Fig 1: Knockdown of KIF18B suppresses migration and invasion of osteosarcoma cells through effects on β-catenin expression. Protein and mRNA expression were analyzed via qPCR and Western blot, respectively. (A) Protein and (B) mRNA expression of β-catenin and Wnt5a in shCtrl and shKIF18B cell lines. KIF18B knockdown resulted in lower mRNA and protein levels of CTNNB1 than shCtrl treatment whereas expression of WNT5A was not affected. (C) Protein and (D) mRNA expression of Myc, and cyclin D, genes downstream of CTNNB1, in shCtrl and shKIF18B cell lines. Protein levels of CTNNB1 (β-catenin), c-MYC, and cyclin D1 were significantly downregulated and the mRNA levels of c-MYC and cyclin D1 decreased to 25.1% and 17.9% in shKIF18B cell lines, respectively. (E) Protein and (F) mRNA expression of KIF18B and β-catenin in shCtrl, shKIF18B, and shKIF18B-OE β-catenin cell lines. Expression of KIF18B in shKIF18B and shKIF18B-OE β-catenin cells was significantly downregulated. Expression of CTNNB was recovered in shKIF18B-OE β-catenin cells, but remained very low in shKIF18B cells. (G) CCK-8 analysis of shCtrl, shKIF18B, and shKIF18B-OE β-catenin cell lines. shKIF18B-OE β-catenin cells showed recovery of proliferative activity to control levels. (H) Transwell assays of shCtrl, shKIF18B, and shKIF18B-OE β-catenin cell lines. (I) Relative migration and invasion activities of shCtrl, shKIF18B, and shKIF18B-OE β-catenin cell lines. Migration and invasion activities of shKIF18B-OE β-catenin cells were recovered to control levels. (J) Proportion of shCtrl, shKIF18B, and shKIF18B-OE β-catenin cells in each cell cycle phase. The proportion of cells in each cell cycle phase in shKIF18B-OE β-catenin cells was similar to that of the control group. (K) Apoptotic cell ratios in shCtrl, shKIF18B, and shKIF18B-OE β-catenin lines. The apoptosis ratio in shKIF18B-OE β-catenin cells was similar to that of the control group. The Mann-Whitney U test was used for statistical analysis in B, E, and I–K and Kolmogorov-Smirnov test applied for G.
Fig 2: Schematic diagram of KIF18B-APC-β-catenin and KIF18B-ATF2-β-catenin pathways. ATF2 is transported into the nucleus and β-catenin mRNA expression is regulated by KIF18B. Simultaneously, β-catenin is protected from degradation through APC binding of KIF18B.
Fig 3: Knockdown of KIF18B affects proliferation of osteosarcoma cells. (A) mRNA and (B) protein expression of KIF18B was downregulated in the shKIF18B-transfected cell line. The KIF18B mRNA level in the shKIF18B group was 18% that in the shCtrl group, as determined using qPCR. The protein level was < 10% that in the shCtrl group, as determined using Western blot. (C) Cell count curves. (D) Cell count fold changes between shCtrl and shKIF18B transfection groups. (E) Distribution of cell cycle phases for shCtrl and shKIF18B cells, determined via flow cytometry. (F) Proportion of each cell cycle phase in shCtrl and shKIF18B cell groups. Compared to shCtrl cells, KIF18B knockdown induced a significant increase in cells in the G1 phase and decrease in cells in S and G2/M phases. (G) KIF18B knockdown enhanced apoptosis in U2OS cells. (H) Proportion of apoptotic cells in shCtrl and shKIF18B lines. The apoptosis rate was 12.5% in shKIF18 cells and 4.7% in shCtrl cells. (I) Western blot analysis of activation of Caspase 3. Pro Caspase3 was expressed in both shCtrl and shKIF18B cell lines and cleaved Caspase 3 detected in the shKIF18B cell line. The Kolmogorov-Smirnov test was used for statistical analysis of data from C and D and Mann-Whitney U test for data from A, F and H.
Fig 4: KIF18B is overexpressed in osteosarcoma tissues and cells. (A) mRNA and (B) protein expression of KIF18B was higher in the three osteosarcoma cell lines than in hFOB1.19. The Mann-Whitney U test was used for statistical analysis. KIF18B mRNA expression in the osteosarcoma cell lines (HOS, U2OS, and Saos-2) was significantly higher than that in hFOB1.19 immortalized human osteoblasts, as determined via RT-qPCR using GAPDH as a reference gene (n = 3). Highest expression of KIF18B was detected in HOS cells (three times that in hFOB1.19). KIF18B protein expression in HOS, U2OS, and Saos-2 cell lines was significantly higher than that in hFOB1.19, consistent with mRNA results. (C) KIF18B is overexpressed in osteosarcoma tissues of patients. Higher KIF18B expression was detected in tumor tissues of 75% (15/20) patients. The Mann-Whitney U test was used for statistical analysis. (D) Immunohistochemical staining for KIF18B in cancer and adjacent tissues from patients with osteosarcoma.
Fig 5: KIF18B regulates β-catenin at the transcriptional level through ATF2. (A) Relative luciferase activity was determined in U2OS cells co-transfected with CTNNB1 promoter-luciferase reporter and shKIF18B or a non-targeting control vector (shCtrl). Upon KIF18B knockdown, luciferase activity was significantly decreased. (B) ChIP-qPCR enrichment for quantification of binding to the KIF18B locus. The CTNNB1 promoter was subdivided evenly into four sections. The second section exhibited binding to the promoter region of β-catenin. (C) qPCR analysis of mRNA expression of TFs that bind the promoter region of β-catenin. (D) mRNA expression of CTNNB1 in five TF siRNA cell lines. Downregulation of ATF2 and RUNX1 affected CTNNB1 mRNA expression. (E) Relative luciferase activity was determined in five TF siRNA cells co-transfected with CTNNB1 promoter-luciferase reporter and shKIF18B or a non-targeting control vector (shCtrl). The luciferase activities of the promoter-reporter vector in the ATF2 siRNA groups were downregulated to different degrees. (F) ChIP-qPCR enrichment to quantify binding to the KIF18B locus in Ctrl siRNA, ATF2 siRNA, and RUNX1 siRNA cells. KIF18B binding to the promoter region was significantly downregulated only when ATF2 expression was decreased. (G) Determination of relative luciferase activity in U2OS cells co-transfected with CTNNB1 promoter-wt and CTNNB1 promoter-mut-luciferase reporter and shKIF18B or a non-targeting control vector (shCtrl). Upon deletion of the potential ATF2 binding site in the promoter region of β-catenin, KIF18B significantly suppressed transcriptional activation of luciferase. (H) Interactions between KIF18B and ATF2 in U2OS cells overexpressing KIF18B with a DDK tag, detected via co-immunoprecipitation. KIF18B interacted with ATF2. (I) qPCR analysis of co-expression of β-catenin and KIF18B or ATF2. A positive relationship between co-expression of β-catenin, KIF18B and ATF2 was evident. (J) Protein and (K) mRNA expression of ATF2 in shCtrl and shKIF18B cell lines. Knockdown of KIF18B did not affect mRNA and protein expression levels. (L) Protein expression of KIF18B and ATF2 in cytoplasmic and nuclear fractions of shCtrl and shKIF18B cell lines. Nuclear distribution of ATF2 was significantly decreased in shKIF18B cell lines. The Mann-Whitney U test was used for statistical analysis in (A–H, K).
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