ABSTRACT

This study focused on the role of methionine (MET) in the autophagy of gastric cancer stem cells (GCSCs) and aims to elaborate its regulatory mechanism.

In the present study, the GCSCs were isolated from human gastric cancer cell lines using an anti-CD44 antibody, and then cultured in MET⁺ homocysteine (HCY)⁻ or MET⁻HCY⁺ medium.

In MET⁺HCY^–treated GCSCs, autophagy was suppressed, the methylation and phosphorylation of RAB37 were elevated, and miR-200b expression was down-regulated. Lentiviral vector (LV-) carrying methionine-γ lyase (an enzyme that could specifically lyse MET; Metase) promoted autophagy, reduced the methylation and phosphorylation of RAB37, and up-regulated miR-200b expression in MET⁺HCY^–-treated GCSCs. Then, we found that miR-200b suppressed the expression of protein kinase C α (PKCα), a protein that could inactivate RAB37 through promoting its phosphorylation. LV-Metase down-regulated RAB37 phosphorylation via miR-200b/PKCα, thus promoting the RAB37-mediated autophagy and suppressing cell viability in MET⁺HCY^–treated GCSCs. Finally, the in vivo study proved that LV-Metase treatment inhibited tumor growth through up-regulating RAB37 expression.

In conclusion, MET suppressed RAB37 expression via enhancing its methylation and suppressed RAB37 activity via miR-200b/PKCα axis, thus repressing RAB37-mediated autophagy in GCSCs. The supplementation of Metase lysed MET, thus inducing the autophagy of GCSCs and inhibiting tumor growth.

摘要

本研究侧重于甲硫氨酸（MET）在胃癌干细胞（GCSCs）自噬中的作用，旨在阐述其调控机制。

在本研究中，GCSCs 是使用抗 CD44 抗体从人胃癌细胞系中分离出来的，然后在 MET ⁺高半胱氨酸 (HCY) ^-或 MET ^- HCY ⁺培养基中培养。

在MET ⁺ HCY ^-处理GCSCs，自噬被抑制，RAB37的甲基化和磷酸化均升高，和miR-200b的表达下调。携带甲硫氨酸-γ裂解酶（一种可以特异性裂解 MET 的酶；Metase）的慢病毒载体 (LV-) 促进自噬，降低 RAB37 的甲基化和磷酸化，并上调 MET ⁺ HCY ^–处理的 GCSC 中的miR-200b 表达。然后，我们发现 miR-200b 抑制蛋白激酶 C α (PKCα) 的表达，PKCα 是一种可以通过促进 RAB37 磷酸化来灭活 RAB37 的蛋白质。LV-Metase 通过 miR-200b/PKCα 下调 RAB37 磷酸化，从而促进 RAB37 介导的自噬并抑制 MET ⁺ HCY 中的细胞活力^–处理过的 GCSC。最后，体内研究证明 LV-Metase 治疗通过上调 RAB37 表达抑制肿瘤生长。

总之，MET通过增强其甲基化抑制RAB37表达并通过miR-200b/PKCα轴抑制RAB37活性，从而抑制GCSC中RAB37介导的自噬。补充 Metase 裂解 MET，从而诱导 GCSCs 的自噬并抑制肿瘤生长。