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Abstract：
Background: Gastric cancer (GC) is one of the most common malignancies worldwide, with high incidence and mortality rate. Tripartite motif-containing 28 (TRIM28) is an important molecule that affects the occurrence and development of tumors, but its function in GC has not been elucidated clearly. The purpose of this study is to explore the molecular mechanism by which TRIM28 affect the GC. Methods: TRIM28 expression was tested in RNA-seq data from TCGA database, tumor tissue samples from patients and GC cell lines. Genes were silenced or overexpressed by siRNA, lentivirus-mediated shRNA, or plasmids. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to explore the proliferation of GC cells after TRIM28 knockdown. RNA-seq and TCGA database were used to identify target genes. Luciferase report assay was employed to detect the possible mechanism between TRIM28 and Indoleamine 2,3-dioxygenase (IDO1). Tryptophan concentration in cell supernatant was measured using a fluorometric assay kit. MGC-803 and 746T cells were injected into mice to establish xenograft animal models. Results: The expression of TRIM28 was positively correlated with tumor size and poorer prognosis. Upregulation of TRIM28 was observed in GC tissues and cells. In vitro, we proved that knockdown of TRIM28 significantly inhibited the proliferation of GC cells. Then TRIM28 was found to be positively correlated with the expression of IDO1 in GC cells. In accordance with this, tryptophan levels in cell supernatants were increased in TRIM28 knockdown GC cells and overexpression of IDO1 could reverse this phenotype. Serum response factor (SRF), a reported regulator of IDO1, was also regulated by TRIM28 in GC cells. And decreased expression of IDO1 induced by TRIM28 knockdown could be partly reversed through overexpression of serum response factor (SRF) in GC cells. Functional research demonstrated that the expression of IDO1 was increased in GC and IDO1 knockdown could also inhibited the proliferation of GC cells. Furthermore, overexpression of IDO1 could partly reverse proliferation inhibited by TRIM28 knockdown in GC cells. In vivo, knockdown of TRIM28 significantly inhibited the tumor growth and overexpression of IDO1 and SRF both could reverse proliferation inhibited by TRIM28 knockdown. Conclusions: TRIM28 is crucial in the development of GC, and may regulate IDO1 through SRF. TRIM28 promote GC cell proliferation through SRF/IDO1 axis.
摘要：
背景:胃癌(GC)是全球范围内最常见的恶性肿瘤之一。发病率和死亡率高。含三方基序28(TRIM28)是影响肿瘤发生发展的重要分子,但其在GC中的功能尚未明确阐明。本研究旨在探讨TRIM28影响GC的分子机制。方法:在TCGA数据库的RNA-seq数据中检测TRIM28表达,来自患者和GC细胞系的肿瘤组织样品。基因被siRNA沉默或过表达，慢病毒介导的shRNA，或质粒。进行细胞计数试剂盒-8(CCK-8)和集落形成测定以探索在TRIM28敲低后GC细胞的增殖。RNA-seq和TCGA数据库用于鉴定靶基因。荧光素酶报告测定法用于检测TRIM28和吲哚胺2,3-双加氧酶（IDO1）之间的可能机制。使用荧光测定试剂盒测量细胞上清液中的色氨酸浓度。将MGC-803和746T细胞注射到小鼠体内以建立异种移植动物模型。结果：TRIM28的表达与肿瘤大小和预后呈正相关。在GC组织和细胞中观察到TRIM28的上调。体外，我们证明敲低TRIM28可以显著抑制GC细胞的增殖。然后发现TRIM28与GC细胞中IDO1的表达呈正相关。据此,在TRIM28敲低GC细胞中，细胞上清液中的色氨酸水平增加，IDO1的过表达可以逆转这种表型。血清反应因子(SRF),据报道，IDO1的调节因子在GC细胞中也受到TRIM28的调节。TRIM28敲低诱导的IDO1表达降低可通过在GC细胞中过表达血清反应因子（SRF）部分逆转。功能研究表明，IDO1在GC中表达增加，IDO1敲低也能抑制GC细胞的增殖。此外，IDO1的过表达可以部分逆转GC细胞中TRIM28敲低抑制的增殖。在体内,TRIM28敲低可显著抑制肿瘤生长，IDO1和SRF的过表达均可逆转TRIM28敲低抑制的增殖。结论：TRIM28在GC的发展中至关重要，并可能通过SRF调节IDO1。TRIM28通过SRF/IDO1轴促进GC细胞增殖。
