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Methylation of the miR‑29b‑3p promoter contributes to angiogenesis, invasion, and migration in pancreatic cancer.

ABSTRACT: The aim of the present study was to investigate the effects of microRNA (miR)‑29b‑3p gene promoter methylation on angiogenesis, invasion, and migration in human pancreatic cancer. Prediction of promoter methylation of miR‑29b‑3p was performed through the MethPrimer tool. Then the methylation levels of miR‑29b‑3p in human pancreatic cancer tissues and cell lines were detected by pyrosequencing, and the relative expression of miR‑29b‑3p was assessed in pancreatic cancer tissues by qPCR. The results were analyzed by linear regression. Western blot analysis was used to detect expression of DNA methyltransferases (DNMTs) in pancreatic cancer tissues and adjacent tissues. The Transwell assay was used to detect the ability of cell migration and invasion. Cells were co‑cultured with human umbilical vein endothelial cells (HUVECs) to detect the ability of angiogenesis. The results revealed that DNMT1 expression in pancreatic cancer tissues was higher than that in adjacent tissues. Further results showed that expression of miR‑29b was negatively correlated with the methylation level of the miR‑29b promoter. Bxpc3 and Capan‑2 cells had higher methylation levels, and the expression level of miR‑29b‑3p in Bxpc3 and Capan‑2 cells was found to be lower than that of other cell lines. Expression of zonula occludens‑1 (ZO‑1) and occludin was significantly increased, and the migration of cancer cells was decreased after cells were treated with siRNA DNMT1. Further results showed that miR‑29b reversed the promotive effect of DNMT1 overexpression on tumor cell malignant properties. Methylation of the miR‑29b‑3p promoter contributes to angiogenesis, invasion, and migration in pancreatic cancer. This study indicated that the alteration of methylation of mR‑19b may be a potential approach for inhibiting the progression of pancreatic cancer.

SUBMITTER: Wang L

PROVIDER: S-EPMC7709820 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIt was previously demonstrated that miR-199a-3p plays an important role in tumor progression; especially, its down-regulation in papillary thyroid cancer (PTC) is associated with cancer cell invasion and proliferation. In the present report, we investigated the mechanism involved in the down-regulation of miR-199a-3p in PTC and how miR-199a-3p regulates PTC invasion both in vivo and in vitro.MethodsqRT-PCR and Western blot assays were used to determine the expression of the investigated genes. Bisulfite sequencing PCR was used to investigate miR-199a-3p methylation. The functions of miR-199a-3p were investigated by a series of in vitro and in vivo experiments.ResultsOur results showed hypermethylation of the miR-199a-3p promoter, which resulted in decreased miR-199a-3p expression both in PTC cell lines and PTC tissues. DNA-methyltransferase 3a (DNMT3a), a target gene of miR-199a-3p, was increased both in PTC cell lines and PTC tissues, while 5-aza-2'-deoxycytidine (methyltransferase-specific inhibitor) or knock-down using DNMT3a Small-Interfering RNA could restore the expression of miR-199a-3p, and the over-expression of miR-199a-3p could decrease the expression of DNMT3a; this suggests that miR-199a-3p/DNMT3a constructs a regulatory circuit in regulating miR-199a-3p/DNMT3a expression. Moreover, gain- and loss-of-function studies revealed that miR-199a-3p is involved in cancer cell migration, invasion, and growth. Meanwhile, we found that RAP2a was also a direct target of miR-199a-3p, which might mediate the tumor-growth-inhibiting effect of miR-199a-3p. To further confirm the tumor-suppressive properties of miR-199a-3p, stable overexpression of miR-199a-3p in a PTC cell line (BCPAP cells) was xenografted to athymic BALB/c nude mice, resulting in delayed tumor growth in vivo. In clinical PTC samples, the expression of RAP2a and DNMT3a was increased significantly, and the expression of RAP2a was inversely correlated with that of miR-199a-3p.ConclusionOur studies demonstrate that an epigenetic change in the promoter region of miR-199a contributes to the aggressive behavior of PTC via the miR-199a-3p/DNMT3a regulatory circuit and directly targets RAP2a.

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Methylation of the miR‑29b‑3p promoter contributes to angiogenesis, invasion, and migration in pancreatic cancer.

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