尿苷基转移酶通过调节miR132/212簇的尿苷化影响食管上皮细胞放射敏感性  

TUT4 affects the radiosensitivity of esophageal epithelial cells by regulating the uridylation of miR132/212 clusters

作　　者：韩阳 于静萍 孙志强 罗居东 Han Yang;Yu Jingping;Sun Zhiqiang;Luo Judong(Department of Radiotherapy,Changzhou No.2 People′s Hospital,Affiliated Hospital of Nanjing Medical University 213003,Changzhou China)

机构地区：[1]南京医科大学附属常州第二人民医院放疗科,213003

出　　处：《中华放射医学与防护杂志》2021年第11期801-807,共7页Chinese Journal of Radiological Medicine and Protection

基　　金：国家自然科学基金(82073339,81773224);江苏省自然科学基金(BK20191157)。

摘　　要：目的验证末端尿苷基转移酶(TUT4)通过影响miR132/212簇的尿苷化作用影响食管上皮细胞(HEEC)的放射敏感性。方法本研究通过PCR的方法检测0、2、4、6和8 Gy X射线照射后0、6、18、24和48 h HEEC中TUT4的表达。将细胞分为阴性对照组、下调TUT4表达组(shTUT4组)、单纯6 Gy照射组(6 Gy组)、6 Gy照射+下调TUT4表达组(6 Gy+shTUT4组)分别检测TUT4对细胞放射敏感性、细胞增殖、细胞周期、线粒体损伤、氧自由基产生的影响;通过PCR检测下调TUT4表达对miR132/212尿苷化的影响;克隆形成和增殖实验检测过表达miR132/212及miR132/212+UUU(末端添加3个尿嘧啶的miR132/212)时HEEC的放射敏感性;增殖实验检测在下调TUT4表达且过表达miR132/212及尿苷化miR132/212时HEEC的增殖情况。结果PCR结果显示,2、4、6和8 Gy X射线照射后TUT4表达增加,差异有统计学意义(t=12.84、62.06、27.86、32.43,P<0.05)。下调TUT4表达可增加HEEC的放射敏感性,D0、Dq、SF2值分别为0.79、1.61、0.47,与对照组比较差异有统计学意义(t=13.2、5.85、7.31,P<0.05),放射增敏比(SER_(D0))为1.41;且经6 Gy照射后,低表达TUT4组细胞增殖减少(t=7.12、13.63,P<0.05)、S期细胞增加(t=11.98,P<0.05)、线粒体损伤增加(t=11.98,P<0.05)、氧自由基产生增加(t=15.65,P<0.05)。进一步研究结果表明,下调TUT4表达可使miR132/212表达增加,miR132/212+UUU表达减少(t=27.90、60.99,P<0.05);而过表达miR132/212和miR132/212+UUU也可影响HEEC的放射敏感性,SER_(D0)分别为1.20、0.71。双重转染实验证明,TUT4低表达且miR132/212过表达细胞的增殖能力较单纯TUT4低表达时更弱(t=4.76、7.65,P<0.05);同时TUT4低表达且过表达miR132/212+UUU时细胞增殖能力较单纯TUT4低表达更强,差异有统计学意义(t=7.22,P<0.05)。结论X射线可增加HEEC中TUT4的表达,TUT4可降低HEEC放射敏感性、减轻放射损伤,其机制与miR132/212的尿苷化相关。Objective To validate the effect of TUT4 on the radiosensitivity of esophageal epithelial cells(HEEC)by regulating the uridylation of miR132/212 clusters.Methods The expression of TUT4 in HEEC at 0,6,18,24 and 48 h after 0,2,4,6 and 8 Gy X-ray irradiation was detected by PCR.The HEEC cells were divided into four groups:NC group,shTUT4 group,6 Gy group,and 6 Gy+shTUT4 group.The effects of TUT4 on cell radiosensitivity,cell proliferation,cell cycle,mitochondrial damage,and oxygen free radical production were detected respectively.The effect of down-regulated TUT4 expression on miR132/212 uridylation was detected by RT-PCR,and the radiosensitivity of HEEC with overexpression of miR132/212 or miR132/212+UUU was detected by clone formation and proliferation assay,respectively.Proliferation assay was performed to detect the proliferation of HEEC when TUT4 expression was down-regulated and miR132/212 or miR132/212+UUU was overexpressed.Results TUT4 expression increased after different doses of X-ray irradiation(t=12.84,62.06,27.86,32.43,P<0.05).Downregulation of TUT4 expression increased the radiosensitivity of HEEC(t=13.2,5.85,7.31,P<0.05)with a SER_(D0)of 1.41 and D0=0.79,Dq=1.61,SF2=0.47.Compared with 6 Gy group,cell proliferation in 6 Gy+shTUT4 group was decreased(t=7.12,13.63,P<0.05),cells in S phase were increased(t=11.98,P<0.05),mitochondrial damage was increased(t=11.98,P<0.05),and ROS level was increased(t=15.65,P<0.05).Down-regulation of TUT4 expression increased miR132/212 expression and decreased miR132/212+UUU expression(t=27.90,60.99,P<0.05).Overexpression of miR132/212 increased the radiosensitivity of HEEC,and overexpression of miR132/212+UUU decreased the radiosensitivity of HEEC,with SER_(D0) of 1.20 and 0.71,respectively.Cell proliferation of shTUT4+miR132/212 group waslower than that of shTUT4 group(t=4.76,7.65,P<0.05),and cell proliferation of shTUT4+miR132/212+UUU group was higher than that of shTUT4(t=7.22,P<0.05).Conclusions X-ray irradiation increased the expression of TUT4 in HEEC,and the down-

关 键 词：食管放射损伤 尿甘基转移酶 miR132/212簇 尿苷化 

分 类 号：R730.55[医药卫生—肿瘤]