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. 2020 Oct 29;8(2):e001588. doi: 10.1136/jitc-2020-001588

Figure 3.

Figure 3

miR-200a directly targets CXCL12 3’UTR region. (A) miR-200a negatively regulated Journal for immunotherapy for cancer CXCL12 protein expression according to WB. (B) Luciferase reporter assay was used to determine direct targeting of CXCL12 3’UTR by miR-200a. (C) IHC detection of CXCL12 expression in various NPC xenograft groups from online supplemental figure 3F1. Scale bars at the bottom right: 75 μm. (D) Assay of migration of Treg cells verified that overexpression of CXCL12 promotes migration of Treg cells, and miR-200a overexpression inhibited movement of Treg cells and significantly suppresses the stimulatory effect of CXCL12 on migration of Treg cells. (E) overexpression of CXCL12 suppressed the miR-200a expression in EBNA1-negative NPC cell lines. (F) overexpression of CXCL12 decreased the suppressive effect of miR-200a on migration of Treg cells, and suppression of CXCL12 by specific siRNA reduced migration of Treg cells in vitro. (G) miR-200a expression level was elevated after transfection with CXCL12-specific siRNA in EBNA1-positive NPC cell lines according to qRT-PCR. (H1) In vivo migration assay verified that overexpression of CXCL12 promotes migration of Treg cells, and suppression of CXCL12 decreases infiltration of Treg cells. Upper: animal experiment flow chart. Lower: representative image and tumor growth curves of various tumor groups in humanized immune reconstruction mouse model (*p<0.05; **p<0.01; ***p<0.001). (H2) (H3) The in vivo tumor infiltration with CD4+ T cells, CD8+ T cells and Treg cells and CXCR4 (alias CD184) receptor expression levels were detected by FACS. (I) Representative CXCL12 expression in NPC patients in the EBNA1 low and i high expression groups according to IHC. Scale bars at the bottom right: 500 μm (low magnification) and 75 μm (high magnification). (J) Kaplan-Meier analysis of recurrence risk and overall survival for concurrent CXCL12 expression and infiltration of Treg cells groups in 105 NPC patients. ANOVA, analysis of variance; NPC, nasopharyngeal carcinoma; Treg, regulatory T; WB, western blot.