Huang Chao, Liu Hong, Gong Xiu-Li, Wu Li-Yun, Wen Bin
Pi-Wei Institute, Guangzhou University of Traditional Chinese Medicine, Guangzhou, Guangdong 510000, P.R. China.
Oncol Rep. 2017 Mar;37(3):1637-1645. doi: 10.3892/or.2017.5379. Epub 2017 Jan 17.
The tissue microenvironment functions as a crucial player in carcinogenesis, and transforming growth factor-β1 (TGF-β1) within the microenvironment stimulates the formation of neoplasms. Using an in vitro model of malignancy induced by TGF-β1, we assessed the effect of evodiamine and berberine on the interaction between DNA methyltransferases (DNMTs) and target microRNAs (miRNAs) in the model. Colon tissues from neonatal rats 7 days of age were cultured and malignancy was induced by TGF-β1 in vitro for 48 h, and then the tissues were respectively treated with evodiamine and berberine for 24 h. Morphological alteration of tissues was observed by an inverted microscope, histological structures were observed using hematoxylin and eosin staining, and the expression levels of DNMTs and targeted miRNAs screened by bioinformatics software combined with Gene chip analysis in our previous study were detected by immunohistochemistry and quantified by real-time PCR. Twenty-four hours after treatment with TGF-β1, expression levels of DNMT1, DNMT3A, DNMT3B and miR-152 (target DNMT1), miR-429 (target DNMT3A) and miR-29a (target DNMT3A/3B) were markedly decreased; however, after 48 h, the expression levels of DNMT1 and DNMT3A were significantly increased, but their target miRNAs were still decreased. After treatment with a DNMT inhibitor (5-Aza-dC), expression levels of the miRNAs were increased to a larger extent, but did not reach normal levels. After treatment with berberine and evodiamine for 24 h, respectively, increased expression of DNMT1, DNMT3A, DNMT3B and miR-152, miR-429, miR-29a was noted. In conclusion, the results of the present study suggest that miRNAs can also be post-transcriptionally regulated by their corresponding DNMTs and that berberine and evodiamine regulate the expression of these genes, which provides early epigenetic evidence for the prevention and therapy of colorectal cancer.
组织微环境在肿瘤发生过程中起着关键作用，微环境中的转化生长因子-β1（TGF-β1）会刺激肿瘤形成。我们利用TGF-β1诱导的体外恶性肿瘤模型，评估了吴茱萸碱和黄连素对该模型中DNA甲基转移酶（DNMTs）与靶微小RNA（miRNAs）相互作用的影响。取7日龄新生大鼠的结肠组织进行培养，体外经TGF-β1诱导恶性肿瘤48小时，然后分别用吴茱萸碱和黄连素处理组织24小时。用倒置显微镜观察组织的形态学改变，用苏木精和伊红染色观察组织结构，通过免疫组织化学检测我们之前研究中利用生物信息学软件结合基因芯片分析筛选出的DNMTs和靶向miRNAs的表达水平，并通过实时PCR进行定量分析。用TGF-β1处理24小时后，DNMT1、DNMT3A、DNMT3B以及miR-152（靶标DNMT1）、miR-429（靶标DNMT3A）和miR-29a（靶标DNMT3A/3B）的表达水平显著降低；然而，48小时后，DNMT1和DNMT3A的表达水平显著升高，但其靶标miRNAs仍降低。用DNA甲基转移酶抑制剂（5-氮杂-2'-脱氧胞苷）处理后，miRNAs的表达水平有更大程度的升高，但未达到正常水平。分别用黄连素和吴茱萸碱处理24小时后，观察到DNMT1、DNMT3A、DNMT3B以及miR-152、miR-429、miR-29a的表达增加。总之，本研究结果表明，miRNAs也可在转录后水平受到其相应DNMTs的调控，且黄连素和吴茱萸碱可调节这些基因的表达，这为结直肠癌的预防和治疗提供了早期表观遗传学证据。
