Zhang Jing, Fu Yan-Chao, Kang Chun-Sheng, Zhang Qing-Yu, Wang Tao, Zhang Jie
Department of Gastroenterology, Tianjin Medical University General Hospital, Tianjin 300052, China.
Zhonghua Nei Ke Za Zhi. 2009 Jul;48(7):557-61.
To construct a short hairpin RNA(shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocarcinoma cells.
P85 and Akt1 shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein expression was identified with real-time PCR and Western blot. The proliferative activity of tumor cells was evaluated with MTT assay and flow cytometry in vitro. rAd5-HK and rAd5-P + A mediated by adenovirus were injected into the established subcutaneous SGC-7901 gastric adenocarcinoma in nude mice. During the observation period of 21 days, tumor volume was measured every 3 days to further testify the anti-tumor effect of rAd5-P + A on the SGC-7901 gastric adenocarcinoma cells and cell in situ apoptosis was detected with TUNEL assay.
The adenovirus vector rAd5-P + A was successfully constructed and it dramatically downregulated P85 and Akt1 mRNA expression in SGC-7901 gastric adenocarcinoma cells. Compared with a control group of SGC-7901 cells and cells transfected with general adenovirus rAd5-HK as control, P85 and Akt1 protein expression 48 h and 72 h after rAd5-P + A transfection was decreased by 57.5% and 63.7%, 67.8% and 75.6% with statistical significance (P = 0.005, P = 0.003). Cell proliferative activity in rAd5-P + A transfected cells was suppressed from the second day (P < 0.001) and the decreased P85 and Akt1 expression was accompanied by 5.9% -7.1% decrease of S phase fraction and 12.1% - 13.7% increase of G0/G1 phase. The tumor volume of rAd5-P + A treated group was smaller than that of the control and rAd5-HK group with statistical significance (F = 9.871, P = 0.025). Moreover, rAd5-P + A could induce cell in situ apoptosis.
Adenovirus-mediated targeting P85 and Akt1 shRNA can inhibit the growth of SGC-7901 human gastric adenocarcinoma cells and this may provide a new strategy of combination gene therapy in gastric adenocarcinoma.
构建靶向P85和蛋白激酶B1（PKB1/Akt1）的短发夹RNA（shRNA）腺病毒载体，并研究其对人胃腺癌细胞SGC-7901生长的影响。
采用同源重组技术将P85和Akt1 shRNA表达框亚克隆至pGSadeno腺病毒载体，构建pGSadeno-P85+Akt1（rAd5-P+A）载体。经筛选和扩增后，用PacI酶切重组腺病毒载体并转染至SGC-7901细胞，然后用荧光显微镜检测其滴度和转染效率。用实时PCR和Western blot鉴定P85和Akt1 mRNA及蛋白表达。体外采用MTT法和流式细胞术评估肿瘤细胞的增殖活性。将腺病毒介导的rAd5-HK和rAd5-P+A注射到已建立的裸鼠皮下SGC-7901胃腺癌模型中。在21天的观察期内，每3天测量一次肿瘤体积，以进一步验证rAd5-P+A对SGC-7901胃腺癌细胞的抗肿瘤作用，并用TUNEL法检测细胞原位凋亡。
成功构建腺病毒载体rAd5-P+A，其显著下调SGC-7901胃腺癌细胞中P85和Akt1 mRNA表达。与SGC-7901细胞对照组及转染普通腺病毒rAd5-HK的细胞对照组相比，rAd5-P+A转染后48 h和72 h时，P85和Akt1蛋白表达分别降低了57.5%和63.7%、67.8%和75.6%，差异有统计学意义（P=0.005，P=0.003）。rAd5-P+A转染细胞的增殖活性从第二天开始受到抑制（P<0.001），P85和Akt1表达降低伴随着S期比例下降5.9%-7.1%，G0/G1期比例升高12.1%-13.7%。rAd5-P+A治疗组的肿瘤体积小于对照组和rAd5-HK组，差异有统计学意义（F=9.871，P=0.025）。此外，rAd5-P+A可诱导细胞原位凋亡。
腺病毒介导的靶向P85和Akt1的shRNA可抑制人胃腺癌细胞SGC-7901的生长，这可能为胃腺癌联合基因治疗提供新策略。
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