环状RNA-VANGL1对胃癌细胞增殖和凋亡的作用及机制研究

Effects of circular RNA VANGL1 on proliferation and apoptosis of gastric cancer cells

目的探讨环状RNA-VANGL1(circ_VANGL1)对胃癌细胞增殖和凋亡的影响,并初步探讨其中的分子机制。方法采用qRT-PCR检测circ_VANGL1在胃癌组织和细胞中的表达水平,并分析circ_VANGL1与患者临床病理因素如年龄、性别、肿瘤体积、病理分型、分期、淋巴结转移之间的相关性。构建携带circ_VANGL1全长序列的慢病毒(Lenti-circ_VANGL1)和携带circ_VANGL1 shRNA的慢病毒(Lenti-circ_VANGL1-shRNA)分别转染胃癌细胞株(NCI-N87和HGC-27)。CCK-8实验检测circ_VANGL1表达上调或下调后NCI-N87和HGC-27细胞的增殖变化。AnnexinⅤ/PI实验检测circ_VANGL1表达上调或下调后NCI-N87和HGC-27细胞凋亡的变化。JC-1法检测circ_VANGL1表达上调或下调后NCI-N87和HGC-27细胞线粒体势能变化。Western印迹法检测Fas、FADD、bax、bcl-2、细胞色素C以及Caspase-3活性片段的蛋白表达变化。结果circ_VANGL1在胃癌组织中表达显著高于癌旁组织,且其表达水平与肿瘤体积、分期和淋巴结转移显著正相关。Lenti-circ_VANGL1可以上调NCI-N87和HGC-27细胞内circ_VANGL1表达并促进细胞增殖;Lenti-circ_VANGL1-shRNA可以下调circ_VANGL1表达并诱导细胞凋亡。Lenti-circ_VANGL1-shRNA可以导致细胞线粒体膜势能降低。Lenti-circ_VANGL1-shRNA可以促进Fas细胞表面死亡受体(Fas cell surface death receptor,Fas)、Fas相关蛋白死亡结构域(Fas associated via death domain,FADD)、bax(BCL2 associated X)、胞质细胞色素C(cytoplasmic cytochrome C)以及Caspase-3活性片段表达增高,降低bcl-2和线粒体细胞色素C蛋白表达。miRDB软件显示circ_VANGL1存在12个潜在的靶miRNAs,其中miR-605-3p、miR-377-5p、miR-4468以及miR-5008-5p等4个miRNAs的表达在circ_VANGL1表达下调后显著增高。结论circ_VANGL1在胃癌中表达异常增高,参与调控胃癌细胞的增值和凋亡,在胃癌发生发展过程中发挥重要作用。

Objective To investigate the effects of circular RNA VANGL1 (circ-VANGL1) on the proliferation and apoptosis of gastric cancer cells and its mechanisms. Methods Real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circ-VANGL1 in the gastric cancer tissues and gastric cancer cell lines. The correlation of circ-VANGL1 expression with clinicopathological features of gastric cancer was analyzed. The lentivirus containing circ-VANGL1 full length transcript (Lenti-circ-VANGL1) and containing circ-VANGL1shRNA (Lenti-circ-VANGL1-shRNA) were constructed to upregulate or downregulate the expression of circ-VANGL1 in gastric cancer NCI-N87 and HGC-27 cells. CCK-8 assay was used to examine the viability of NCI-N87 and HGC-27 cells after transfection. Annexin Ⅴ/PI double staining assay was used to examine the apoptosis of NCI-N87 and HGC-27 cells after transfection. JC-1 assay was used to examine the mitochondrial membrane potential of NCI-N87 and HGC-27 cells after transfection. Western blotting was used to examine the expression of Fas, FADD, bax, bcl-2, cytochrome C and cleaved caspase-3 after transfection. Results The expression of circ-VANGL1 was upregulated in gastric cancer tissues, and it was positively correlated with tumor size, TNM stage and lymph node metastasis. Lenti-circ-VANGL1 promoted the proliferation of NCI-N87 and HGC-27 cells, while Lenti-circ-VANGL1-shRNA promoted apoptosis of NCI-N87 and HGC-27 cells. Lenti-circ-VANGL1-shRNA decreased the mitochondrial membrane potential of NCI-N87 and HGC-27 cells. Lenti-circ-VANGL1-shRNA increased the expression of Fas, FADD, bax, cytosolic cytochrome C and cleaved caspase-3, and decreased the expression of bcl-2 and mitochondrial cytochrome C. The prediction with miRDB software showed that circ-VANGL1 had 12 target miRNAs, and miR-605-3p, miR-377-5p, miR-4468, miR-5008-5p expression was significantly increased after circ-VANGL1 silencing. Conclusion The abnormal expression of circ-VANGL1 in gastric cancer may be involved in regulating the proliferation and apoptosis of gastric cancer cells.