Abstract：
Lung cancer (LC) is a major inducer of cancer-related death. We aim to reveal the effect of Calsequestrin2 (CASQ2) on macrophage polarization and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in LC. Hub genes were determined from protein-protein interaction networks based on GSE21933 and GSE1987 data sets using bioinformatic analysis. Expression of hub genes was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Cell Counting Kit-8, 5-ethynyl-2\'-deoxyuridine, wound-healing, colony formation, and transwell assays were performed to assess the impact of CASQ2 on LC cells. A xenograft mouse model was evaluated using hematoxylin-eosin, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining to investigate the effect of CASQ2 on LC. The role of CASQ2 in regulating macrophage polarization and JAK/STAT pathway was evaluated by western blot andRT-qPCR. We screened out 155 common differentially expressed genes in GSE21933 and GSE1987 data sets. Myomesin-2, tyrosine kinase, sex determining region Y-box 2, platelet and endothelial cell adhesion molecule 1, matrix metallopeptidase 9, claudin-5, caveolin-1, CASQ2, recombinant ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (ATP2A2), and ankyrin repeat domain 1 were identified as the hub genes with high prediction value. CASQ2 was selected as a pivotal regulator of LC. In vitro experiments and xenograft models revealed that CASQ2 overexpression suppressed proliferation, colony formation, migration, invasion of LC cells, and tumor growth in vivo. Additionally, overexpression of CASQ2 promoted the expression of M1 macrophage markers (cluster of differentiation 80 [CD80], interleukin [IL]-12, inducible nitric oxide synthase [iNOS]), while decreasing the expression of M2 macrophage markers (CD163, IL-10, Arg1) in tumor-associated macrophages and xenograft tissues. Finally, we found that overexpression of CASQ2 inhibited JAK/STAT pathway. CASQ2 is a novel biomarker, which can alleviate LC via inhibiting M2 tumor-associated macrophage polarization and JAK/STAT pathway.
摘要：
肺癌（LC）是癌症相关死亡的主要诱因。我们旨在揭示Calsequestrin2（CASQ2）对LC中巨噬细胞极化和Janus激酶/信号转导子和转录激活因子（JAK/STAT）途径的影响。使用生物信息学分析，基于GSE21933和GSE1987数据集，从蛋白质-蛋白质相互作用网络中确定了Hub基因。通过实时定量聚合酶链反应(RT-qPCR)验证hub基因的表达。细胞计数试剂盒-8，5-乙炔基-2'-脱氧尿苷，伤口愈合,菌落形成,进行和transwell测定以评估CASQ2对LC细胞的影响。使用苏木精-伊红评估异种移植小鼠模型，免疫组织化学，和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色，以研究CASQ2对LC的影响。通过蛋白质印迹和RT-qPCR评估了CASQ2在调节巨噬细胞极化和JAK/STAT通路中的作用。我们在GSE21933和GSE1987数据集中筛选出155个常见的差异表达基因。Myomesin-2,酪氨酸激酶,性别决定区Y-box2,血小板和内皮细胞黏附分子1,基质金属肽酶9,claudin-5,caveolin-1,CASQ2,重组ATP酶,Ca++传输，心肌,慢抽搐2(ATP2A2),和锚蛋白重复结构域1被鉴定为具有高预测值的hub基因。选择CASQ2作为LC的关键调节器。体外实验和异种移植模型显示CASQ2过表达抑制增殖,菌落形成,迁移,侵入LC细胞，和体内肿瘤生长。此外,CASQ2的过表达促进M1巨噬细胞标记物的表达(分化簇80[CD80]，白细胞介素[IL]-12，诱导型一氧化氮合酶[iNOS]），同时降低M2巨噬细胞标志物（CD163，IL-10，Arg1）在肿瘤相关巨噬细胞和异种移植组织中的表达。最后,我们发现CASQ2过表达抑制JAK/STAT通路。CASQ2是一种新型生物标志物，通过抑制M2型肿瘤相关巨噬细胞极化和JAK/STAT通路减轻LC。
