Overexpression of DNA methyltransferase 1 (DNMT-1) is observed mostly in pancreatic cancer and it can cause tumor suppressor genes silencing in this disease. Recent studies suggest that abnormal expressions of microRNAs (miRs) are involved in pathogenesis of different types of human cancers including pancreatic cancer. In this study we aimed to investigate the effect of miR-148b and -152 on reverting the tumorigenic phenotype of pancreatic cancer cell lines. In order to investigate whether miR-148b and -152 are involved in the regulation of DNMT-1, luciferase reporter assay was used and confirmed that the DNMT-1 mRNA could be a target for miR-148b and miR-152. Furthermore, overexpression of miR-148b and -152 in pancreatic cancer cell lines (MIA PaCa-2 and AsPC-1) decreased DNMT-1 expression (53% and 59% respectively), returned DNA methylation to normal patterns and induced re-expression of tumor suppressor genes, like BNIP3 (4.7- and 3.8-fold) and SPARC (5.3- and 2.9-fold) for miR-148b and -152 respectively. Moreover, the introduced miR-148b and -152 could inhibit the proliferation of MIA PaCa-2 (35% and 37% respectively) and AsPC-1 (39% and 40% respectively) cell lines. The apoptosis rates of MIA PaCa-1 after treatment with miR-148b and -152 were 10% and 8% respectively; while these rates in AsPC-1 were 16% and 11% respectively. Conclusively these findings mean that miRs that are targeting DNMT-1 and modifying methylation status of tumor suppressor genes such as BNIP3 and SPARC can be applied in killing the pancreatic cancer cells and decreasing the tumorigenicity of these cells.

Oxaliplatin as a component of (Neo-) adjuvant chemotherapeutic regimens is administered to colorectal cancer patients. Unfortunately, the acquisition of resistance to this drug in nearly 90% of metastatic patients rendered it as an ineffective drug. Therefore, resistance mechanisms to this drug should be elucidated. There are different genes like GSTP1 and ABCB1 which are responsible for oxaliplatin resistance. We hypothesized that miR-129-5p, miR-302c-5p, miR-3664-5p, mir-3714 and miR-513a-3p are targeting ABCB1 gene, while GSTP1 was predicted to be the potential target of miR-3664-5p, mir-3714 and miR-513a-3p. In order to study this hypothesis, resistant colorectal cell lines were generated through intermittent exposure of HCT116, SW480 and HT29 to the increasing doses of oxaliplatin. MTT assays validated this resistance induction. Expression of ABCB1 and GSTP1 in addition to their targeting miRNAs in different cell lines were studied by quantitative real time PCR in the cell lines. Even though in comparison with HCT116 and SW480 cell lines, GSTP1 expression was reduced in resistant cells, ABCB1 expression was upregulated in these cell lines. On the other hand, HT-29 resistant cells showed elevated GSTP1 and unchanged ABCB1 levels. While miR-302c-5p level was downregulated in resistant cell lines, miR-129-5p and miR-3664-5p level showed different pattern of reduction in the resistant SW480 and HCT116 cell lines. GSTP1 level was correlated directly with miR-513a-3p and miR-3664-5p in all SW480 and HCT116 derived cell lines, however in HT-29-OXR1, GSTP1 level was correlated inversely with miR-3664-5p. In conclusion, upregulation of ABCB1 can be considered as the crucial component of poor response to oxaliplatin which is likely controlled by miR-302c-5p.

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.

Aim: The aim was to investigate the effect of miR-377 on DNMT1 expression and cancer phenotype in pancreatic cancer cells. Materials & methods: Real-time PCR, luciferase assay, MTT and Annexin-PI staining were used. Results: Decreased miR-377 and increased DNMT1 (verified as a target for mir-377) levels in pancreatic cancer tissues and cell lines in comparison with normal tissues was confirmed to be influenced by promoter methylation. Also hypermethylation of BNIP3, SPARC, TFPI2 and PENK promoters was observed in tumor samples but not in normal tissues which negatively correlated with their expression. Restoration of miR-377 resulted in a reduction of the expression of DNMT1 and reactivation of BNIP3 and SPARC genes via promoter demethylation. Furthermore, enhanced expression of miR-377 could significantly inhibit cell proliferation and induce apoptosis. Conclusion: Our findings showed that miR-377 through targeting DNMT1 could reduce DNA methylation of some tumor suppressor genes and restore their expres...