CAJ | 학술논문

目的观察应用RNA干扰(RNAi)技术敲低PIK3R1、AKT1表达后在体外对胃腺癌SGC7901细胞侵袭的抑制作用.方法将重组腺病毒质粒表达载体rAd5-A-P转染至SGC7901细胞.Realtime PCR和Western blot分别检测转染前后目的基因mRNA和蛋白的表达水平,酶联免疫吸附试验(ELISA)检测细胞外基质金属蛋白酶(MMP)-2、MMP-9的浓度变化.划痕、Transwell、3-DMatrigel基质生长实验评价肿瘤细胞侵袭能力的变化.结果 rAd5-A-P可以有效抑制PIK3R1、AKT1的表达,MMP-2、MMP-9表达下调,基质金属蛋白酶组织抑制因子(TIMP)-2表达上调,ELISA证实细胞外MM9-2、MMP-9浓度在治疗组明显减低.划痕实验显示rAd5-A-P转染组细胞转移运动能力明显减弱,Transwell穿过细胞数结果:对照组(105.0±4.0)和无义序列组(102.5±6.4),rAd5-A-P转染组(67.0±3.9),3-D的Matrigel基质生长实验显示与对照组和无义序列组比较,rAd5-A-P转染组细胞形成的细胞团块较小.结论靶向AKT1、PIK3R1的RNAi技术可以序列特异性地抑制PIK3R1、AKT1表达,在体外明显抑制SGC7901细胞的侵袭能力.
목적 관찰응용RNA간우(RNAi)기술고저PIK3R1、AKT1표체후재체외대위선암SGC7901세포침습적억제작용.방법 장중조선병독질립표체재체rAd5-A-P전염지SGC7901세포.Realtime PCR화Western blot분별검측전염전후목적 기인mRNA화단백적표체수평,매련면역흡부시험(ELISA)검측세포외기질금속단백매(MMP)-2、MMP-9적농도변화.화흔、Transwell、3-DMatrigel기질생장실험평개종류세포침습능력적변화.결과 rAd5-A-P가이유효억제PIK3R1、AKT1적표체,MMP-2、MMP-9표체하조,기질금속단백매조직억제인자(TIMP)-2표체상조,ELISA증실세포외MM9-2、MMP-9농도재치료조명현감저.화흔실험현시rAd5-A-P전염조세포전이운동능력명현감약,Transwell천과세포수결과:대조조(105.0±4.0)화무의서렬조(102.5±6.4),rAd5-A-P전염조(67.0±3.9),3-D적Matrigel기질생장실험현시여대조조화무의서렬조비교,rAd5-A-P전염조세포형성적세포단괴교소.결론 파향AKT1、PIK3R1적RNAi기술가이서렬특이성지억제PIK3R1、AKT1표체,재체외명현억제SGC7901세포적침습능력.
Objective To observe the inhibitory effects on the invasion of gastric adenocarcinoma SGC7901 cells by small hairpin RNA targeting PIK3R1 and AKT1 in vitro. Methods The recombinant adenovirus vector plasmid expression vector which contained PIK3R1 and AKT1 shRNA was transfected into SGC7901 cells. Real time PCR and Western blot were used to detect the expression of P1K3R1 and AKT1. ELESA was used to measure the change in the MMP-2 and MMP-9 expression. The invasion ability of the tumor ceils was examined by Scarification, Transwell and 3-dimensional matrigel matrix tests. Re-sults rAdS-A-P mediated shRNA targeting PIK3R1 ,and AKT1 dramatically down-regulated their expres-sion in SGC7901 cells. MMP-2 and MMP-9 were downregulated,and TIMP-2 was upregulated. The extra-cellular levels of MMP-2 and MMP-9 were decreased. Scarification test indicated that the invision ability was decreased obviously. Transwell showed that the number of cells invading through the matrigel in con-trol. nonsense sequence, and rAd5-A-P transfection groups was 105.0±4.0,102.5±6.4, and 67.0±3.9 respectively. In the rAdS-A-P transfection group, cells formed only small aggregates as compared with the control and nonsense sequence groups in 3-dimensional matrigel matrix growth test. Conclusion ShRNA targeting PIK3R1 ,and AKTI downregulated significantly their expression in a sequence-specific manner, and inhibited the invasion of gastric adenocarcinoma SGC7901 cells in vitro.