环状RNA-UBXN7促进肝癌细胞增殖和迁移并抑制其凋亡

目的探讨环状RNA-UBXN7(circ_UBXN7)对肝癌细胞增殖、迁移以及凋亡的影响,并初步探讨其中的分子机制。方法采用qRT-PCR检测circ_UBXN7在肝癌组织和细胞中的表达水平,并分析circ_UBXN7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_UBXN7全长序列的慢病毒(Lenti-circ_UBXN7)和携带circ_UBXN7 shRNA的慢病毒(Lenti-circ_UBXN7-shRNA)分别转染肝癌细胞株(HepG2和HUH-7)。CCK-8实验检测circ_UBXN7表达上调或下调后对HepG2和HUH-7细胞的增殖能力的影响。Annexin V/PI实验检测circ_UBXN7表达上调或下调后HepG2和HUH-7细胞的凋亡变化。JC-1法检测circ_UBXN7表达上调或下调后HepG2和HUH-7细胞线粒体势能的变化。Transwell检测circ_UBXN7表达上调或下调后HepG2和HUH-7细胞迁移能力变化。蛋白质印迹法检测细胞上皮间质化标志蛋白TWIST、E-钙黏蛋白(E-cadherin)、N-cadherin、Vimentin的表达变化。circ_UBXN7表达水平与临床病理因素采用χ2检验,两组比较采用t检验,三组及以上比较采用单因素方差分析且组间比较采用LSD法。结果circ_UBXN7在肝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(P<0.05)。Lenti-circ_UBXN7可以上调HepG2和HUH-7细胞内circ_UBXN7表达并促进细胞增殖;Lenti-circ_UBXN7-shRNA可以下调circ_UBXN7表达并诱导细胞凋亡。Lenti-circ_UBXN7-shRNA可以导致细胞线粒体膜势能降低。Lenti-circ_UBXN7可以促进细胞迁移,而Lenti-circ_UBXN7-shRNA可以抑制细胞迁移。Lenti-circ_UBXN7可以诱导Twist、N-cadherin、Vimentin蛋白表达增高,并降低E-cadherin蛋白表达;而Lenti-circ_UBXN7-shRNA对各蛋白表达水平的影响则相反。Starbase V2.0软件显示miR-203a与circ_UBXN7存在潜在结合位点,并且在HepG2和HUH-7细胞中miR-203a与circ_UBXN7表达水平呈负相关。结论circ_UBXN7在肝癌发生发展中发挥重要促癌作用,有望成为肝癌治疗的潜在靶点。

Circular RNA-UBXN7 promotes proliferation,migration and suppresses apoptosis in hepatocellular cancer

Objective To investigate the effect and molecular mechanism of circular RNA-UBXN7(circ_UBXN7)on the proliferation,migration and apoptosis of hepatocellular carcinoma cells.Methods Circ_UBXN7 expression in the tissues and cells of hepatocellular cancer was detected by quantitative real-time polymerase chain reaction(qRT-PCR),and the relationship between circ_UBXN7 expression and clinicopathological features,including age,gender,tumor volume,pathological classification,staging,and lymph node metastasis was analyzed.The full-length sequence of circ_UBXN7 with lentivirus carrying lenti circ_UBXN7 and lenti circ_UBXN7 shRNA was constructed to transfect hepatocellular cell lines(HepG2 and Huh-7),respectively.CCK-8 experiments were performed to detect the ability of up-or down-regulation of circ_UBXN7 on the proliferation of HEPG2 and HUH-7 cells.Annexin V/PI experiment was used to detect the changes in apoptosis of HEPG2 and HUH-7 cells after up-regulation or down-regulation of circ_UBXN7 expression.JC-1 assay was used to detect the changes in mitochondrial potential energy of HEPG2 and HUH-7 cells after up-regulation or down-regulation of circ_UBXN7 expression.Transwell was used to detect the migration ability of HEPG2 and HUH-7 cells after up-regulation or down-regulation of circ_UBXN7 expression.Western blotting was used to detect the expressional change of TWIST,E-cadherin,N-cadherin and vimentin.Statistical analysis:The expression levels of circ_UBXN7 and clinicopathological features were measured by chi-square test.Two groups were compared by t-test and three groups and above were compared by single factor analysis of variance.LSD method was used for comparison between groups.Results The expression of circ_UBXN7 in liver cancer tissues was significantly higher than adjacent tissues,and its expression level was significantly positively correlated with tumor volume,stage,and lymph node metastasis(P<0.05).Lenti-circ_UBXN7 had up-regulated the expression of circ_UBXN7 in HEPG2 and HUH-7 cells and promoted cell proliferation.Lenti-circ_UBXN7-shRNA had down-regulated the expression of circ_UBXN7 and induced apoptosis.Lenti-circ_UBXN7-shRNA had reduced the mitochondrial membrane potential of cells.Lenti-circ_UBXN7 had promoted cell migration,while lenti-circ_UBXN7-shRNA had inhibited cell migration.Lenti-circ_UBXN7 had induced increased expression of Twist,N-cadherin,and Vimentin proteins,and reduced the expression of E-cadherin protein.Lenti-circ_UBXN7-shRNA had opposite effects on the expression levels of each protein.Starbase V2.0 software showed that miR-203a and circ_UBXN7 had potential binding sites,and miR-203a and circ_UBXN7 expression levels were negatively correlated in HEP??G2 and HUH-7 cells.Conclusion circ_UBXN7 plays an important role in promoting the occurrence and development of liver cancer,and is expected to become a potential target for the treatment of liver cancer.