Fig 2: Effects of DNMT3B- and TET3-3'UTR overexpression on apoptosis and cell migration in HCT116 cells. (A and D) Caspase-Glo 3/7 and (B and E) wound healing assays were used to monitor the effects of 3'UTR overexpression on apoptosis and migratory capacity, respectively. (C and F) The migration rates corresponding to (B) and (E). Set-ups A-C and D-F were performed in full-serum (10%) and low-serum (4%) conditions, respectively. Data are representative of three independent trials and expressed as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. GLO, pmirGLO construct; empty, GLO empty, pmirGLO empty vector control; DNMT3B, DNA methyltransferase 3ß; TET3, TET methylcytosine dioxygenase 3.

Fig 3: miR-4465 negatively regulates the expression of endogenous DNMT3B, TET3 and PTEN. (A) RT-qPCR demonstrated the effects of increasing amounts of transfected miR-4465 on the endogenous transcript levels of DNMT3B, TET3 and PTEN. (B) Western blot analyses demonstrated the effects of miR-4465 overexpression on endogenous protein levels of DNMT4B, TET3 and PTEN. (C) Relative quantification of the protein expression levels of DNMT3B, TET3 and PTEN in western blots normalized to GAPDH (n=5). (D) Dual-luciferase assays demonstrated derepression of endogenous DNMT3B, TET3 and PTEN following co-transfection with increasing amounts of gene-specific TPs. (E-G) Dual-luciferase assays demonstrated direct targeting of (E) DNMT3B-, (F) TET3- and (G) PTEN-3'UTR by miR-4465 and rescue by gene-specific target protectors. (H) RT-qPCR demonstrated the effects of transfected miR-4465 and co-transfected gene-specific target TPs on the endogenous transcript levels of DNMT3B, TET3 and PTEN. All experiments were performed in cells maintained in 10% serum. Data presented are representative of three independent trials and expressed as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. miR, microRNA; UTR, untranslated region; GLO Empty, empty pmirGLO vector; Zs Empty, empty pmR-ZsGreen1 vector; TP, target protector; RT-qPCR, reverse transcription-quantitative PCR; DNMT3B, DNA methyltransferase 3ß; TET3, TET methylcytosine dioxygenase 3.

Fig 4: Oxidative DNA damage alters DNA methylation patterns in the promoter and transcribed regions of the BRCA1 gene through the crosstalk between pol ß and DNMT3b. Oxidized DNA base lesions such as 8-oxoGs can occur at the CpGs located in the promoter and transcribed regions of the BRCA1 gene. The lesions can be removed by a DNA glycosylase, leaving an abasic (AP) site. The AP site is subsequently cleaved by APE1, generating the 1-nt gap intermediates. Subsequently, pol ß is recruited and performs nucleotide insertion. In the scenario where pol ß inserted a correct nucleotide to bypass an 8-oxoG, DNMT3b is recruited to the CpG dinucleotides through its interaction with pol ß. This allows DNMT3b to create a new 5mC on the adjacent CpG site but not at the damaged site because of the inhibition of the methyltransferase activity by the base lesion. This then results in the loss of 5mC and a new methylation site (subpathway 1). In the scenario where pol ß inserted an incorrect nucleotide to bypass an 8-oxoG, DNMT3b is also recruited to the damage site through its interaction with pol ß leading to a mutation, loss of 5mC, and addition of a new 5mC at the adjacent CpG site (subpathway 2).

Fig 5: Silencing of DNMT3B Enhances Radiosensitivity via Promoting Superior Apoptosis in NPC Cells(A) Apoptotic rates determined by flow cytometry. Compared to control groups, the apoptotic rate significantly increased in the DNMT3B-knockdown group after irradiation. (B and D) Representative images from colony formation assays in transfected NPC cells after irradiation (B, CNE2 cells; D, HNE1 cells). (C and E) Survival fraction curves of clone formation assays in transfected NPC cells with radiation (C, CNE2 cells; E, HNE1 cells). *p < 0.05, **p < 0.01, ***p < 0.001.