| 韩阳,于静萍,孙志强,罗居东.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[J].中华放射医学与防护杂志,2021,41(11):801-807 |
| 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 |
| 投稿时间：2021-05-18 |
| DOI：10.3760/cma.j.issn.0254-5098.2021.11.001 |
| 中文关键词: 食管放射损伤 尿甘基转移酶 miR132/212簇 尿苷化 |
| 英文关键词:Radiation esophagitis TUT4 miR132/212 Uridylation |
| 基金项目:国家自然科学基金（82073339；81773224）；江苏省自然科学基金（BK20191157） |
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| 中文摘要: |
| 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（SERD0）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，SERD0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。 |
| 英文摘要: |
| 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 SERD0 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 SERD0 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-regulation of TUT4 reduced HEEC radiosensitivity and radiation damage, where the uridylation of miR132/212 was involved in. |
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