中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
10期
1917-1920
,共4页
陈辉%瞿丽%黄永刚%谢达飞
陳輝%瞿麗%黃永剛%謝達飛
진휘%구려%황영강%사체비
RNA干扰%RRM2基因%癌,肝细胞
RNA榦擾%RRM2基因%癌,肝細胞
RNA간우%RRM2기인%암,간세포
RNA interference%RRM2 gene%Carcinoma,hepatocellular
目的 观察RNA干扰沉默RRM2基因表达对人肝癌细胞HepG2细胞生物学影响,并探讨其分子机制.方法 利用小RNA干扰(siRNA)技术沉默HepG2细胞中RRM2基因的表达,用采用实时定量聚合酶链反应(Real-time PCR)及Western blot技术检测RRM2 mRNA及蛋白的表达；用细胞计数试剂盒(CCK-8)方法检测siRNA-RRM2对HepG2细胞增殖的影响；用Transwell细胞迁移系统检测siRNA-RRM2对HepG2细胞迁移能力的影响；采用Western blot技术检测抗凋亡蛋白B淋巴细胞/白血病-2(bcl-2),促凋亡蛋白bax和细胞色素C(Cyt-C)蛋白水平变化；建立成瘤模型每组10只,观察RRM2基因沉默后对移植瘤生长的影响.结果 siRNA-RRM2有效沉默HepG2细胞中RRM2表达；CCK-8法结果显示与对照组比较下调RRM2基因抑制HepG2增殖[(41.20±2.13)％比(91.35±10.22)％]；Transwell细胞迁移系统检测显示下调RRM2基因后,显著抑制了HepG2的迁移能力(21.20±1.28比132.50±7.88)；Western blot结果显示抗凋亡蛋白bcl-2下调,促凋亡蛋白bax上调,Cyt-C上调；体内实验证实下调RRM2组肿瘤体积明显小于对照组,到第8周时,siRNA-RRM2组肿瘤体积为(171.13 ±28.12) mm3,对照组为(1487.41±168.20) mm3.结论 siRNA-RRM2能显著抑制RRM2基因在人肝癌细胞HepG2中的表达,部分逆转HepG2的恶性生物学行为并明显抑制HepG2在裸鼠体内的成瘤和生长.
目的 觀察RNA榦擾沉默RRM2基因錶達對人肝癌細胞HepG2細胞生物學影響,併探討其分子機製.方法 利用小RNA榦擾(siRNA)技術沉默HepG2細胞中RRM2基因的錶達,用採用實時定量聚閤酶鏈反應(Real-time PCR)及Western blot技術檢測RRM2 mRNA及蛋白的錶達；用細胞計數試劑盒(CCK-8)方法檢測siRNA-RRM2對HepG2細胞增殖的影響；用Transwell細胞遷移繫統檢測siRNA-RRM2對HepG2細胞遷移能力的影響；採用Western blot技術檢測抗凋亡蛋白B淋巴細胞/白血病-2(bcl-2),促凋亡蛋白bax和細胞色素C(Cyt-C)蛋白水平變化；建立成瘤模型每組10隻,觀察RRM2基因沉默後對移植瘤生長的影響.結果 siRNA-RRM2有效沉默HepG2細胞中RRM2錶達；CCK-8法結果顯示與對照組比較下調RRM2基因抑製HepG2增殖[(41.20±2.13)％比(91.35±10.22)％]；Transwell細胞遷移繫統檢測顯示下調RRM2基因後,顯著抑製瞭HepG2的遷移能力(21.20±1.28比132.50±7.88)；Western blot結果顯示抗凋亡蛋白bcl-2下調,促凋亡蛋白bax上調,Cyt-C上調；體內實驗證實下調RRM2組腫瘤體積明顯小于對照組,到第8週時,siRNA-RRM2組腫瘤體積為(171.13 ±28.12) mm3,對照組為(1487.41±168.20) mm3.結論 siRNA-RRM2能顯著抑製RRM2基因在人肝癌細胞HepG2中的錶達,部分逆轉HepG2的噁性生物學行為併明顯抑製HepG2在裸鼠體內的成瘤和生長.
목적 관찰RNA간우침묵RRM2기인표체대인간암세포HepG2세포생물학영향,병탐토기분자궤제.방법 이용소RNA간우(siRNA)기술침묵HepG2세포중RRM2기인적표체,용채용실시정량취합매련반응(Real-time PCR)급Western blot기술검측RRM2 mRNA급단백적표체；용세포계수시제합(CCK-8)방법검측siRNA-RRM2대HepG2세포증식적영향；용Transwell세포천이계통검측siRNA-RRM2대HepG2세포천이능력적영향；채용Western blot기술검측항조망단백B림파세포/백혈병-2(bcl-2),촉조망단백bax화세포색소C(Cyt-C)단백수평변화；건립성류모형매조10지,관찰RRM2기인침묵후대이식류생장적영향.결과 siRNA-RRM2유효침묵HepG2세포중RRM2표체；CCK-8법결과현시여대조조비교하조RRM2기인억제HepG2증식[(41.20±2.13)％비(91.35±10.22)％]；Transwell세포천이계통검측현시하조RRM2기인후,현저억제료HepG2적천이능력(21.20±1.28비132.50±7.88)；Western blot결과현시항조망단백bcl-2하조,촉조망단백bax상조,Cyt-C상조；체내실험증실하조RRM2조종류체적명현소우대조조,도제8주시,siRNA-RRM2조종류체적위(171.13 ±28.12) mm3,대조조위(1487.41±168.20) mm3.결론 siRNA-RRM2능현저억제RRM2기인재인간암세포HepG2중적표체,부분역전HepG2적악성생물학행위병명현억제HepG2재라서체내적성류화생장.
Objective To investigate the effect of small interference RNA (siRNA) silencing RRM2 on the biological behaviors of human hepatoma cancer cell line HepG2 and and the molecular mechanisms.Methods RRM2 expression was knocked down in human hepatoma cancer cell line HepG2 by RRM2 siRNA.The expression level of RRM2 was detected in human hepatoma cancer cell line HepG2 and human normal liver cell line HL-7702 at mRNA and protein levels by real-time polymerase chain reaction (PCR) or Western blotting.The cell proliferation was measured by cell counting Kit-8 (CCK-8).The migration was observed by using Transwell cell migration system.The expression levels of B lymphocytes/leukemia-2 (bcl-2),bax and cytochrome C (Cyt-C) proteins were detected by using Western blotting.Ten male BALB/C nude mice were subcutaneously injected with siRNA-RRM2/HepG2,siRNA-NC/HepG2 or NEG/HepG2 cells respectively to study the growth of tumor in each group.Results siRNA-RRM2 could down-regulate the expression of RRM2 in HepG2 remarkably,and siRNA-RRM2 could inhibit both the proliferation [(41.20 ± 2.13) ％ vs (91.35 ± 10.22) ％] and migration ability of HepG2 cells (21.20 ±1.28 vs 132.50 ±7.88) as compared with the negative control group.siRNA-RRM2 could down-regulate the expression of bcl-2 and up-regulate the expression of bax and Cyt-C.Additionally,the volume of xenograft tumors in siRNA-RRM2 group was decreased significantly as compared with control group.The volume of xenograft tumors in siRNA-RRM2 group was (171.13 ± 28.12) mm3 and that in siRNA-NC group was (1487.41 ± 168.20) mm3 at 8th week.Conclusion siRNA-RRM2 can down-regulate the expression of RRM2 in the HepG2 cells and may play an important role in the regulation of biological behaviors of HepG2 cells,and also inhibit the tumor growth of nude mice model.
