微小RNA-425在胃癌组织的表达及对胃癌细胞株BGC-823凋亡、自噬的影响

Expression of microRNA -425 in gastric cancer tissues and its effect on apoptosis and autophagy of human gastric cancer cell line BGC823

目的观察微小RNA（miRNA，miR）-425在胃癌组织和细胞株的表达，并探讨miR-425对胃癌BGC823细胞凋亡、自噬的影响。方法采用实时定量聚合酶链反应（Real-timePCR）检测60例胃癌组织和癌旁组织miR-425表达，分析其与临床病理参数的关系，并检测miR-425在7种胃癌细胞株（HGC-27、BGC-803、MKN45、MGC-823、SGC-7901、NCL-N87、AGS）及正常胃黏膜细胞GES-1的表达。选取miR425表达水平最高的胃癌细胞瞬时转染miR-425inhibitors。Real-timePCR检测miR-425mRNA表达。流式细胞仪检测细胞周期、凋亡率及线粒体膜电位。Westernblot检测凋亡、自噬相关蛋白表达。结果胃癌组织miR-425相对表达水平为0．804-0．13，较癌旁组织（0．28±0．04）显著升高（P〈0．05），且其表达与TNM分期、细胞分化程度、肿瘤浸润深度及有无淋巴结转移密切相关（P〈0．05）。BGC-803细胞中miR-425表达水平明显高于HGC-27、MKN-45、MGC-823、SGC-7901、NCL-N87、AGS及GES-1细胞（P〈0．05）。与对照组和空白转染组比较，转染miR-425inhibitors能够明显诱导BGC-803细胞凋亡并出现G1期阻滞，同时线粒体膜电位明显降低（P〈0．05）；且B细胞淋巴瘤／白血病-2（bcl-2）、p62蛋白表达明显下调，B细胞淋巴瘤／白血病-2相关x蛋白（bax）、半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-9、微管相关蛋白1轻链3（LC3）、Beclinl蛋白表达明显上调（P〈0．05）。结论miR-425在胃癌组织和细胞株呈过表达，下调miR-425表达能够通过线粒体途径诱导胃癌BGC-803细胞凋亡和自噬。

Objective To investigate the expression of microRNA （miRNA, miR） -425 in gas- tric cancer tissues and cell lines as well as its effect on apoptosis and autophagy of human gastric cancer cells BGC - 823. Methods The expression of miR - 425 in 60 cases of gastric cancer tissues and their ad- jacent tissues was detected by real -time quantitative polymerase chain reaction （Real -time PCR）. The re- lationship between miR -425 expression and clinicopathological factors of gastric carcinoma was analyzed. The expression of miR - 425 was detected in 7 strains of gastric cancer cells （ HGC - 27, BGC - 803, MKN-45, MGC- 823, SGC -7901, NCL- N87 and AGS） and the normal gastric mucosa cell line, GES - 1, was used as the control. The gastric cancer cells with the highest level of miR - 425 were chosen for the transfection with miR -425 inhibitors plasmids by liposomes transient transfection method. Real - time PCR was used to detect the expression of miR - 425 mRNA of transfected BGC - 803 cells. Flow cytometry was used to detect the cell apoptosis, cell cycle progression and mitochondrial trans -mem- brane potential. Western blotting was used to detect the expression of proteins associated with apoptosis and autophagy. Results The relative expression of miR - 425 in gastric carcinoma tissues was 0. 80 ~ 0. 13, significantly higher than that （0. 28± 0.04 ） of the adjacent tissues （P 〈 0. 05 ）. Its expression was related to TNM clinical stage, differentiation, depth of invasion and lymph node metastasis （ P 〈 0. 05 ）. The degree of expression of miR - 425 in BGC - 803 was obviously higher than that in HGC - 27, MKN - 45, MGC - 823, SGC - 7901, NCL - N87, AGS and GES - 1 cells and the difference was significant （ P 〈 0. 05 ）. As compared with negative control group and blank control group, miR -425 inhibitors promoted the apoptosis of BGC - 803 and the cell cycle analysis revealed that miR - 425 inhibitors could induce a G1 - phase arrest and greatly decreased the mitochondrial trans - membrane potential （ P 〈 0. 05 ）. Mean- while the expression of B cell lymphoma/leukemia - 2 （ bel - 2） and p62 proteins were down - regulated, and the expression of bc1-2 associated X protein （bax） , Caspase -9, microtubule -associated protein 1 light chain 3 （ LC3 ） and Beclinl proteins was up - regulated （ P 〈 0. 05 ）. Conclusion MiR - 425 was o- ver - expressed in gastric cancer tissues and ceils, and down - regulation of miR - 425 can induce apopto- sis and autophag＇y via the mitochondrial pathway in BGC -803 cells.