Department of Orthopedics, Chengdu Fifth People's Hospital, Chengdu, Sichuan, 611130, China.
PLoS One. 2024 May 31;19(5):e0303593. doi: 10.1371/journal.pone.0303593. eCollection 2024.
Rheumatoid arthritis (RA) is a common inflammatory and autoimmune disease. Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) is a crucial and a rate-limiting enzyme responsible for deoxynucleotide triphosphate(dNTP) production. We have found a high expression level of RRM2 in patients with RA, but the molecular mechanism of its action remains unclear.
We analyzed the expression of hub genes in RA using GSE77298 datasets downloaded from Gene Expression Omnibus database. RRM2 and insulin-like growth factor-2 messenger ribonucleic acid (mRNA)-binding protein 3 (IGF2BP3) gene knockdown was achieved by infection with lentiviruses. The expression of RRM2, IGF2BP3, matrix metalloproteinase (MMP)-1, and MMP-9 were detected via western blotting assay. Cell viability was detected via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MeRIP-qRT-PCR was performed to test the interaction of IGF2BP3 and RRM2 mRNA via m6A modification. Cell proliferation was determined by clone formation assay. Migration and invasion assays were performed using transwell Boyden chamber.
RRM2 and IGF2BP3 were highly expressed in clinical specimens and tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β-stimulated synovial cells. RRM2 and IGF2BP3 knockdown inhibited the proliferation, migration, and invasion of MH7A cells. The inhibitory effects of IGF2BP3 knockdown were effectively reversed by simultaneously overexpressing RRM2 in MH7A cells. By analyzing N6-methyladenosine (m6A)2Target database, five m6A regulatory target binding sites for IGF2BP3 were identified in RRM2 mRNA, suggesting a direct relationship between IGF2BP3 and RRM2 mRNA. Additionally, in RRM2 small hairpin (sh)RNA lentivirus-infected cells, the levels of phosphorylated Akt and MMP-9 were significantly decreased compared with control shRNA lentivirus-infected cells.
The present study demonstrated that RRM2 promoted the Akt phosphorylation leading to high expression of MMP-9 to promote the migration and invasive capacities of MH7A cells. Overall, IGF2BP promotes the expression of RRM2, and regulates the migration and invasion of MH7A cells via Akt/MMP-9 pathway to promote RA progression.
类风湿关节炎（RA）是一种常见的炎症性和自身免疫性疾病。核核苷酸还原酶调节亚单位 M2（RRM2）是一种关键的限速酶，负责脱氧核苷酸三磷酸（dNTP）的产生。我们发现 RA 患者的 RRM2 表达水平较高，但作用的分子机制尚不清楚。
我们使用从基因表达综合数据库下载的 GSE77298 数据集分析 RA 中的枢纽基因表达。通过感染慢病毒实现 RRM2 和胰岛素样生长因子 2 信使 RNA（mRNA）结合蛋白 3（IGF2BP3）基因的敲低。通过 Western blot 检测 RRM2、IGF2BP3、基质金属蛋白酶（MMP）-1 和 MMP-9 的表达。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）检测细胞活力。通过 MeRIP-qRT-PCR 检测 IGF2BP3 和 RRM2 mRNA 通过 m6A 修饰的相互作用。通过集落形成试验测定细胞增殖。使用 Transwell Boyden 室进行迁移和侵袭试验。
RRM2 和 IGF2BP3 在临床标本和肿瘤坏死因子-α（TNF-α）和白细胞介素（IL）-1β刺激的滑膜细胞中高表达。RRM2 和 IGF2BP3 敲低抑制 MH7A 细胞的增殖、迁移和侵袭。在 MH7A 细胞中同时过表达 RRM2 可有效逆转 IGF2BP3 敲低的抑制作用。通过分析 N6-甲基腺苷（m6A）2Target 数据库，在 RRM2 mRNA 中鉴定出五个 IGF2BP3 的 m6A 调节靶结合位点，表明 IGF2BP3 与 RRM2 mRNA 之间存在直接关系。此外，在 RRM2 短发夹（sh）RNA 慢病毒感染的细胞中，与对照 shRNA 慢病毒感染的细胞相比，磷酸化 Akt 和 MMP-9 的水平显著降低。
本研究表明，RRM2 促进 Akt 磷酸化，导致 MMP-9 高表达，从而促进 MH7A 细胞的迁移和侵袭能力。总的来说，IGF2BP 通过 Akt/MMP-9 通路促进 MH7A 细胞的迁移和侵袭，从而促进 RA 的进展。
