环状RNA_0023990/微小RNA-873-5p/ANXA2对甲状腺癌放射敏感性的影响及机制研究

The mechanism of circ_0023990/miR-873-5p/ANXA2 axis regulating radiosensitivity and development of thyroid carcinoma

目的探究circ_0023990对甲状腺癌细胞放射敏感性的影响及可能机制。方法采用逆转录聚合酶链反应(qRT-PCR)法检测2016年8月至2018年11月于河南省人民医院就诊的55例甲状腺癌患者[其中男12例,女43例,年龄(53.3±7.3)岁]癌组织及甲状腺癌细胞系(TPC-1、KTC-1、FTC-133和CAL-62)中circ_0023990的表达,分析癌组织中circ_0023990表达与患者临床病理特征的关系。甲状腺癌细胞TPC-1和KTC-1均分为circ_0023990小干拢RNA(sh-circ_0023990)组、无意义阴性序列(sh-NC)组、sh-circ_0023990+miR-873-5p抑制剂(anti-miR-873-5p)组、sh-circ_0023990+阴性对照序列(anti-miR-NC)组、miR-873-5p组、对照序列(miR-NC)组、miR-873-5p+pcDNA-ANXA2组和miR-873-5p+pcDNA组,克隆形成实验检测细胞放射敏感性;且各组细胞用4 Gy放射线照射后,蛋白质印迹法检测细胞中γH2AX蛋白表达。双荧光素酶报告基因实验验证circ_0023990与miR-873-5p及miR-873-5p与ANXA2的靶向关系。结果circ_0023990在甲状腺癌组织表达高于正常组织(2.15±0.09比0.97±0.05,P<0.05),且其表达与甲状腺癌患者肿瘤大小、淋巴结转移及TNM分期密切相关(P<0.05)。甲状腺癌TPC-1、KTC-1、FTC-133和CAL-62细胞中circ_0023990表达高于正常甲状腺细胞HTori-3(3.16±0.38、2.63±0.28、1.82±0.24、1.71±0.22比1.00±0.10,P均<0.05)。sh-circ_0023990组TPC-1和KTC-1细胞存活分数明显低于sh-NC组(P<0.05),增敏比分别为2.482、1.643;sh-circ_0023990+anti-miR-873-5p组TPC-1和KTC-1细胞存活分数高于sh-circ_0023990+anti-miR-NC组(P<0.05),增敏比分别为0.305、0.441。miR-873-5p组TPC-1、KTC-1细胞存活分数低于miR-NC组(P<0.05),增敏比分别为2.044、1.653;miR-873-5p+pcDNA-ANXA2组TPC-1、KTC-1细胞存活分数高于miR-873-5p+pcDNA组(P<0.05),增敏比分别为0.496、0.686。4 Gy+sh-circ_0023990组TPC-1和KTC-1细胞中γH2AX蛋白表达高于4 Gy+sh-NC组(2.68±0.27比1.87±0.25,2.46±0.19比1.77±0.14;P均<0.05),4Gy+sh-circ_0023990+anti-miR-873-5p组TPC-1和KTC-1细胞中γH2AX蛋白表达低于4 Gy+sh-circ_0023990+anti-miR-NC组(1.13±0.09比1.69±0.09,1.11±0.08比1.60±0.08,P均<0.05);4 Gy+miR-873-5p组TPC-1和KTC-1细胞中γH2AX蛋白表达高于4 Gy+miR-NC组(2.35±0.16比1.84±0.14,2.26±0.12比1.77±0.13,P均<0.05),4Gy+miR-873-5p+pcDNA-ANXA2组TPC-1和KTC-1细胞中γH2AX蛋白表达低于4 Gy+miR-873-5p+pcDNA组(1.96±0.12比2.41±0.12,1.92±0.07比2.28±0.12,P均<0.05)。circ_0023990靶向负调控miR-873-5p,而ANXA2是miR-873-5p的靶基因。结论circ_0023990在甲状腺癌组织和细胞系中呈高表达,其可能通过靶向调控miR-873-5p/ANXA2轴促进体外甲状腺癌细胞放疗抗性。

Objective To explore the effect and possible mechanism of circ_0023990 on the radiosensitivity of thyroid cancer cells.Methods qRT-PCR was used to detect the expression of circ_0023990 in the cancer tissues of 55 patients with thyroid cancer and thyroid cancer cell lines(TPC-1,KTC-1,FTC-133 and CAL-62),and the relationship between the expression of circ_0023990 in cancer tissues and the clinical characteristics of the patients were analyzed.Thyroid cancer cells TPC-1 and KTC-1 were divided into sh-circ_0023990 group,sh-NC group,sh-circ_0023990+anti-miR-873-5p group,sh-circ_0023990+anti-miR-NC group,miR-873-5p group,miR-NC group,miR-873-5p+pcDNA-ANXA2 group and miR-873-5p+pcDNA group,and then clone formation experiment was used to detect cell radiosensitivity.After each group of cells was irradiated with 4Gy radiation,the expression of γH2AX protein in the cells was detected by Western Blot.The dual luciferase reporter gene experiment verified the targeting relationship between circ_0023990 and miR-873-5p or miR-873-5p and ANXA2.Results The expression of circ_0023990 in thyroid cancer tissues was higher than that in normal tissues(2.15±0.09 vs.0.97±0.05,P<0.05),and its expression was closely related to tumor size,lymph node metastasis and TNM staging of patients with thyroid cancer(P<0.05).The expression of circ_0023990 in thyroid cancer cell lines(TPC-1,KTC-1,FTC-133 and CAL-62)were higher than that of normal thyroid cells HTori-3(3.16±0.38,2.63±0.28,1.82±0.24,1.71±0.22 vs.1.00±0.10,all P<0.05).The survival scores of TPC-1 and KTC-1 cells in the sh-circ_0023990 group were significantly lower than those in the sh-NC group(P<0.05),and the sensitization ratios were 2.482,1.643;The survival scores of TPC-1 and KTC-1 cells in the sh-circ_0023990+anti-miR-873-5p group were higher than those in the sh-circ_0023990+anti-miR-NC group(P<0.05),and the sensitization ratios were 0.305,0.441,respectively.The survival scores of TPC-1 and KTC-1 cells in the miR-873-5p group were lower than those in the miR-NC group(P<0.05),and the sensitization ratios were 2.044,1.653 respectively.The survival scores of TPC-1 and KTC-1 cells in the miR-873-5p+pcDNA-ANXA2 group was higher than that in the miR-873-5p+pcDNA group(P<0.05),and the sensitization ratios were 0.496,0.686,respectively.The expression ofγH2AX protein in TPC-1 and KTC-1 cells of the 4 Gy+sh-circ_0023990 group were higher than that in the 4 Gy+sh-NC group(2.68±0.27 vs.1.87±0.25,2.46±0.19 vs.1.77±0.14;all P<0.05),but the expression ofγH2AX protein in TPC-1 and KTC-1 cells of the 4 Gy+sh-circ_0023990+anti-miR-873-5p group were lower than that in the 4 Gy+sh-circ_0023990+anti-miR-NC group(1.13±0.09 vs.1.69±0.09,1.11±0.08 vs.1.60±0.08;both P<0.05).The expression ofγH2AX protein in TPC-1 and KTC-1 cells in the 4 Gy+miR-873-5p group were higher than that in the 4 Gy+miR-NC group(2.35±0.16 vs.1.84±0.14,2.26±0.12 vs.1.77±0.13;both P<0.05),but the expression ofγH2AX protein in TPC-1 and KTC-1 cells of the 4 Gy+miR-873-5p+pcDNA-ANXA2 group were lower than that in the 4 Gy+miR-873-5p+pcDNA group(1.96±0.12 vs.2.41±0.12,1.92±0.07 vs.2.28±0.12;both P<0.05).circ_0023990 targeted the negative regulation of miR-873-5p,and ANXA2 was the target gene of miR-873-5p.Conclusion circ_0023990 was highly expressed in thyroid cancer tissues and cell lines,and it may promote the radiotherapy resistance of thyroid cancer cells in vivo through regulating miR-873-5p/ANXA2 axis.