P〈0．05）。HMGBlsiRNA转染组细胞迁移穿膜细胞的数量为（293．7±4．4）个／视野，与未转染组[（445．5±5．6）个／视野]比较，差异有统计学意义（P〈0．05）。HMGBlsiRNA转染组的细胞黏附率为（33．4±0．03）％，与未转染组[（57．4±4．2）％]比较，差异有统计学意义（P〈0．05）。HMGBlsiRNA转染组MGC-803细胞MMP-9mRNA的相对表达水平（0．2±0．1）低于未转染组（1．4±0．4，P〈0．05）。HMGBlsiRNA转染组细胞MMP-9蛋白的相对表达水平（0．4±0．1）低于未转染组（2．34±0．7，P〈0．05）。HMGBlsiRNA转染组NF．KB活性明显低于未转染组。结论HMGBl基因沉默可有效抑制MGC-803细胞的侵袭转移，其机制可能与调控NF-KB的活性和MMP-9表达有关。
Objective To investigate the effect of high mobility group box-1 （high mobility group box B 1, HMGB1 ） on the invasive and metastatic abilities of gastric cancer cell line MGC-803 and analyze the possible mechanisms. Methods HMGB1 gene targeting siRNA was designed and synthesized, and HMGBI siRNA oligonucleotides were transfected into the MGC-803 cells with Lipofectamine 2000. The invasive and migratory abilities were detected by transwell assay and scratch assay. The Matrigel matrix glue adhesive ability of MGC-803 cells was evaluated by MTF assay. NF-KB activity was detected by electrophoretic mobility shift assay. The mRNA and protein levels of HMGB1 and MMP-9 were determined by RT-PCR and Western blot, respectively. Results The siRNA down-regulated the levels of HMGB1 mRNA and protein. Compared with that of the control group, the number of invasive （ 142.7 ± 3.4 /view vs. 303.5 ±4.3/view ） and migratory （ 293.7 ± 4.4/view vs. 445.5 ± 5.6/view） cells was significantly increased （P 〈 0. 05） and the adhesive ability of MGC-803 cells to Matrige] was significantly elevated （33.4 ± 0.03% vs. 57.4 ± 4.2% , P 〈 0.05 ）. In addition, silencing of HMGB1 gene significantly inhibited the activity of NF-kB and the relative expression folds of mRNA （ 0.2 ＋ 0. 1 vs. 1.4 ±0.4, P 〈 0.05 ） and protein （0.4±0.1 vs. 2.3±0.7, P〈0. O5）ofMMP-9. Conclusion Silencing of HMGB1 can effectively inhibit the invasion and migration of gastric cancer cells and this effect of HMGB1 may be partly due to itsregulation of NF-KB and MMP-9 expressions.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2013年第4期244-248,共5页
Chinese Journal of Oncology
基金
山东省自然科学基金（ZR2009CMl38）
关键词
胃肿瘤
肿瘤侵润
肿瘤转移
RNA干扰
高迁移率族蛋白1
Stomach neoplasms
Neoplasm invasiveness
Neoplasm metastasis
RNAinterference
High mobility group box-1
