Cancer Research Institute, Experimental Education/Administration Center, School of Basic Medical Science, Southern Medical University, Shatai South Road, Baiyun District, 16, Guangzhou, 510515, China.
First Affiliated Hospital of Zhengzhou University, Zhengzhou University, Zhengzhou, 450001, China.
J Transl Med. 2022 May 10;20(1):205. doi: 10.1186/s12967-022-03403-w.
NOS1 expression predicts poor prognosis in patients with melanoma. However, the molecular function of NOS1 in the type I IFN response and immune escape of melanoma is still unknown.
The CRISPR/Cas9 system was used to generate NOS1-knockout melanoma cells and the biological characteristics of NOS1-knockout cells were evaluated by MTT assay, clonogenic assay, EdU assay, and flow cytometric assay. The effect on tumor growth was tested in BALB/c-nu and C57BL/6 mouse models. The gene expression profiles were detected with Affymetrix microarray and RNA-seq and KEGG (Kyoto Encyclopedia of Genes and Genomes) and CLUE GO analysis was done. The clinical data and transcriptional profiles of melanoma patients from the public database TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus, GSE32611) were analyzed by Qlucore Omics Explorer.
NOS1 deletion suppressed the proliferation of melanoma A375 cells in culture, blocked cell cycling at the G0/G1 phase, and decreased the tumor growth in lung metastasis nodes in a B16 melanoma xenograft mouse model. Moreover, NOS1 knockout increased the infiltration of CD3+ immune cells in tumors. The transcriptomics analysis identified 2203 differential expression genes (DEGs) after NOS1 deletion. These DEGs indicated that NOS1 deletion downregulated mostly metabolic functions but upregulated immune response pathways. After inhibiting with NOS1 inhibitor N-PLA, melanoma cells significantly increased the response to IFN[Formula: see text] by upregulation expression of IFN[Formula: see text] simulation genes (ISGs), especially the components in innate immune signaling, JAK-STAT, and TOLL-LIKE pathway. Furthermore, these NOS1-regulating immune genes (NOS1-ISGs) worked as a signature to predict poor overall survival and lower response to chemotherapy in melanoma patients.
These findings provided a transcriptional evidence of NOS1 promotion on tumor growth, which is correlated with metabolic regulation and immune escape in melanoma cells.
NOS1 表达可预测黑色素瘤患者预后不良。然而，NOS1 在黑色素瘤 I 型 IFN 反应和免疫逃逸中的分子功能仍不清楚。
利用 CRISPR/Cas9 系统生成 NOS1 敲除黑色素瘤细胞，并通过 MTT 检测、集落形成检测、EdU 检测和流式细胞术评估 NOS1 敲除细胞的生物学特性。在 BALB/c-nu 和 C57BL/6 小鼠模型中测试其对肿瘤生长的影响。利用 Affymetrix 微阵列和 RNA-seq 检测基因表达谱，并进行 KEGG（京都基因与基因组百科全书）和 CLUE GO 分析。从公共数据库 TCGA（癌症基因组图谱）和 GEO（基因表达综合数据库，GSE32611）分析黑色素瘤患者的临床数据和转录谱，并利用 Qlucore Omics Explorer 进行分析。
NOS1 缺失抑制黑色素瘤 A375 细胞在培养中的增殖，阻断细胞在 G0/G1 期的周期，并减少 B16 黑色素瘤异种移植小鼠模型中肺转移结节的肿瘤生长。此外，NOS1 敲除增加了肿瘤中 CD3+免疫细胞的浸润。转录组学分析发现 NOS1 缺失后有 2203 个差异表达基因（DEGs）。这些 DEGs 表明，NOS1 缺失主要下调代谢功能，但上调免疫反应途径。用 NOS1 抑制剂 N-PLA 抑制后，黑色素瘤细胞通过上调 IFN[Formula: see text]模拟基因（ISGs）的表达，对 IFN[Formula: see text]的反应显著增加，特别是先天免疫信号、JAK-STAT 和 Toll 样途径的组成部分。此外，这些 NOS1 调节的免疫基因（NOS1-ISGs）作为一个特征，可以预测黑色素瘤患者总体生存率差和对化疗反应低。
这些发现提供了 NOS1 促进肿瘤生长的转录证据，这与黑色素瘤细胞中的代谢调节和免疫逃逸有关。
