KIF18B promotes tumor progression through activating the Wnt/β-catenin pathway in cervical cancer

Abstract

Background: KIF18B was identified as a potential oncogene by analysis of The Cancer Genome Atlas database.

Materials and methods: We assessed KIF18B expression and explored its clinical significance in cervical cancer tissues. We have also evaluated the effects of KIF18B on cervical cancer cell proliferation, migration, and invasion both in vitro and in vivo.

Results: Our results show that KIF18B is overexpressed in cervical cancer tissues and is associated with a large primary tumor size, an advanced FIGO stage, and an advanced tumor grade. Knockdown of KIF18B induces cell cycle G1-phase arrest and inhibits the proliferation, migration, and invasion of cervical cancer cells, whereas its overexpression promotes proliferation, migration, and invasion in these cells. Moreover, silencing of KIF18B reduces expression of CyclinD1, β-catenin, C-myc, and p-GSK3β expression.

Conclusion: These data suggest that KIF18B can serve as a novel oncogene that promotes the tumorigenicity of cervical cancer cells by activating Wnt/β-catenin signaling pathway.

Keywords: CyclinD1; KIF18B; Wnt/β-catenin signaling pathway; cervical cancer.

Figure 1

KIF18B is overexpressed in cervical cancer tumor tissues and correlates with aggressive clinical characteristics. Notes: (A) KIF18B closely associated with a larger primary tumor size in TCGA database. (B) KIF18B is closely associated with the FIGO stage in TCGA database. (C) Normal cervical tissues do not express KIF18B, but several cervical cancer tissues are positive for KIF18B. (D) KIF18B is overexpressed in 87% (26 of 30) of cervical cancer tissues. (E) Representative images of KIF18B staining in normal cervical tissue and at different histological differentiation levels (from well to poor); magnification ×40. (F–H) KIF18B overexpression is associated with primary tumor size (P=0.018), an advanced tumor grade (P<0.001), and staging (P=0.025) in cervical cancer tissue. Abbreviations: TCGA, The Cancer Genome Atlas; IHC, immunohistochemistry.

Figure 2

Knockdown of KIF18B inhibits cervical cancer cell proliferation, invasion, and migration in vitro. Notes: (A) KIF18B mRNA and protein are hyperexpressed in HeLa and Siha cell lines. (B and C) Three specific siRNAs (siRNA-1, siRNA-2, and siRNA-3) were designed and synthesized, with siRNA-1 showing better efficiency. (D) CCK-8 assays showed that knock down of KIF18B inhibited the proliferation of both HeLa and Siha cells. (E) Colony numbers of HeLa and Siha cells transfected with si-KIF18B are fewer than cells transfected with si-NC. (F) Migration and invasion rates of HeLa and Siha cells transfected with si-KIF18B are decreased compared with those in NC cells. Scale bar 100 μm. (G) HeLa and Siha cells transfected with si-KIF18B display more arrest at G1 phase than do cells transfected with si-NC. (H) No significant difference for apoptosis was observed between the si-NC and si-KIF18B groups for both HeLa and Siha cells. *P<0.05, **P<0.01, ***P<0.001. Error bars indicate the SEM. Abbreviations: siRNA, small interfering RNA; NC, negative control; AV, annexin V.

Figure 2

Knockdown of KIF18B inhibits cervical cancer cell proliferation, invasion, and migration in vitro. Notes: (A) KIF18B mRNA and protein are hyperexpressed in HeLa and Siha cell lines. (B and C) Three specific siRNAs (siRNA-1, siRNA-2, and siRNA-3) were designed and synthesized, with siRNA-1 showing better efficiency. (D) CCK-8 assays showed that knock down of KIF18B inhibited the proliferation of both HeLa and Siha cells. (E) Colony numbers of HeLa and Siha cells transfected with si-KIF18B are fewer than cells transfected with si-NC. (F) Migration and invasion rates of HeLa and Siha cells transfected with si-KIF18B are decreased compared with those in NC cells. Scale bar 100 μm. (G) HeLa and Siha cells transfected with si-KIF18B display more arrest at G1 phase than do cells transfected with si-NC. (H) No significant difference for apoptosis was observed between the si-NC and si-KIF18B groups for both HeLa and Siha cells. *P<0.05, **P<0.01, ***P<0.001. Error bars indicate the SEM. Abbreviations: siRNA, small interfering RNA; NC, negative control; AV, annexin V.

Figure 2

Knockdown of KIF18B inhibits cervical cancer cell proliferation, invasion, and migration in vitro. Notes: (A) KIF18B mRNA and protein are hyperexpressed in HeLa and Siha cell lines. (B and C) Three specific siRNAs (siRNA-1, siRNA-2, and siRNA-3) were designed and synthesized, with siRNA-1 showing better efficiency. (D) CCK-8 assays showed that knock down of KIF18B inhibited the proliferation of both HeLa and Siha cells. (E) Colony numbers of HeLa and Siha cells transfected with si-KIF18B are fewer than cells transfected with si-NC. (F) Migration and invasion rates of HeLa and Siha cells transfected with si-KIF18B are decreased compared with those in NC cells. Scale bar 100 μm. (G) HeLa and Siha cells transfected with si-KIF18B display more arrest at G1 phase than do cells transfected with si-NC. (H) No significant difference for apoptosis was observed between the si-NC and si-KIF18B groups for both HeLa and Siha cells. *P<0.05, **P<0.01, ***P<0.001. Error bars indicate the SEM. Abbreviations: siRNA, small interfering RNA; NC, negative control; AV, annexin V.

Figure 3

Overexpression of KIF18B promotes cervical cancer cell proliferation, invasion, and migration in vitro. Notes: (A) The wound-healing assay showed that the migration of oe-KIF18B C33A cells was increased. (B) CCK-8 assays revealed that overexpression of KIF18B promoted the proliferation of C33A cells. (C) oe-KIF18B-transfected C33A cells had more colonies than did cells transfected with the control, pcDNA3.1. (D) Migration and invasion assays showed that oe-KIF18B treatment impaired the migration and invasion capacities of C33A cells. Scale bar 100 μm. **P<0.01, ***P<0.001. Error bars indicate the SEM.

Figure 4

KIF18B plays a carcinogenic role by activating the Wnt/β-catenin signaling pathway. Notes: (A) Gene Ontology analysis indicates enrichment of cell cycle pathways among genes co-expressed with KIF18B. (B) CyclinD1 mRNA expression was reduced after transfection with si-KIF18B, but the expression of p21, p27, and CyclinE were not altered both in HeLa and Siha cells. (C) CyclinD1 protein expression was decreased after transfection with si-KIF18B, with no difference in the expression of p21, p27, or CyclinE expression both in HeLa and Siha cells. (D) In HeLa cell, compared to si-NC treatment, si-KIF18B treatment resulted in decreased protein levels of C-myc, β-catenin, and p-GSK3β, but the total level of GSK3β has no obvious difference. ***P<0.001, #no significance. Error bars indicate the SEM. Abbreviation: NC, negative control.

Figure 4

KIF18B plays a carcinogenic role by activating the Wnt/β-catenin signaling pathway. Notes: (A) Gene Ontology analysis indicates enrichment of cell cycle pathways among genes co-expressed with KIF18B. (B) CyclinD1 mRNA expression was reduced after transfection with si-KIF18B, but the expression of p21, p27, and CyclinE were not altered both in HeLa and Siha cells. (C) CyclinD1 protein expression was decreased after transfection with si-KIF18B, with no difference in the expression of p21, p27, or CyclinE expression both in HeLa and Siha cells. (D) In HeLa cell, compared to si-NC treatment, si-KIF18B treatment resulted in decreased protein levels of C-myc, β-catenin, and p-GSK3β, but the total level of GSK3β has no obvious difference. ***P<0.001, #no significance. Error bars indicate the SEM. Abbreviation: NC, negative control.

Figure 5

Knockdown of KIF18B suppresses tumor growth in vivo. Notes: (A) Tumor nodules from mice injected with sh-KIF18B cells are significantly smaller than those from mice injected with NC cells. (B) sh-KIF18B tumors generated from both HeLa and Siha cell lines have less dense Ki-67 staining. Magnification ×40.(C) Compared with the NC group, the sh-KIF18B group showed reduced tumor sizes. (D) Compared with the NC group, the sh-KIF18B group showed reduced tumor weight. **P<0.01, ***P<0.001. Error bars indicate the SEM. Abbreviations: shRNA, small hairpin RNA; NC, negative control.

Figure 5

Knockdown of KIF18B suppresses tumor growth in vivo. Notes: (A) Tumor nodules from mice injected with sh-KIF18B cells are significantly smaller than those from mice injected with NC cells. (B) sh-KIF18B tumors generated from both HeLa and Siha cell lines have less dense Ki-67 staining. Magnification ×40.(C) Compared with the NC group, the sh-KIF18B group showed reduced tumor sizes. (D) Compared with the NC group, the sh-KIF18B group showed reduced tumor weight. **P<0.01, ***P<0.001. Error bars indicate the SEM. Abbreviations: shRNA, small hairpin RNA; NC, negative control.