circRNA_103809调节胃癌干样细胞特性和线粒体功能的研究

Study of circRNA_103809 Regulating Stem-like Cell Characteristics of Gastric Cancer and Mitochondrial Function

目的初步探究环状RNA103809(circRNA_103809)调节胃癌干样细胞特性和线粒体功能的方法。方法设计3组干扰circRNA_103809的shRNA转染胃癌(SGC-7901)细胞株,选择干扰效果最好的shRNA进行后续研究;将SGC-7901细胞分为Control组、shRNA-NC组和sh-circ103809组;采用Western blot法和RT-PCR检测干细胞标志物细胞黏附分子44(CD44)、胚胎干细胞关键蛋白(SOX2)和八聚体结合转录因子4(OCT4)水平;观察肿瘤细胞成球情况;采用流式细胞术检测线粒体膜电位变化;采用Western blot法检测线粒体损伤标志物兔抗人单克隆抗体(Bax)/B淋巴细胞瘤-2(Bcl-2)、裂解半胱天冬酶9/半胱氨酸蛋白酶9(Caspase9)、裂解半胱天冬酶3/半胱氨酸蛋白酶3(Caspase3)表达。结果sh-circ103809组CD44、SOX2和OCT4蛋白及mRNA水平明显低于Control组和shRNA-NC组(P<0.05)。与Control组和shRNA-NC组相比,sh-circ103809组细胞成球直径减小、成球率降低(P<0.05)。sh-circ103809组线粒体膜电位低于Control组和shRNA-NC组(P<0.05)。sh-circ103809组Bax/Bcl-2、Caspase9/β-actin、Caspase3/β-actin蛋白表达水平明显高于Control组和shRNA-NC组(P<0.05)。结论干扰circRNA_103809表达可抑制干细胞标志物蛋白表达及胃癌细胞体外成瘤能力,降低线粒体膜电位,进而促进细胞凋亡、抑制肿瘤的发展进程。

Objective To explore the method of RNA103809(circRNA_103809)regulating the stem-like cell characteristics of gastric cancer and mitochondrial function.Methods Three groups of interfering circRNA_103809 were transfected into gastric cancer(SGC-7901)cell line,and the shRNA with the optimal interference effect was selected for follow-up study.SGC-7901 cells were divided into control group,shRNA-NC group and sh-circ103809 group.The levels of cell adhesion molecule 44(CD44),embryonic stem cell key protein(SOX2)and octamer-binding transcription factor 4(OCT4)were detected by Western blot and RT-PCR.The tumor cell spheroidization was observed,and the changes of mitochondrial membrane potential were detected by flow cytometry.Western blot was used to detect the expression of Rabbit anti human monoclonal antibody(Bax)/blymphoma-2(Bcl-2),cleaved caspase 9(cleaved cas9)/caspase 9,cleaved caspase 3(cleaved cas3)/caspase 3 as markers of mitochondrial damage.Results The protein and mRNA levels of CD44,SOX2 and OCT4 in sh-circ103809 group were significantly lower than those in control group and shRNA-NC group(P<0.05).Compared with control group and shRNA-NC group,the spheroid diameter and the rate of spheroidization in sh-circ103809 group decreased(P<0.05).The mitochondrial membrane potential of sh-circ103809 group was lower than that of control group and shRNA-NC group(P<0.05).The protein expression levels of Bax/Bcl-2,Caspase9/β-actin and Caspase3/β-actin in sh-circ103809 group were significantly higher than those in the control group and shRNA-NC group(P<0.05).Conclusion Interference the expression of circrna_103809 can inhibit the expression of stem cell marker protein and the tumorigenic ability of gastric cancer cells in vitro,reduce the mitochondrial membrane potential,promote cell apoptosis and inhibit the development of tumor.