中文摘要：

目的探讨短发夹（sh）RNA芳香烃受体核易位蛋白（ARNT）2i慢病毒表达载体对高转移肝癌细胞HCCLM6侵袭和迁移的影响。方法以ARNT2为目的基因，化学合成4条干扰RNA，与经MluⅠ和ClaⅠ酶切后的pLVTHM载体连接过夜，筛选并鉴定阳性克隆。利用脂质体2000将ShRNA—ARNT2i，pCMV—dR8．74，pMD2G共转染293T细胞，获得shRNA—ARNT2i慢病毒颗粒，进一步感染HCCLM6靶细胞，并分为转染shRNAARNT2i组、未转染的空白对照组和转染阴性对照序列组。用荧光实时定量PCR和Westernblot检测ARNT2基因的干扰效率。应用划痕和Boyden小室法观察ARNT2表达下调对HCCLM6侵袭和迁移能力的影响。用SPSS16．0统计软件对数据进行独立样本t检验和方差分析，其中多组资料两两比较采用LSD法。结果荧光实时定量PCR检测转染shRNAARNT2i组，未转染的空白对照组和转染阴性对照序列组3组ARNT2mRNA相对表达值分别为0．154±0．024、0．860±0．145、1．004±0．009，F=113．14，P〈0．01，差异有统计学意义；Westernblot显示转染stLRNA—ARNT2i组ARNT2蛋白的表达量为16．45±1．6，转染阴性对照序列组ARNT2蛋白的表达量为44．56±2．07，两组比较，t=18．58，P〈0．01，差异有统计学意义；转染shRNA—ARNT2i组细胞在划痕48h后基本愈合，而转染阴性对照序列组和未转染的空白对照组划痕依然可见；转染shRNAARNT2i组穿膜细胞数为（13．25±1．04）个，而未转染的空白对照组和转染阴性对照序列组的穿膜细胞数分别为（6．50±2．56）个、（6．75±2．05）个，F=29．645，P〈0．01，差异有统计学意义。结论shRNA—ARNT2i载体能显著下调HCCLM6细胞ARNT2基因和蛋白的表达，促进HCCLM6细胞的运动和侵袭。

英文摘要：

Objective To investigate the effects of ARNT2 on invasion and migration of HCCLM6 cells. Methods Four short hairpin oligoes targeting to ARNT2 were s cloned into the pLVTHM vecto. 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. Results The relative rnRNA 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 〈 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 〈 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 〈 0.01）. Conclusion Inhibition of ARNT2 gene promotes the invasion and migration of HCCLM6 cells.