2023 年第 10 期 第 18 卷

长链非编码RNA DLGAP1-AS2在肾透明细胞癌中的作用机制与预后价值

Mechanism and prognostic value of long non-coding RNA DLGAP1-AS2 in clear cell renal cell carcinoma

作者：乔丽文王惠明

英文作者：Qiao Liwen Wang Huiming

单位：武汉大学人民医院肾病内科，武汉430060

英文单位：Department of Nephrology Renmin Hospital of Wuhan University Wuhan 430060 China

关键词：肾透明细胞癌；长链非编码RNADLGAP1-AS2；免疫逃逸；磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白信号通路

英文关键词：Clearcellrenalcellcarcinoma;Longnon-codingRNADLGAP1-AS2;Immuneescape;Phosphatidylinositol3-kinase/proteinkinaseB/mammaliantargetofrapamycinpathway

• 摘要：

• 目的  探讨长链非编码RNA(lncRNA)DLGAP1-AS2在肾透明细胞癌（ccRCC）中的作用及分子机制，以及DLGAP1-AS2表达在ccRCC患者中的预后价值。方法  首先分别在人肾小管上皮细胞系HK-2和人ccRCC细胞系786-O中检测DLGAP1-AS2的表达水平，随后，通过细胞转染技术将过表达DLGAP1-AS2（Ov-DLGAP1-AS2）和过表达对照物（Ov-NC）转染到人ccRCC细胞系786-O细胞中，并检测细胞增殖活性和免疫逃逸能力的变化。检测Ov-DLGAP1-AS2对786-O细胞中磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路成员表达的影响。使用PI3K/Akt/mTOR通路激动剂740Y-P刺激786-O细胞，进一步探讨DLGAP1-AS2的作用机制。最后，在ccRCC患者中进行生存分析，并构建基于DLGAP1-AS2的ccRCC患者预后判断列线图。结果  786-O细胞中DLGAP1-AS2表达水平低于HK-2细胞［（0.50±0.08）比（0.95±0.05）］（P=0.002）。接种细胞48、72 h后，Ov-DLGAP1-AS2组细胞增殖活性低于Ov-NC组和对照组（均P＜0.05）。Ov-DLGAP1-AS2组细胞上清液中可溶性人程序性死亡配体1（sPD-L1）浓度低于Ov-NC组和对照组（均P＜0.05），将786-O细胞与T细胞共培养后，Ov-DLGAP1-AS2/T组细胞上清液中γ干扰素、肿瘤坏死因子α（TNF-α）、白细胞介素2（IL-2）浓度高于Ov-NC/T组和T细胞组（均P＜0.05）。Ov-DLGAP1-AS2组中PI3K、Akt和mTOR的表达水平低于Ov-NC组和对照组（均P＜0.05）。加入740Y-P刺激786-O细胞，接种细胞48、72 h后，Ov-DLGAP1-AS2+740Y-P组细胞增殖活性高于Ov-DLGAP1-AS2组（均P＜0.05），且Ov-DLGAP1-AS2+740Y-P组细胞上清液中sPD-L1的浓度高于Ov-DLGAP1-AS2组，与T细胞共培养后，Ov-DLGAP1-AS2/T+740Y-P组细胞上清液中γ干扰素、TNF-α、IL-2浓度低于Ov-DLGAP1-AS2/T组（均P＜0.05）。Kaplan-Meier生存曲线表明DLGAP1-AS2高表达患者预后差于DLGAP1-AS2低表达患者（Log-rank P＜0.001），且DLGAP1-AS2高表达为ccRCC患者预后的独立危险因素（均P＜0.05）。基于DLGAP1-AS2构建了列线图，经验证可以较好地预测ccRCC患者生存率。结论  过表达DLGAP1-AS2能够通过抑制PI3K/Akt/mTOR通路，抑制786-O细胞的增殖活性和免疫逃逸。DLGAP1-AS2高表达预示ccRCC患者预后不良。DLGAP1-AS2是ccRCC潜在的治疗靶点和预后标志物。

• Objective  To explore the effects and molecular mechanism of long non-coding RNA (lncRNA) DLGAP1-AS2 in clear cell renal cell carcinoma (ccRCC), and the prognostic value of DLGAP1-AS2 expression in ccRCC patients. Methods  The expression levels of DLGAP1-AS2 in human tubular epithelial cell line HK-2 cells and human ccRCC cell line 786-O cells were measured. Overexpression of DLGAP1-AS2 (Ov-DLGAP1-AS2) and overexpression control (Ov-NC) were transfected into 786-O cells, and proliferation activity and immune escape ability were detected. The effects of DLGAP1-AS2 overexpression on the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway in 786-O cells were examined. The PI3K/Akt/mTOR pathway agonist 740Y-P was added to further explore the underlying mechanism. Finally, survival analysis was performed in ccRCC patients, and a nomogram based on DLGAP1-AS2 was constructed. Results  The expression level of DLGAP1-AS2 in 786-O cells was lower than that in HK-2 cells［（0.50±0.08） vs （0.95±0.05）］（P=0.002）. At 48 and 72 h after cell inoculation, Ov-DLGAP1-AS2 group had lower cell proliferation activity than Ov-NC group and control group (all P＜0.05). The concentration of soluble human programmed death ligand-1 (sPD-L1) in the cell supernatant of Ov-DLGAP1-AS2 group was lower than that of Ov-NC group and control group (both P＜0.05). After co-cultured with T cells, the concentrations of interferon-γ, tumor necrosis factor α (TNF-α) and interleukin 2 (IL-2) in the cell supernatant of Ov-DLGAP1-AS/T group were higher than those of Ov-NC/T group and T cells group (all P＜0.05). The expression levels of PI3K, Akt and mTOR of Ov-DLGAP1-AS2 group were lower than those of Ov-NC group and control group (all P＜0.05). Adding 740Y-P to stimulate 786-O cells, at 48 and 72 h after cell inoculation, Ov-DLGAP1-AS2+740Y-P group had higher cell proliferation activity than Ov-DLGAP1-AS2 group (both P＜0.05). The concentration of sPD-L1 in the cell supernatant of Ov-DLGAP1-AS2+740Y-P group was higher than that in Ov-DLGAP1-AS2 group; after co-cultured with T cells, the concentrations of interferon-γ, TNF-α and IL-2 in the cell supernatant of Ov-DLGAP1-AS2/T+740Y-P group were lower than those in Ov-DLGAP1-AS2/T group (all P＜0.05). Kaplan-Meier survival curves indicated that patients with high DLGAP1-AS2 expression had a worse prognosis than those with low DLGAP1-AS2 expression (Log-rank P＜0.001), and high DLGAP1-AS2 expression is an independent risk factor for patient outcome (both P＜0.05). The nomogram constructed based on DLGAP1-AS2 had high predictive power in ccRCC patients survival rate. ConclusionsDLGAP1-AS2 overexpression can inhibit the proliferation activity and immune escape of 786-O cells by inhibiting the PI3K/Akt/mTOR pathway. High expression of DLGAP1-AS2 predicts poor prognosis in ccRCC patients. DLGAP1-AS2 is a potential therapeutic target and prognostic marker for ccRCC.