Li Wei-Wei, Wu Wei-Zhong, Liang Ying, Xiao Chuan-Li, Tao Zhong-Hua, Wang Lu, Sun Hui-Chuan, Fan Jia
Liver Cancer Institute and Zhongshan Hospital, Fudan Unviersity, Shanghai 200032, China.
Zhonghua Gan Zang Bing Za Zhi. 2010 Jan;18(1):27-31. doi: 10.3760/cma.j.issn.1007-3418.2010.01.007.
To investigate the effects of ARNT2 on invasion and migration of HCCLM6 cells.
Four short hairpin oligos targeting to ARNT2 were s cloned into the pLVTHM vector. Lentiviral vectors shRNA-ARNT2i, pCMV-dR8.74 and pMD2G were cotransfected into 293T cells using Lipofectamine 2000. HCCLM6 was infected with virus supernatant. ARNT2 mRNA and protein expressions were detected using quantitative Real time-PCR and Western blot, respectively. The invasion and migration of HCCLM6 cells were evaluated using wound healing assay and cell invasion assay in vitro. Statistical analysis was performed with SPSS 16.0.
The relative mRNA levels of ARNT2 were 0.154+/-0.024, 0.860+/-0.145, 1.004+/-0.009 in shRNA-ARNT2i virus infected HCCLM6 cells, mock-infected cells and control vector virus infected cells (F = 113.14, P more than 0.01). The expression of ARNT2 at protein level was 16.45+/-1.6, 44.56+/-2.07 in the HCCLM6 cells infected with shRNA-ARNT2i virus and negative control vector virus, respectively (t = 18.58, P less than 0.01). The scrape wound of HCCLM6 cells infected with shRNA-ARNT2i virus healed faster than cells infected with control vector virus or mock-infected cells. The number of cells invading through Matrigel was higher in the HCCLM6 cells infected with shRNA-ARNT2i virus (13.25+/-1.04) than that in mock-infected HCCLM6 cells and the HCCLM6 cells infected with negative control vector virus (6.50+/-2.56, 6.75+/-2.05) (F = 29.645, P less than 0.01).
Inhibition of ARNT2 gene promotes the invasion and migration of HCCLM6 cells.
研究芳烃受体核转运蛋白2（ARNT2）对肝癌细胞系HCCLM6侵袭和迁移能力的影响。
设计4条靶向ARNT2的短发夹寡核苷酸序列，克隆至pLVTHM载体。将慢病毒载体shRNA - ARNT2i、pCMV - dR8.74和pMD2G用脂质体2000共转染至293T细胞，收集病毒上清感染HCCLM6细胞。分别采用实时定量聚合酶链反应（qRT - PCR）和蛋白质免疫印迹法（Western blot）检测ARNT2 mRNA和蛋白表达水平。采用划痕实验和体外细胞侵袭实验评价HCCLM6细胞的侵袭和迁移能力。应用SPSS 16.0软件进行统计学分析。
shRNA - ARNT2i病毒感染的HCCLM6细胞、空载体病毒感染的细胞及未感染病毒的细胞中，ARNT2相对mRNA水平分别为0.154±0.024、0.860±0.145、1.004±0.009（F = 113.14，P＜0.01）。shRNA - ARNT2i病毒感染的HCCLM6细胞和阴性对照载体病毒感染的HCCLM6细胞中，ARNT2蛋白表达水平分别为16.45±1.6、44.56±2.07（t = 18.58，P＜0.01）。shRNA - ARNT2i病毒感染的HCCLM6细胞划痕愈合速度快于阴性对照载体病毒感染的细胞和未感染病毒的细胞。shRNA - ARNT2i病毒感染的HCCLM6细胞穿过基质胶的细胞数（13.25±1.04）高于未感染病毒的HCCLM6细胞及阴性对照载体病毒感染的HCCLM6细胞（6.50±2.56、6.75±2.05）（F = 29.645，P＜0.01）。
抑制ARNT2基因可促进HCCLM6细胞的侵袭和迁移。
