TCF4 silencing sensitizes the colon cancer cell line to oxaliplatin as a common chemotherapeutic drug.

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.

Among autophagy genes, ATG16L1 but not IRGM is associated with Crohn's disease in Iranians.

BACKGROUND: The prevalence of inflammatory bowel diseases is uprising in countries like Iran. Genetic predisposing elements play prominent role in the pathogenesis of Crohn's disease. In this study we studied the role of autophagy genes like IRGM (Immunity related GTPase M) and ATG16L1 (Autophagy related 16 like 1) in the pathogenesis of Crohn's Disease in Iranian patients. METHODS: One hundred thirty-eight patients and 99 normal controls were recruited in this study. Polymorphisms in -1644 and -308 upstream of IRGM gene were studied by PCR-sequencing and 20 kb CNVdel/insertion was studied by specific PCR. Rs10065171, rs4958847 in IRGM gene and rs2241880 in ATG16L1 were studied by Taqman genotyping assays. RESULTS: None of the so-called predisposing alleles of IRGM gene predispose Iranians to Crohn's disease while the prevalence of some of them like CNV deletion was higher in normal controls. Surprisingly all the so-called predisposing alleles in IRGM were linked to each other (especially rs4958847 with rs10065172 and polymorphisms in -308 region with rs4958847). Patients harboring A allele in rs4958847 site showed higher ratio of fibrostenotic type of disease while in patients with C/T genotype in rs4958847, colonic involvement was seen more frequently. G allele in ATG16L1 was associated with Crohn's disease though it was not associated with any phenotypic manifestation. CONCLUSION: In our study the association of ATG16L1 to Crohn's disease in Iranian patients was confirmed while it was shown that the studied polymorphisms in IRGM was not associated with Crohn's disease. Therefore in order to have a better picture about the genetics of Crohn's disease in Iranian patients, it is recommended to study other clinically effective polymorphisms in IRGM and ATG16L1 in addition to other genes which are responsible for the process of autophagy.

MiR-339 and especially miR-766 reactivate the expression of tumor suppressor genes in colorectal cancer cell lines through DNA methyltransferase 3B gene inhibition.

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'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.