沉默circBANP通过上调miR-338-3p的表达增强结直肠癌细胞和裸鼠皮下移植瘤的放射敏感性  

Silence of circBANP increases radiosensitivity of colorectal cancer cells and inhibits growth of subcutaneous xenografts by up-regulating miR-338-3p expression

作　　者：谢毅[1] 刘建波[2] 李俊蒙 张超[1] 卢创新[1] 文泽军[3] Xie Yi;Liu Jianbo;Li junmeng;Zhang Chao;Lu Chuangxin;Wen Zejun(Department of Gastrointestinal Surgery,Henan People′s Hospital,Zhengzhou 450000,China;Department of Oncology,Henan People′s Hospital,Zhengzhou 450000,China;Department of Radiotherapy,Henan People′s Hospital,Zhengzhou 450000,China)

机构地区：[1]河南省人民医院胃肠外科,郑州450000 [2]河南省人民医院肿瘤科,郑州450000 [3]河南省人民医院放疗科,郑州450000

出　　处：《中华肿瘤杂志》2021年第5期533-540,共8页Chinese Journal of Oncology

摘　　要：目的探讨circBANP对结直肠癌细胞和裸鼠皮下移植瘤放射敏感性的影响及潜在分子机制。方法选取2018年1月至2019年1月于河南省人民医院手术切除的20例结直肠癌患者的癌及癌旁正常黏膜组织,建立放射抵抗结直肠癌细胞LoVo/R,采用实时荧光定量聚合酶链反应(RT-qPCR)检测circBANP和miR-338-3p的表达,克隆形成实验检测细胞的放射敏感性,四甲基偶氮唑蓝(MTT)法检测细胞活力,Western blot检测细胞中自噬相关蛋白LC3和p62的表达,免疫荧光法检测细胞中微管相关蛋白轻链3(LC3)的荧光强度。通过Circular RNA Interactome网站预测circBANP的下游微小RNA(miRNA),采用双荧光素酶报告基因实验进一步验证。建立LoVo/R细胞的裸鼠移植瘤模型,观察circBANP对放射后移植瘤生长的影响。结果结直肠癌组织中circBANP和miR-338-3p的表达水平分别为3.21+0.29和0.47+0.04,较癌旁组织(分别为1.00+0.07和1.00+0.05)显著升高(均P<0.05)。成功建立了放射抵抗结直肠癌细胞LoVo/R,其circBANP表达水平为3.21±0.34,高于LoVo细胞(1.00±0.07,P<0.05),miR-338-3p表达水平为0.33±0.04,低于LoVo细胞(1.00±0.08,P<0.05)。4 Gy照射后,与沉默对照组比较,沉默circBANP组LoVo/R细胞活力降低[分别为(34±4)%和(62±6)%,P<0.05],细胞存活分数亦降低(分别为0.07±0.02和0.27±0.04,P<0.05),放射增敏比为1.843。放射诱导后,LoVo/R细胞中LC3Ⅱ/Ⅰ表达增加,p62表达降低,细胞发生自噬。自噬抑制剂氯喹可逆转放射对LoVo/R细胞中LC3Ⅱ/Ⅰ表达的诱导和p62表达的抑制作用,促进放射对LoVo/R细胞活力和存活的抑制,其放射增敏比为1.780。4 Gy照射后,与沉默对照组比较,沉默circBANP组LoVo/R细胞中LC3相对荧光强度降低(分别为0.11±0.01和1.00±0.12,P<0.05),LC3-Ⅱ/Ⅰ表达水平降低(分别为1.25±0.13和3.84±0.39,P<0.05),p62表达增加(分别为2.76±0.29和1.00±0.08,P<0.05)。经Circular RNA Interactome网站预测、双荧光素酶报告基因实验Objective To investigate the effect of circBANP on radiosensitivity of colorectal cancer cells and subcutaneous transplanted tumor in nude mice and its potential molecular mechanism.Methods The carcinoma and adjacent normal mucosal tissues of 20 patients with colorectal cancer who were surgically resected in Henan People′s Hospital from January 2018 to January 2019 were selected.The radio-resistant colorectal cancer cell LoVo/R was established.Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expressions of circBANP and miR-338-3p.The radiation sensitivity was determined by cell clone formation experiment.Cell vitality was detected by using methyl thiazolyl tetrazolium(MTT).The expressions of autophagy-related protein microtubule-associated protein light chain 3(LC3)and p62 were detected by western blot.The fluorescence intensity of LC3 in cells was detected by immunofluorescence assay.The downstream microRNAs(miRNAs)of circBANP were predicted by Circular RNA Interactome website and further verified by dual luciferase reporter gene assay.The transplanted tumor model of LoVo/R cells in nude mice was established,and the effect of circBANP on the growth of transplanted tumor after radiation was observed.Results The expression levels of circBANP and miR-338-3p in colorectal cancer tissues were 3.21+0.29 and 0.47+0.04,respectively,which were significantly higher than 1.00+0.07 and 1.00+0.05 in adjacent tissues(P<0.05).The circBANP expression level of LoVo/R cells was 3.21±0.34,higher than 1.00±0.07 of LoVo cells(P<0.05),and the expression level of miR-338-3p of LoVo/R cells was 0.33±0.04,lower than 1.00±0.08 of LoVo cells(P<0.05).After 4 Gy irradiation,compared with the control group,the viability of LoVo/R cells in the circBANP silencing group[(34±4)%vs(62±6)%,P<0.05],the cell survival fraction(0.07±0.02 vs 0.27±0.04,P<0.05)were decreased,and the radiation sensitization ratio was 1.843,the expression of LC3Ⅱ/Ⅰin LoVo/R cells increased while p62 expression decreased,the cell

关 键 词：结直肠肿瘤 circBANP miR-338-3p LOVO细胞 放射敏感性 

分 类 号：R735.34[医药卫生—肿瘤]