目的：

主动DNA去甲基化酶TET家族介导的主动DNA去甲基化途径是否参与以及如何调控肿瘤放疗敏感性知之甚少。本研究致力于探究主动DNA去甲基化酶在结直肠癌辐射增敏中的作用及作用机制。以期阐明主动DNA去甲基化介导辐射增敏的新机制。

方法：

检测电离辐射和CPT处理结直肠癌细胞后TET酶家族成员（TET1、TET2、TET3）蛋白水平和mRNA表达。

检测5种不同结直肠癌相关细胞系TET3表达与细胞辐射敏感性的关系。

分析TCGA数据库中TET3在肿瘤组织中的表达及TET3表达与结直肠癌病理分级、临床预后的关系。

分别构建TET3过表达HCT-8细胞系和TET3过表达HT-29细胞系，检测外源过表达TET3的结直肠癌细胞在辐射诱导后的细胞凋亡水平、细胞周期、DNA损伤程度、克隆形成率及ROS水平。

siRNA技术分别构建TET3敲降的HCT 116细胞系和TET3敲降的LS 180细胞系，检测外源敲降TET3的结直肠癌细胞在辐射后的细胞凋亡水平、细胞周期、DNA损伤程度和克隆形成率。

采用慢病毒构建稳定过表达TET3的HCT-8细胞系并构建了皮下移植瘤模型。局部15 Gy照射后，监测肿瘤生长。

免疫荧光和细胞质细胞胞核蛋白检测TET3与SUMO1，SUMO2/3的共定位，CoIP实验检测TET3是否可被SUMO1，SUMO2/3修饰。

构建3个截短突变质粒3×FLAG-TET3-1#、3×FLAG-TET3-2#、3×FLAG-TET3-3#，和潜在SUMO化修饰位点突变质粒3×FLAG-TET3m，分别进行突变后CoIP实验。

免疫荧光和细胞质细胞胞核蛋白检测TET3野生型和TET3m突变体的亚细胞定位和蛋白稳定性。

检测TET3的SUMO1修饰增加或缺失的结直肠癌细胞在辐射后的DNA损伤程度，细胞凋亡水平，细胞周期分布。

结果：

TET3蛋白在结直肠癌细胞电离辐射处理后响应性增加，而mRNA没有变化；TET1、TET2蛋白和mRNA无显著变化。

结直肠癌细胞系中TET3蛋白和mRNA表达与其恶性表型和辐射敏感性呈正相关。

TET3在23/27种肿瘤组织中显著高表达，包括结直肠癌。TET3与结直肠癌的病理恶性分级呈正相关。结肠癌患者中高TET3表达者的无病间隔（DFI）明显缩短

在结直肠癌细胞系中，外源过表达TET3增加了辐射诱导的细胞凋亡，G2/M期阻滞，DNA损伤及克隆抑制。

在结直肠癌细胞系中，siRNA敲降TET3减少了辐射诱导的细胞凋亡，G2/M期阻滞，DNA损伤及克隆抑制。

过表达TET3的结直肠肿瘤辐射后肿瘤体积、重量均受到显著抑制。

TET3可被SUMO1和SUMO2/3修饰。并且，辐射只增加TET3的SUMO1修饰水平。

TET3-2#，TET3-3#可被SUMO1修饰。只有TET3-3#检测到SUMO2/3的修饰。TET3蛋白上6个潜在的SUMO化修饰位点突变体TET3m导致TET3的SUMO1修饰不再发生，而不影响TET3的SUMO2/3修饰。

外源TET3野生型和TET3m均表达和定位于结直肠肿瘤细胞核中。TET3m相比TET3野生型蛋白半衰期明显缩短。

过表达SUMO1进一步增加TET3野生型促进的辐射诱导后γH2AX和53BP1灶累积，细胞凋亡，G2/M期阻滞；而TET3m失去了这一效应。

结论：

TET3是结直肠癌细胞中响应辐射的关键主动DNA去甲基化酶。

TET3在结直肠癌细胞中具有潜在的辐射增敏作用。

TET3与结直肠癌病理分级及不良预后呈正相关，TET3可作为潜在的结直肠癌预后预测因子。

体外过表达TET3增加了结直肠癌细胞的放射敏感性。

体外敲降TET3减弱了结直肠癌细胞的放射敏感性。

过表达TET3增加体内结直肠癌的放射敏感性。

TET3是SUMO1和SUMO2/3的靶蛋白；辐射只增加TET3的SUMO1修饰。

TET3中K479，K758，K1012，K1188，K1397，K1623位点是TET3发生SUMO1修饰的主要位点。TET3被SUMO2/3修饰位于833-1795 AA之间除K1012，K1188，K1397，K1623以外的其他位点 。

TET3的SUMO化修饰增加TET3蛋白的稳定性，不改变其核定位。

TET3的SUMO1修饰是其介导结直肠癌细胞放射增敏的重要机制。

Objective:

Little is known regarding whether and how the TET enzyme family's active DNA demethylation pathway is involved in tumor radiosensitivity regulation. Here, we aim to explore more about the role and mechanism of active DNA demethylase in colo cancer radiation sensitization, and better understand the function of DNA demethylation in tumor radiosensitization.

Methods:

The protein level and mRNA expression of TET enzyme family members (TET1, TET2, TET3) in colorectal cancer cells following ionizing radiation and CPT treatment were detected.

The relationship between TET3 expression and cellular radiosensitivity in five distinct colorectal cancer-related cells was detected.

The relationship between TET3 expression and the pathological grade and clinical prognosis of colorectal cancer were investigated.

TET3-overexpressing HCT-8 cell line and TET3-overexpressing HT-29 cell line were constructed. And, the effect of exogenous overexpression of TET3 on radiation-induced apoptosis level, cell cycle arrest, DNA damage, and clonogenicity inhibition were detected in colorectal cancer cells.

TET3 knockdown HCT 116 and TET3 knockdown LS 180 cell lines were constructed by siRNA technology, and the effect of exogenous knockdown of TET3 on radiation-induced apoptosis, cell cycle arrest, DNA damage, and clone formation in colorectal cancer cells were detected.

The HCT-8 stably transfected cell line was constructed by overexpressing TET3 lentivirus, and a subcutaneous transplanted tumor model was constructed to observe the radiosensitizing effect of TET3 protein in vivo.

The co-localization of TET3 and SUMO1, SUMO2/3 was detected by immunofluorescence and cytoplasmic, nuclear extraction. And the interaction between TET3 and SUMO1, SUMO2/3 was detected by CoIP assay.

Three truncation mutants, TET3-1#, TET3-2#, TET3-3#, and a potential SUMOylation modification site mutant TET3m were constructed. Furthermore, respectively, CoIP experiments were performed to find the combination site of TET3 protein and SUMO.

Immunofluorescence, cytoplasmic nuclear extraction, and CHX were used to detect the localization and stability of TET3 wild-type and TET3m mutants.

The effects of increased or absent SUMO1 modification of TET3 on radiation-induced DNA damage, apoptosis, and cell cycle arrest in colorectal cancer cells were detected.

Results:

In response to radiation therapy for colorectal cancer cells, the TET3 protein increased, but the mRNA barely changed; the protein and mRNA of TET1 and TET2 barely changed.

The malignant phenotype and radiosensitivity of colorectal cancer cell lines were favorably linked with TET3 protein and mRNA expression.

TET3 is upregulated in 23 of the 27 tumor types investigated, including colorectal cancer. TET3 was shown to positively correlate with the colorectal cancer pathological malignancy grade. In colorectal cancer patients with highly TET3 expression, the disease-free interval (DFI) was significantly reduced.

Overexpression of TET3 in colorectal cancer cell lines increased radiation-induced apoptosis, G2/M phase arrest, DNA damage, and clonal suppression in vitro.

The intervention of TET3 in colorectal cancer cell lines in vitro reduced radiation-induced apoptosis, G2/M phase arrest, DNA damage, and clonal suppression.

Overexpression of TET3 significantly increased radiation-induced inhibition of colorectal tumor volume and tumor weight in vivo.

Immunofluorescence experiments, cytoplasmic and nuclear component analysis confirmed that TET3 co-localized with SUMO1 and SUMO2/3 in the nucleus. The CoIP results confirmed that TET3 interacts with SUMO1 and SUMO2/3, respectively. Moreover, radiation only increased the interaction between TET3 and SUMO1 and had no significant effect on the interaction between TET3 and SUMO2/3.

SUMO1 interacted with TET3-2# and TET3-3#, while SUMO2/3 could only detect interaction with TET3-3#. TET3m disrupted the interaction of TET3 with SUMO1 but did not affect the interaction of TET3 with SUMO2/3.

Immunofluorescence stain, cytoplasmic and nuclear fraction experiments showed that TET3 wild-type and TET3m were localized in the nucleus. And TET3m showed more unstable than TET3 wild-type protein.

Overexpression of SUMO1 in the TET3 wild-type cell line further increased the radiation-induced γH2AX and 53BP1 foci accumulation, cell apoptosis,  G2/M phase arrest. However, these effects were absent in the TET3m cell line.

Conclusions:

TET3 was the crucial DNA demethylase in colorectal cancer cells responding to radiation.

TET3 was a potential radiosensitizing factor in colorectal cancer cells.

TET3 was positively correlated with the pathological malignancy grade and poor prognosis of colorectal cancer, and TET3 is a potential predictor of colorectal cancer prognosis.

Overexpression of TET3 in vitro increased the radiosensitivity of colorectal cancer cells.

Interference with TET3 in vitro attenuated the radiosensitivity of colorectal cancer cells.

Overexpression of TET3 increased the radiosensitivity of colorectal cancer in vivo.

TET3 was the target protein of SUMO1 and SUMO2/3; Radiation only increased the SUMO1 modification of TET3.

K479, K758, K1012, K1188, K1397, and K1623 were the main bind sites between TET3 and SUMO1. The binding region of TET3 and SUMO2/3 was located at 833-1795 AA except for K1012, K1188, K1397, and K1623.

SUMOylation of TET3 increased the stability of TET3 protein without changing its nuclear localization.

The SUMO1 modification of TET3 was an essential mechanism mediating the radiosensitization of colorectal cancer cells.