Fig 1: Silencing LINC01021 promotes CDX2 expression and stability(A) qRT-PCR analysis of CDX2 expression in cancer tissues and adjacent normal tissues (n = 78); ∗p < 0.05. (B) IHC of CDX2 expression in cancer tissues and adjacent normal tissues. (C) Western blot analysis of CDX2 protein expression in BGC823 cell lines after CHX treatment. CDX2 expression was quantified relative to GAPDH expression at indicated times and normalized to the 0-hour time point (before CHX treatment); ∗p < 0.05, ∗∗p < 0.01 versus si-NC treatment. Measurement data were expressed as mean ± standard deviation. The data between two groups were analyzed by paired t test.
Fig 2: Molecular schematic diagram concerning LINC01021 in gastric cancerLINC01021 was upregulated in gastric cancer and could promote phosphorylation and nuclear export of CDX2 by CDK2. Silencing LINC01021 inhibited the binding of CDK2 and CDX2, as well as CDX2 phosphorylation. Additionally, silencing LINC01021 suppressed cell invasion, migration, and angiogenesis by upregulating the expression of KISS1.
Fig 3: LINC01021 plays roles in CDX2 phosphorylation, nuclear export, and transcriptional activity of downstream target genes via CDK2(A) The website (http://gps.biocuckoo.org/links.php#l1) predicted sites that CDK2 promoted CDX2 phosphorylation while highest score was observed in S100 and S60. (B) The website (http://www.phosphonet.ca/) predicted the phosphorylation enzymes at the S100 and S60 sites of CDX2, including CDK2. (C) coIP showed that CDK2 can bind to CDX2 in BGC823 cells. ∗p < 0.05 versus IgG. (D) Western blot analysis of CDX2 expression in BGC823 after IP with anti-phosphoserine. ∗p < 0.05 versus oe-NC treatment; #,∗p < 0.05 versus si-NC treatment. (E) Western blot of CDX2 expression in cytoplasm and nucleus after overexpressing CDK2 in cytoplasm and nucleus with GAPDH used as internal reference in neoplasm and Lamin A/C in nucleus. (F) qRT-PCR analysis of KISS1 expression and western blot analysis of CDX2 in BGC823 cells treated with oe-CDK2. ∗p < 0.05 versus oe-NC; #p < 0.05 versus oe-CDK2 treatment. (G) Western blot analysis of CDX2 expression in BGC823 cells treated with si-LINC01021 after IP with anti-phosphoserine. ∗p < 0.05 versus IgG. (H) CoIP of binding between CDK2 and CDX2 in BGC823 cells treated with si-LINC01021. ∗p < 0.05 versus IgG.
Fig 4: LINC01021 inhibits KISS1 expression through CDX2(A) LncMAP prediction of the target genes of CDX2 modulated by LINC01021. (B) RNA pull-down assay of binding between LINC01021 and CDX2 in BGC823 and SGC-7901 cells. (C) RIP assay of binding between LINC01021 and CDX2; ∗p < 0.05 versus IgG. (D) qRT-PCR analysis of KISS1 in gastric cancer tissues and adjacent normal tissues (n = 78). (E) qRT-PCR analysis of CDX2 and KISS1 expression in cells treated with oe-CDX2. ∗p < 0.05 versus oe-NC treatment. (F) Dual-luciferase reporter gene assay of binding between CDX2 and KISS1. ∗p < 0.05 versus oe-NC treatment. (G) ChIP assay of binding between CDX2 and KISS1. ∗p < 0.05 versus IgG. (H) ChIP assay of binding between CDX2 and KISS1 in BGC823 and SGC-7901 cells at the presence of si-LINC01021 or si-NC. ∗p < 0.05 versus IgG; #p < 0.05 versus si-NC treatment. (I) qRT-PCR of KISS1 expression in BGC823 and SGC-7901 cells after silencing LINC01021. ∗p < 0.05 versus si-NC treatment; #p < 0.05 versus si-LINC01021 + si-NC. Measurement data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted by one-way ANOVA. The data between two groups were analyzed by paired t test.
Fig 5: LINC01021 downregulates the expression of KISS1 through CDX2, thereby promoting cell migration, invasion, and angiogenesis in gastric cancer(A) qRT-PCR of LINC01021, CDX2, and KISS1 expression. (B) Western blot analysis of CDX2 and KISS1 expression. (C) Transwell assay to detect cell migration. (D) Transwell assay to detect cell invasion. (E) Tube formation assay to measure blood vessels. (F) Western blot analysis of VEGF and CD34 protein expression. ∗p < 0.05 versus si-NC + oe-NC treatment; #p < 0.05 versus si-LINC01021 + si-CDX2 treatment; &p < 0.05 versus si-LINC01021 + oe-KISS1 treatment. Measurement data were expressed as mean ± standard deviation. Comparisons among multiple groups were conducted by one-way ANOVA. The data between two groups were analyzed by paired t test.
Supplier Page from Abcam for Anti-CDX2 antibody