Expression of CCL18 in glioblastoma and its effect on proliferation and migration of human U87MG cells

doi:10.11958/20221791

Abstract

Abstract:

Objective To analyze the expression chemokine (C-C motif) ligand 18 (CCL18) on the clinical prognosis of glioblastoma (GBM) and its effects on the proliferation and migration of U87MG cells. Methods The differential expression of CCL18 gene in GBM and normal brain tissue was analyzed based on the GBM data from Cancer Genome Atlas (TGGA) database and the normal brain tissue data from Genotype-Tissue Expression (GTEx) database. Immunohistochemical staining was used to verification. Kaplan-Meier survival analysis, univariate and multivariable Cox regression analysis were used to evaluate the impact of CCL18 expression on the prognosis in GBM patients. A nomogram was established based on the results of multivariate Cox analysis. The calibration curve and receiver-operating characteristic (ROC) curve were draw for validation. Human glioblastoma U87MG cells were divided into the siRNA-CCL18 group, the siRNA-NC group and the Mock-siRNA group. qPCR was used to detect the content of CCL18 mRNA in the different U87MG cell groups. The CCK-8 assay and cell scratch test were used to detect the cell proliferation and migration ability of U87MG cells in each group. Results CCL18 was significantly upregulated in GBM tissue (P<0.05). The immunohistochemistry results suggested that CCL18 protein was significantly increased in GBM tissue (P<0.05). Survival analysis indicated that high expression of CCL18 was associated with poor prognosis in GBM patients (P<0.05). Multivariate Cox regression analysis revealed that IDH wild-type and high level of CCL18 expression were the two major risk factors affecting the poor prognosis of GBM (P<0.05). The calibration curve showed that the actual survival rate of patients was consistent with the predicted survival rate. The ROC curve demonstrated that the model had a good predictive for predicting the survival of GBM patients. siRNA reduced the expression of CCL18 in human U87MG cells. The results of CCK-8 assay showed that the proliferation of U87MG cells was significantly inhibited (P<0.05), and the wound healing scratch ability of U87MG cells with low expression of CCL18 decreased (P<0.01). Conclusion CCL18 is over-expressed in GBM tissue, and high CCL18 expression is associated with poor prognosis in GBM patients．CCL18 promotes GBM cell proliferation and migration and may act as a relevant predictive biomarker for GBM.

Key words: glioblastoma, chemokine CCL18, cell proliferation, cell movement, prognosis

CLC Number:

R739.41

Figures/Tables 12

References 20

[1]	KARAMI FATH M, BABAKHANIYAN K, ANJOMROOZ M, et al. Recent advances in glioma cancer treatment:conventional and epigenetic realms[J]. Vaccines, 2022, 10(9):1448. doi:10.3390/vaccines10091448.
[2]	ALLAMI P, HEIDARI A, REZAEI N. The role of cell membrane-coated nanoparticles as a novel treatment approach in glioblastoma[J]. Front Mol Biosci, 2022, 9:1083645. doi:10.3389/fmolb.2022.1083645.
[3]	RODRÍGUEZ-CAMACHO A, FLORES-VÁZQUEZ J G, MOSCARDINI-MARTELLI J, et al. Glioblastoma treatment:state-of-the-art and future perspectives[J]. Int J Mol Sci, 2022, 23(13):7207. doi:10.3390/ijms23137207.
[4]	PARK J H, KANG I, LEE H K. The immune landscape of high-grade brain tumor after treatment with immune checkpoint blockade[J]. Front Immunol, 2022, 13:1044544. doi:10.3389/fimmu.2022.1044544.
[5]	KHAN F, PANG L Z, DUNTERMAN M, et al. Macrophages and microglia in glioblastoma:heterogeneity,plasticity,and therapy[J]. J Clin Invest, 2023, 133(1):e163446. doi:10.1172/jci163446.
[6]	HUANG X J, LAI S Q, QU F L, et al. CCL18 promotes breast cancer progression by exosomal miR-760 activation of ARF6/Src/PI3K/Akt pathway[J]. Mol Ther Oncolytics, 2022, 25:1-15. doi:10.1016/j.omto.2022.03.004.
[7]	SHEFLER I, SALAMON P, ZITMAN-GAL T, et al. Tumor-derived extracellular vesicles induce CCL18 production by mast cells:A possible link to angiogenesis[J]. Cells, 2022, 11(3):353. doi:10.3390/cells11030353.
[8]	ARAVIND A, PALOLLATHIL A, REX D A B, et al. A multi-cellular molecular signaling and functional network map of C-C motif chemokine ligand 18 (CCL18):a chemokine with immunosuppressive and pro-tumor functions[J]. J Cell Commun Signal, 2022, 16(2):293-300. doi:10.1007/s12079-021-00633-3.
[9]	VIVIAN J, RAO A A, NOTHAFT F A, et al. Toil enables reproducible,open source,big biomedical data analyses[J]. Nat Biotechnol, 2017, 35(4):314-316. doi:10.1038/nbt.3772.
[10]	DEMOGEOT N, SALLERON J, RECH F, et al. Impact of fractionated stereotactic radiotherapy on activity of daily living and performance status in progressive/recurrent glioblastoma:a retrospective study[J]. Radiat Oncol, 2022, 17(1):201. doi:10.1186/s13014-022-02169-1.
[11]	LOUIS D N, PERRY A, WESSELING P, et al. The 2021 WHO classification of tumors of the central nervous system:a summary[J]. Neuro Oncol, 2021, 23(8):1231-1251. doi:10.1093/neuonc/noab106.
[12]	TAKACS G P, FLORES-TORO J A, HARRISON J K. Modulation of the chemokine/chemokine receptor axis as a novel approach for glioma therapy[J]. Pharmacol Ther, 2021, 222:107790. doi:10.1016/j.pharmthera.2020.107790.
[13]	KORBECKI J, OLBROMSKI M, DZIĘGIEL P. CCL18 in the progression of cancer[J]. Int J Mol Sci, 2020, 21(21):7955. doi:10.3390/ijms21217955.
[14]	ZENG W F, XIONG L X, WU W, et al. CCL18 signaling from tumor-associated macrophages activates fibroblasts to adopt a chemoresistance-inducing phenotype[J]. Oncogene, 2023, 42(3):224-237. doi:10.1038/s41388-022-02540-2.
[15]	LIU X Q, XU X Y, DENG W, et al. CCL18 enhances migration,invasion and EMT by binding CCR8 in bladder cancer cells[J]. Mol Med Rep, 2019, 19(3):1678-1686. doi:10.3892/mmr.2018.9791.
[16]	WANG B, XIAO H, YANG X, et al. A novel immune-related microRNA signature for prognosis of thymoma[J]. Aging, 2022, 14(11):4739-4754. doi:10.18632/aging.204108.
[17]	GAO W Q, LI Y Y, ZHANG T, et al. Systematic analysis of chemokines reveals CCL18 is a prognostic biomarker in glioblastoma[J]. J Inflamm Res, 2022, 15:2731-2743. doi:10.2147/jir.S357787.
[18]	GROCHANS S, KORBECKI J, SIMIŃSKA D, et al. CCL18 expression is higher in a glioblastoma multiforme tumor than in the peritumoral area and causes the migration of tumor cells sensitized by hypoxia[J]. Int J Mol Sci, 2022, 23(15):8536. doi:10.3390/ijms23158536.
[19]	MA L, WANG H, LI Z, et al. Chemokine (C-C motif) ligand 18 is highly expressed in glioma tissues and promotes invasion of glioblastoma cells[J]. J Cancer Res Ther, 2019, 15(2):358-364. doi:10.4103/jcrt.JCRT_360_17.
[20]	HUANG Y, MOTTA E, NANVUMA C, et al. Microglia/macrophage-derived human CCL18 promotes glioma progression via CCR8-ACP5 axis analyzed in humanized slice model[J]. Cell Rep, 2022, 39(2):110670. doi:10.1016/j.celrep.2022.110670.

组别	引物序列（5′→3′）
siRNA-CCL18组	上游：AGUUCAUAGUUGACUAUUC
	下游：AAAGUUCAUAGUUGACUAU
Mock-siRNA组	上游：UAUCAAGAUGACGCUGCAA
	下游：AAUAUCAAGAUGACGCUGC
siRNA-NC组	上游：GAUGACGCUGCAAUGCAUA
	下游：AAGAUGACGCUGCAAUGCA

组别	引物序列（5′→3′）
siRNA-CCL18组	上游：AGUUCAUAGUUGACUAUUC
	下游：AAAGUUCAUAGUUGACUAU
Mock-siRNA组	上游：UAUCAAGAUGACGCUGCAA
	下游：AAUAUCAAGAUGACGCUGC
siRNA-NC组	上游：GAUGACGCUGCAAUGCAUA
	下游：AAGAUGACGCUGCAAUGCA

基因名称	引物序列（5′→3′）	产物大小/ bp
CCL18	上游：TCGTCTATACCTCCTGGCAGATTCC	137
CCL18	下游：CCACTTCTTATTGGGGTCAGCACAG	137
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	197
GAPDH	下游：GGCTGTTGTCATACTTCTCATGG	197

基因名称	引物序列（5′→3′）	产物大小/ bp
CCL18	上游：TCGTCTATACCTCCTGGCAGATTCC	137
CCL18	下游：CCACTTCTTATTGGGGTCAGCACAG	137
GAPDH	上游：GGAGCGAGATCCCTCCAAAAT	197
GAPDH	下游：GGCTGTTGTCATACTTCTCATGG	197

变量	B	SE	Wald χ²	P	HR	HR 95%CI
年龄	0.311	0.173	3.241	0.072	1.365	0.973~1.915
性别	0.026	0.182	0.020	0.887	1.026	0.719~1.466
KPS评分	-0.177	0.226	0.611	0.434	0.838	0.538~1.305
IDH状态	-1.201	0.396	9.182	0.002	0.301	0.138~0.654
CCL18表达	0.464	0.174	7.114	0.008	1.870	1.303~2.683