腺病毒介导的靶向蛋白激酶B1和环氧合酶2短发夹RNA抑制人胃腺癌细胞的侵袭和转移

目的 构建靶向性蛋白激酶B1(Aktl)和环氧合酶2(COX-2)短发夹RNA(shRNA)腺病毒载体,研究其对SGC-7901人胃腺癌细胞侵袭和转移的抑制效果.方法 构建腺病毒载体pGSadeno-Aktl+COX-2(rAd5-A+C)并体外转染SGC-7901人胃癌细胞株后,用实时定量PCR和蛋白印迹分别检测对照组SGC-7901、空载组rAd5-HK和治疗组rAd5-A+C中Aktl、COX-2 mRNA和蛋白质的表达,其中以对照组SGC-7901、空载组rAd5-HK作为阴性对照.用ELISA法分别检测它们的MMP-2和MMP-9含量;用Transwell法分析它们的侵袭和转移能力.结果 构建的rAd5-A+C重组腺病毒载体在转染SGC-7901细胞48 h后可显著抑制Aktl和COX-2 mRNA的表达,而治疗组rAd5-A+C的Aktl和COX-2蛋白表达量与对照组和空载组相比分别下调68.4%和60.2%(均P<0.01);rAd5-A+C组中MMP-2和MMP-9含量分别为(39.7±1.7)ng/ml(31.3±3.6)ng/ml,明显低于对照组SGC-7901(278.4±15.5)ng/ml(225.4±15.1)ng/ml和卒载组rAd5-HK(275.5±2.1)ng/ml、(226.0±23.3)ng/ml(P=0.01、P=0.021).结论 腺病毒介导的靶向性Aktl和COX-2shRNA可抑制人胃腺癌细胞的侵袭和转移.

Inhibitory effects of adenovirus-mediated shRNA targeting Akt1 and COX-2 on invasion and metastasis of SGC-7901 cells

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting Aktl (protein kinase B1, PKBI/Aktl) and cyclooxygenase-2 (COX-2) and study its effects on the invasion and metastasis of SGC-7901 human gastric adenocarcinoma cells. Methods Aktl and COX-2 shRNA expression frames were subcloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno-Aktl+COX-2 (rAdS-A+C) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells, the titer and transfection efficiency were detected by fluorescent microscopy. Aktl and COX-2 mRNA and protein expression was identified by real-time PCR and Western blot. MMP-2 and MMP-9 contents in control group SGC-7901、rAd5-HK and treatment group rAdS-A+C were detected by ELISA assay and transwell assay analyzed cell invasion and metastasis ability. Results Adenovirus vector rAdS-A+C was successfully constructed and it dramatically down-regulated Aktl and COX-2 mRNA and protein expression in SGC-7901 gastric cancer cells. MMP-2 and MMP-9 contents in treatment group rAd5-A+C were respectively (39.7± 1.7) ng/ml, (31.3±3.6) ng/ml, and they were lower than those in control group SGC-7901 (278.4± 15.5) ng/ml, (225.4±15.1) ng/ml and rAd5-HK (275.5±2.1) ng/ml, (226.0±23.3) ng/ml (P= 0.01, P=0.021). Transwell assay showed treatment group rAd5-A+C significantly inhibited the invasion and metastasis of SGC-7901 gastric adenocacinoma cells. Conclusions Adenovirus-mediated targeting Aktl and COX-2 shRNA can inhibit the invasion and metastasis of SGC-7901 human gastric adenocarcinoma cells.