hsa_circ_0005777在肝母细胞瘤中的表达及对细胞增殖周期的影响研究

Expression of hsa_circ_0005777 in hepatoblastoma and its effect on cellular proliferation and cycle

目的探索环状RNA hsa_circ_0005777在肝母细胞瘤中的表达及对肝母细胞瘤细胞增殖周期的影响。方法实验细胞包括人肝母细胞瘤细胞株HepG2和HUH6,将实验细胞培养在含10%胎牛血清及1%双抗的DMEM培养基中,置于37℃、5%CO2的恒温培养箱中。实验组织标本包括肝母细胞瘤肿瘤及癌旁组织标本,来源于中山大学孙逸仙纪念医院小儿外科住院患儿手术切除的组织。所有肿瘤组织标本和癌旁组织标本在手术切除后,均立即浸泡在RNA later保存液中防止RNA降解,随后放在-80℃冰箱保存。在肝母细胞瘤细胞株中转染hsa_circ_0005777过表达质粒,为过表达组;未转染过表达质粒的细胞株作为阴性对照,为对照组。检测内容包括:①鉴定hsa_circ_0005777在肝母细胞瘤细胞株中的性质及分布情况;②检测hsa_circ_0005777在肝母细胞瘤肿瘤、癌旁组织组织标本和肝母细胞瘤细胞株中的表达情况;③检测过表达组对肝母细胞瘤细胞株细胞增殖能力的影响;④检测过表达组对肝母细胞瘤细胞株细胞周期的影响及过表达组对细胞周期蛋白Cyclin D1表达水平的影响。结果①hsa_circ_0005777在肝母细胞瘤细胞株中成环并富集在细胞质中;②hsa_circ_0005777在肝母细胞瘤组织标本中的表达显著高于癌旁组织,在肝母细胞瘤肿瘤组织标本及细胞株中明显高表达;③过表达组可明显提高肝母细胞瘤细胞株的增殖能力;④过表达组可明显促进细胞周期蛋白Cyclin D1的表达,可促进肝母细胞瘤细胞株细胞G1/S期转化。结论hsa_circ_0005777可促进肝母细胞瘤肿瘤细胞增殖,有望成为治疗肝母细胞瘤的新靶点。

Objective To explore the expression of circRNA hsa_circ_0005777 in hepatoblastoma(HB)and its effect on proliferative cycle of HB cells.Methods Human HB cell lines of HepG2 and HUH6 were cultured in DMEM medium containing 10%fetal bovine serum and 1%double antibody in a constant temperature incubator at 37℃and 5%CO2.Experimental tissue samples included HB tumor and adjacent non-cancerous tissue samples obtained from surgically resected tissues of hospitalized children.All samples were immediately soaked in RNA later for preventing RNA degradation after surgical resection and then stored in a-80℃freezer.HB cell line was transfected with hsa_circ_0005777 overexpression plasmid(overexpression group);cell line not transfected with overexpression plasmid served as negative control.The detection contents included identification of nature and distribution of hsa_circ_0005777 in HB cell lines;detection of the expression of hsa_circ_0005777 in HB tumors,tissue samples and HB cell lines;detection of the effect of overexpression group on cellular proliferative capability;detection of the effect of overexpression group on cell cycle of HB cell lines and the effect of overexpression group on the expression level of Cyclin D1.Results Looped hsa_circ_0005777 became enriched in cytoplasm.The expression of hsa_circ_0005777 was significantly higher in HB tissue than that in adjacent non-cancerous tissues.The overexpression group could significantly improve the cellular proliferative capability,significantly boost the expression of Cyclin D1 and promote the G1/S phase transformation.Conclusions hsa_circ_0005777 can promote the proliferation of HB cells and may be a new therapeutic target for HB.