烟酰胺核糖对胰腺癌细胞DNA甲基化调控机制研究

Pancreatic cancer（PC）is a malignant tumor with strong potential of metastasis and poor prognosis. Tumorigenesis and therapeutic targets of PC are inconclusive. We exploited nicotinamide riboside (NR) as an intervention molecule to investigate the tumor inhibition effect, the results showed that the proliferation rate was significantly inhibited and the 5-Hydroxymethylcytosine (5hmC) level was markedly elevated. Additionally, the expression of Euchromatic histone-lysine N-methyltransferase 2 (EHMT2), which was reported to be highly expressed in a series of tumors, was dramatically inhibited. However, the mechanism behind the association between NR and PC remains unclear. We hypothesize that TET proteins, which play an important role in the demethylation process in mammals, can be regulated by NR through its effect on histone deacetylase (SIRT) and EHMT2 activities. We will use the ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) to precisely detect genomic 5hmC level. In addition, we will perform the Cas9 gene editing technology coupled with high-throughput DNA sequencing analysis to investigate the regulation mechanism of NR on PC. This research will provide important significance on PC therapy and drug development.

胰腺癌恶性程度高，转移性强，预后效果差，其发病机理和治疗靶点仍然较为模糊。我们采用烟酰胺核糖（NR）作为暴露分子，实验表明NR可以抑制胰腺癌细胞增殖速度，提高细胞基因组5-羟甲基胞嘧啶（5hmC）含量，同时降低组蛋白甲基化酶EHMT2在细胞内的表达，有报道EHMT2在多种肿瘤呈现异常高表达状态，但分子机制仍然不清楚。根据实验结果我们提出假设，NR是否可通过调节细胞能量代谢影响组蛋白去乙酰化酶（SIRT）或组蛋白甲基化酶（EHMT2）活性，从而影响TET蛋白活性或调控TET蛋白表达进而影响细胞甲基化调控，使某些关键基因表达发生改变从而抑制胰腺癌细胞增殖。本研究从表观遗传学角度出发，利用高效液相色谱串联质谱、高通量测序以及Cas9敲除技术等技术手段，考察烟酰胺核糖对胰腺癌细胞甲基化调控机制，同时结合表型分析，提出胰腺癌治疗的潜在靶点，对胰腺癌治疗与抗癌药物研发具有重要参考意义。

胰腺癌恶性程度高，转移性强，预后效果差，其发病机理和治疗靶点仍然较为模糊。我们采用烟酰胺核糖(NR)作为暴露分子，发现在烟酰胺核糖暴露胰腺癌细胞后显著抑制跨膜蛋白TSPAN1的表达。然而，TSPAN1在胰腺癌进展中的作用及其潜在的分子机制尚未完全阐明。在本研究中，我们验证了TSPAN1在胰腺癌中的致癌作用， TSPAN1促进细胞增殖、迁移、侵袭和肿瘤发生。我们发现miR-216a通过直接与TSPAN1的3 ' -UTR结合，负调控TSPAN1的表达，阐明了胰腺癌中TSPAN1上调的原因。之后，我们通过RNA-Seq分析，首次揭示TSPAN1转录调控ITGA2。在胰腺癌细胞中，在过表达TSPAN1基础上敲低ITGA2可以消减TSPAN1促进的细胞增殖和侵袭。此外，我们阐明了TSPAN1是通过调控TET2 ，DNMT3B和DNMT1的水平，导致ITGA2启动子CpG岛的低甲基化，进而表观调控ITGA2的表达。综上，我们提出了miR-216a/TSPAN1/ITGA2轴，并验证了它在胰腺癌发展过程中的作用，为更好的研究胰腺癌的致癌机理以及寻找新的胰腺癌治疗方法提供理论基础。