Nicotinamide N-methyltransferase is related to MELF pattern invasion in endometrioid carcinoma [RNA-seq]. Nicotinamide N-methyltransferase is related to MELF pattern invasion in endometrioid carcinoma [RNA-seq]

In endometrioid carcinoma (EC), grade 1 (G1) EC is generally associated with a good prognosis. However, in a minority of G1, we often find a more aggressive histological pattern: MELF (microcystic, elongated, and fragmented) pattern. We previously revealed that EC with high expression of S100A4 and serum deprivation-response protein (SDPR) was related to MELF pattern invasion. However, the molecular feature of the invasive front area of MELF pattern has not been investigated. In the current study, we searched for genes preferentially expressed in the invasive front area of EC with MELF pattern by using laser microdissection and RNA sequencing, and revealed that Nicotinamide N-methyltransferase (NNMT) is related to invasiveness of MELF pattern. We confirmed that NNMT expression was high in the invasive front area of MELF pattern in immunohistochemical analysis. Moreover, using endometrioid carcinoma cell lines, we showed that NNMT promotes migration, invasion, colony formation, epithelial-mesenchymal transition (EMT), and chemoresistance. We speculated that depletion of NNMT promotes histone methylation and leads to tumor suppression because NNMT consumes S-adenosyl methionine (SAM), which is an essential methylation cofactor. We showed that NNMT-knockout cells the expression of H3K9me2 was enhanced. We speculate that methylation of H3K9 lead to repress the transcription of various oncogenic genes by performing RNA sequencing using NNMT-knockout cell lines. Our findings showed the possibility that NNMT inhibitors, which are expected to be used for the treatment of metabolic disorders, is effective for the treatment of an aggressive EC. This is the first report of the gene analysis focused on the morphological changes of MELF pattern invasion of EC. Overall design: To compare the levels of gene expression profiles between NNMT-knockout AN3CA cells and control cells, total RNA was extracted from KO1, KO2, and EV cells.

子宫内膜样癌（endometrioid carcinoma, EC）中，1级（G1）EC通常预后良好。但在少数G1病例中，常可见更具侵袭性的组织学表型：MELF（微囊、细长型、碎片状，microcystic, elongated, and fragmented）模式。我们前期研究发现，S100A4与血清剥夺应答蛋白（serum deprivation-response protein, SDPR）高表达的EC与MELF模式侵袭相关。然而，目前尚未有针对MELF模式侵袭前沿区域分子特征的研究。
本研究通过激光显微切割联合RNA测序，筛选MELF型EC侵袭前沿区域的优先表达基因，发现烟酰胺N-甲基转移酶（Nicotinamide N-methyltransferase, NNMT）与MELF模式的侵袭性相关。免疫组化分析证实，MELF模式的侵袭前沿区域NNMT表达水平较高。此外，通过子宫内膜样癌细胞系实验，我们证实NNMT可促进细胞迁移、侵袭、集落形成、上皮间质转化（epithelial-mesenchymal transition, EMT）以及化疗耐药。
我们推测，NNMT会消耗甲基化必需辅因子S-腺苷甲硫氨酸（S-adenosyl methionine, SAM），因此敲除NNMT可促进组蛋白甲基化并发挥肿瘤抑制作用。我们发现，NNMT敲除细胞中H3K9me2的表达水平升高。通过对NNMT敲除细胞系进行RNA测序，我们推测H3K9甲基化可抑制多种致癌基因的转录。
本研究结果提示，原本用于代谢紊乱治疗的NNMT抑制剂，有望用于侵袭性EC的治疗。本研究是首个聚焦EC的MELF模式侵袭形态学变化的基因分析报道。
整体实验设计：为比较NNMT敲除AN3CA细胞与对照细胞的基因表达谱差异，本研究从KO1、KO2及空载体（EV）细胞中提取总RNA进行测序。