No abstract

It is observed that upregulation of DNMT3B enzyme in some cancers, including colon cancer, could lead to silencing of tumor suppressor genes. MiR-339 and miR-766 have been predicted to target 3 0 UTR of DNMT3B gene. Luciferase reporter assay validated that individual and co-transfection of miR-766 and miR-339 into the HEK293T cell reduced luciferase activity to 26% § 0.41%, 43% § 0.42 and 64% § 0.52%, respectively, compared to the control (P < 0.05). Furthermore, transduction of miR-339 and miR-766 expressing viruses into colon cancer cell lines (SW480 and HCT116) decreased DNMT3B expression (1.5, 3-fold) and (3, 4-fold), respectively. In addition, DNA methylation of some tumor suppressor genes decreased. Expression of these genes such as SFRP1 (2 and 1.6-fold), SFRP2 (0.07 and 4-fold), WIF1 (0.05 and 4-fold), and DKK2 (2 and 4-fold) increased in SW-339 and SW-766 cell lines; besides, expression increments for these genes in HCT-339 and HCT-766 cell lines were (2.8, 4-fold), (0.005, 1.5-fold), (1.7 and 3-fold) and (0.04, 1.7-fold), respectively. Also, while in SW-766, cell proliferation reduced to 2.8% and 21.7% after 24 and 48 hours, respectively, SW-339 showed no reduced proliferation. Meanwhile, HCT-766 and HCT-339 showed (3.5%, 12.8%) and (18.8%, 33.9%) reduced proliferation after 24 and 48 hours, respectively. Finally, targeting DNMT3B by these miRs, decreased methylation of tumor suppressor genes such as SFRP1, SFRP2, WIF1 and DKK2 in the mentioned cell lines, and returned the expression of these tumor suppressor genes which can contribute to lethal effect on colon cancer cells and reducing tumorigenicity of these cells.

Colon cancer is among the most prevalent cancers worldwide. Although the main modality of treatment is surgery, resistance to chemoradiotherapy raises concerns. Hence, we aimed to determine the effect of RNA-mediated silencing of tcf4, the downstream effector of the wnt signaling pathway, on the response of the SW480 cell line to oxaliplatin, a common chemotherapeutic drug. For this, two different silencing sequences against TCF4 mRNA were selected and cloned into pSilencer neo2.1. The SW480 cell line was stably transfected with the silencing constructs (namely p1396, p1874, and p silencer containing a scrambled sequence) and labeled SW1396, SW1874, and SW-Sc, respectively. Subsequently, the effect of oxaliplatin (from 0 to 11.25 μmol/l) on these cells was studied using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide proliferation assay. Suppression of tcf4 expression in stable transfected cells with p1396 and p1874 was confirmed by quantitative reverse transcription-PCR and western blot analysis. Although oxaliplatin was not toxic to SW480 and SW-sc in the range tested, in SW1396 and SW1874 cells, a toxic effect was evident at 3.75 and 4.375 μmol/l. Also, SW1396 and SW1874 cells appeared to have a round shape in comparison with SW480 and SW-Sc cells. Only for SW1396, the number of apoptotic cells was significantly different before and after the addition of oxaliplatin (LC50 of oxaliplatin). The proliferating cells in SW480, SW1874, and SW-Sc increased after treatment with oxaliplatin; however, this was not observed in SW1396. Although silencing the tcf4 gene would confer sensitivity to oxaliplatin in SW1874 and especially SW1396, in SW480 and SW-Sc, the lethal effect of oxaliplatin was compensated by its effect in increasing the proliferation of cells. This sensitization effect may be because of different mechanisms including TCF4 motifs in the ABCB1 promoter or defects in nucleotide excision repair or double-strand break repair systems after tcf4 silencing.