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. 2018 Dec 14;13(12):e0207139.
doi: 10.1371/journal.pone.0207139. eCollection 2018.

Effects of a single transient transfection of Ten-eleven translocation 1 catalytic domain on hepatocellular carcinoma

Yuying Liu  1 , Hui Zhu  1 , Zhenxue Zhang  1 , Changchun Tu  1 , Dongyuan Yao  1 , Bin Wen  2 , Ru Jiang  3 , Xing Li  4 , Pengfei Yi  5 , Jiejie Zhan  5 , Jiaping Hu  2 , Jianwu Ding  6 , Liping Jiang  1 , Fanglin Zhang  1
Affiliations

Effects of a single transient transfection of Ten-eleven translocation 1 catalytic domain on hepatocellular carcinoma

Yuying Liu et al. PLoS One. .

Abstract

Tumor suppressor genes (TSGs), including Ten-eleven translocation 1 (TET1), are hypermethylated in hepatocellular carcinoma (HCC). TET1 catalytic domain (TET1-CD) induces genome-wide DNA demethylation to activate TSGs, but so far, anticancer effects of TET1-CD are unclear. Here we showed that after HCC cells were transiently transfected with TET1-CD, the methylation levels of TSGs, namely APC, p16, RASSF1A, SOCS1 and TET1, were distinctly reduced, and their mRNA levels were significantly increased and HCC cells proliferation, migration and invasion were suppressed, but the methylation and mRNA levels of oncogenes, namely C-myc, Bmi1, EMS1, Kpna2 and c-fos, were not significantly change. Strikingly, HCC subcutaneous xenografts in nude mice remained to be significantly repressed even 54 days after transient transfection of TET1-CD. So, transient transfection of TET1-CD may be a great advance in HCC treatment due to its activation of multiple TSGs and persistent anticancer effects.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. The expression of TET1 is downregulated in the SMMC 7721 cells.
(A). Structure of TET1 protein. TET1 protein contains a CXXC domain and a catalytic domain (CD) including a Cys-rich part and a DSBH part. TET1-mCD (TET1-CD mutant) harbours two mutated amino acid in the DSBH part of the CD. (B). Expression of TET1 mRNA in the SMMC 7721 cells and LO2 cells was analyzed by Quantitative RT-PCR, and the results were represented as mean ± SD of three independent experiments.**p<0.01. (t-test). (C). Western blot was used to analyze the expression of TET1 protein in the SMMC 7721 cells and LO2 cells. β-actin was used as an internal control.
Fig 2
Fig 2. TET1-CD up-regulates TSGs expression.
(A). The SMMC 7721 cells were transiently transfected with either TET1-CD plasmids or TET1-mCD plasmids. Expression of TET1-CD and TET1-mCD proteins was analysed by Western blot, and β-actin was used as an internal control. (B)&(C). The SMMC 7721 cells were cultured in the 6 mm plate for 24h and then transiently transfected with either pflag-CMV4 vector (control), TET1-CD or TET1-mCD. Expression of TSGs and oncogenes was analyzed by Quantitative RT-PCR 48h after transient transfection, and the results were represented as mean ± SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. (one-way ANOVA).
Fig 3
Fig 3. TET1-CD inhibits proliferation, migration and invasion of the SMMC 7721 cells.
(A). After transfection of empty vector, TET1-CD and TET1-mCD plasmids into the SMMC 7721 cells respectively for 24h, 48h and 72h, and incubation of these cells with CCK-8 solution at 37°C for 1 h, the absorbance of these cells at 450 nm was measured to assess their proliferation. (B). The SMMC 7721 cells were inoculated into the 24-well plate, and fluorescence staining was performed 48h after inoculation. (C). Microscopy was used to capture the cell scratched area at 0h, 24h and 48h. (D). The cells, which migrated from the upper chamber to the lower chamber, were counted to assess their migration and invasion ability. The results were represented as mean ± SD of three independent experiments. One-way ANOVA, *p<0.05 and **p<0.01.
Fig 4
Fig 4. TET1-CD suppresses HCC xenografts growth in nude mice.
(A). The SMMC 7721 cells, which were transiently transfected with empty vector, TET1-CD plasmids or TET1-mCD plasmids, were subcutaneously injected into the nude mice to establish a HCC subcutaneous xenograft model. The nude mice were sacrificed 54 days after the injection. (B). Photographs of excised xenograft tumors. (C). Tumor weight in the control, TET1-CD and TET1-mCD groups are shown. (D). From day 18 and 54 after the injection, the relative tumor volume was measured once every four days. The results were represented as mean ± SD (n = 5). (E). Ki-67 positive tumor cells were detected by immunohistochemistry (IHC). The results were represented as mean ± SD (n = 10). The scale bar is 50μm. One-way ANOVA, **p<0.01.
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Funded by National Natural Science Foundation of China: 81260498, zhan fanglin; National Natural Science Foundation of China: 81060273, zhan fanglin.