Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jun 22:13:848926.
doi: 10.3389/fgene.2022.848926. eCollection 2022.

KIFC3 Promotes Proliferation, Migration, and Invasion in Colorectal Cancer via PI3K/AKT/mTOR Signaling Pathway

Huiling Liao  1   2 , Lan Zhang  1   2 , Shimin Lu  1   2 , Wei Li  1   2 , Weiguo Dong  1
Affiliations

KIFC3 Promotes Proliferation, Migration, and Invasion in Colorectal Cancer via PI3K/AKT/mTOR Signaling Pathway

Huiling Liao et al. Front Genet. .

Abstract

Background: KIFC3, belongs to kinesin superfamily proteins (KIFs), is well known for its role in intracellular cargo movement. KIFC3 has been identified as a docetaxel resistance gene in breast cancer cells, however, the role of KIFC3 and its potential mechanism in colorectal cancer (CRC) remains elusive. Objectives: We aims to investigate the effects of KIFC3 in proliferation, migration, and invasion in CRC as well as the potential mechanism inside. Methods: We investigated the expression of KIFC3 in the Oncomine, Gene Expression Profiling Interactive Analysis databases. The KIFC3 protein expression and mRNA level in CRC cells were evaluated by western blot and qRT-PCR. Cell proliferation ability was detected by CCK-8, EdU, colony formation assay and xenograft tumor in nude mice. Flow cytometry was used to detect the cell cycle. The effect of KIFC3 on the epithelial-to-mesenchymal transition (EMT) was investigated by transwell and wound healing assay. The association of KIFC3 with EMT and PI3K/AKT/mTOR signaling pathway were measured by western blot and immunofluorescence staining. Results: The expression of KIFC3 was higher in CRC tissues than normal colorectal tissue, and was negatively correlated with the overall survival of patients with CRC. KIFC3 silencing inhibited the proliferation, migration and invasion of CRC cells. Meanwhile, it could decrease the number of cells in S phase. KIFC3 silencing inhibited the expression of proliferating cell nuclear antigen, Cyclin A2, Cyclin E1, and CDK2 and increased the expression of p21 and p53. KIFC3 overexpression promoted the G1/S phase transition. KIFC3 silencing inhibited the EMT process, which decreased the level of N-cadherin, Vimentin, SNAIL 1, TWIST, MMP-2, MMP-9 and increased E-cadherin, while KIFC3 overexpression show the opposite results. Furthermore, the knockdown of KIFC3 suppressed the EMT process by modulating the PI3K/AKT/mTOR signaling pathway. KIFC3 silencing decreased the expression of phosphorylated PI3K, AKT, mTOR, but total PI3K, AKT, mTOR have no change. Inversely, the upregulation of KIFC3 increased the expression of phosphorylated PI3K, AKT and mTOR, total PI3K, AKT, mTOR have no change. In a xenograft mouse model, the depletion of KIFC3 suppressed tumor growth. the increased expression levels of KIFC3 could enhance the proliferation, migration and invasion of CRC cells, and enhance the EMT process through the PI3K/AKT/mTOR pathway. Conclusion: Our study substantiates that KIFC3 can participate in the regulation of CRC progression by which regulates EMT via the PI3K/AKT/mTOR axis.

Keywords: KIFC3; PI3K/Akt/mTOR signal pathway; epithelial-to-mesenchymal transition; invasion; migration; proliferation.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Analysis of KIFC3 mRNA expression in normal colorectal and colorectal cancer (CRC) tissues from public databases. (A) Data from the Oncomine databases showed that KIFC3 mRNA expression was upregulated in CRC tissues compared to normal colorectal tissues. (B,C) Data from the GEPIA databases showed that KIFC3 mRNA level was higher in CRC tissues compared to normal colorectal tissues. (D,E) Data from the GEPIA database showed that the expression level of KIFC3 could affect the OS of CRC (p = 0.00069), but did not affect the disease-free survival (p = 0.056). The OS of patients with high expression of KIFC3 was shorter than that of patients with low expression of KIFC3.
FIGURE 2
FIGURE 2
Prognostic analysis of gene signature in the ICGC database. (A) KIFC3 expression and survival time and survival status in ICGC data set. (B) Kaplan-Meier survival curve distribution of KIFC3 in ICGC data database. (C) ROC curve and AUC of KIFC3 at different times. The abscissas of the upper, middle and lower three graphs in Figure 2A all represent samples, and the order of the samples is consistent.
FIGURE 3
FIGURE 3
KEGG pathway and GO enrichment analyses of KIFC3-associated genes. (A) The bubble chart of KEGG pathway. (B) The bubble chart of GO enrichment in biological processes. (C) The bubble chart of GO enrichment in cell component. (D) The bubble chart of GO enrichment in molecular function.
FIGURE 4
FIGURE 4
KEGG pathway and GO enrichment analyses of KIFC3-associated genes. (A) The bar chart of KEGG pathway. (B) The bar chart of GO enrichment in biological processes. (C) The bar chart of GO enrichment in cell component. (D) The bar chart of GO enrichment in molecular function.
FIGURE 5
FIGURE 5
KEGG pathway and GO enrichment analyses of KIFC3-associated genes. (A) The network chart of KEGG pathway. (B) The network chart of GO enrichment in biological processes. (C) The network chart of GO enrichment in cell component. (D) The network chart of GO enrichment in molecular function.
FIGURE 6
FIGURE 6
KIFC3 was expressed at higher levels in CRC cell lines. (A–C) Western blot and qRT-PCR analysis of KIFC3 expression in CRC cell lines (HT29, HCT116, SW480, DLD-1) and normal colon mucosal epithelial cells (NCM460), and quantification analysis results. (D–G) Western blot analysis of the efficiency of sh-KIFC3 and sh-NC transfection compare with Control group in SW480 and HT29 cells, and the efficiency of KIFC3-overexpress and over-Control transfection in DLD-1 cell, and quantification analysis results. (H–J) qRT-PCR analysis of the efficiency of the best knockdown effect in SW480 and HT29 cells, and the efficiency of KIFC3-overexpress, NC and Control transfection in DLD-1 cell. The transfected cells above are all stable transfectants. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 7
FIGURE 7
KIFC3 promotes the proliferation of CRC cells. (A) The transfection efficiency of SW480, HT29 and DLD-1 were observed under microscope. (B–G) EdU assay of KIFC3 silencing or overexpression on CRC cells’ proliferation, and quantification analysis results. (H–J) The effects of KIFC3 silencing or overexpression on the proliferation ability of SW480, HT29 and DLD-1 cells were measured by the CCK-8 assay. (K–P) Colony formation assay of KIFC3 silencing or overexpression on CRC cells’ proliferation, and quantification analysis results. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 8
FIGURE 8
KIFC3 affects the expression of cell cycle-mediated factors. (A–D) Effects of KIFC3 silencing on the cell cycle distribution in SW480 and HT29 cells as showed by flow cytometry assay, and quantification analysis results. (E–H) Effect of KIFC3 silencing on the expression of cell cycle-mediated factors like p21, p53, Cyclin A2, Cyclin E1, and CDK2, as measured by western blot, and quantification analysis results. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 9
FIGURE 9
KIFC3 promotes the proliferation and migration of CRC cells. (A–F) The expression of PCNA in SW480, HT29 and DLD-1 cells after lentivirus vectors delivery in each group as measured by western blot, and quantification analysis results. (G–L) Effects of KIFC3 silencing or overexpression on the wound healing of SW480, HT29 and DLD-1 cells, and quantification analysis results. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 10
FIGURE 10
KIFC3 promotes the migration and invasion of CRC cells. (A–F) Effects of KIFC3 silencing or overexpression on the migration ability of SW480, HT29 and DLD-1 cells, and quantification analysis results. (G–L) Effects of KIFC3 silencing or overexpression on the invasion ability of SW480, HT29 and DLD-1 cells, and quantification analysis results. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 11
FIGURE 11
KIFC3 affected the expression of the related factors in the EMT progress. (A–C) Effects of KIFC3 silencing or overexpression on the expression of the related factors in the EMT progress like E-cadherin, N-cadherin, Vimentin, TWIST1, SNAI1, MMP-2, and MMP-9, as measured by western blot. (D–F) Quantification to the protein level of the related factors in the EMT progress. (G–I) Immunofluorescence staining of E-cadherin and N-cadherin. (J–L) Statistical analysis of the relative mean fluorescence of E-cadherin and N-cadherin. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 12
FIGURE 12
KIFC3 affected the expression of the related factors in the PI3K/AKT/mTOR pathway. (A–C) Effects of KIFC3 silencing or overexpression on the expression of the related factors in the PI3K/AKT/mTOR pathway like PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR, as measured by western blot. (D–F) Quantification to the protein level of the related factors in the PI3K/AKT/mTOR pathway. (G–I) Immunofluorescence staining of p-PI3K and p-AKT. (J–L) Statistical analysis of the relative mean fluorescence of p-PI3K and p-AKT. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 13
FIGURE 13
KIFC3 knockdown inhibited tumor growth and proliferation. (A–D) Transplanted tumor mice image, tumor xenograft image, tumor xenograft volume, tumor xenograft weight of sh-NC and sh-KIFC3 SW480 cells in nude mice. (E,F) Analysis of KIFC3 and Ki67 in tumor xenografts derived from sh-NC and sh-KIFC3 SW480 cells by Immunohistochemistry. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 14
FIGURE 14
LY294002 and Triciribine attenuates the effect of KIFC3 on PI3K/AKT/mTOR pathway in CRC cells. (A) The expression of the related factors in the PI3K/AKT/mTOR pathway was reversed to some extent under the effect of LY294002 and Triciribine. (B,C) Quantification to the protein level of the related factors in the PI3K/AKT/mTOR pathway. The data are presented as the mean ± standard deviation of triplicate independent experiments and were normalized to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
FIGURE 15
FIGURE 15
The relationship between KIFC3 and thePI3K/AKT/mTOR pathway.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Almeida A. C., Maiato H. (2018). Chromokinesins. Curr. Biol. 28 (19), R1131–r1135. 10.1016/j.cub.2018.07.017 - DOI - PMC - PubMed
    1. Ashar Y. V., Zhou J., Gupta P., Teng Q.-X., Lei Z.-N., Reznik S. E., et al. (2020). BMS-599626, a Highly Selective Pan-HER Kinase Inhibitor, Antagonizes ABCG2-Mediated Drug Resistance. Cancers 12 (9), 2502. 10.3390/cancers12092502 - DOI - PMC - PubMed
    1. Chen X., Wei B., Han X., Zheng Z., Huang J., Liu J., et al. (2014). LGR5 Is Required for the Maintenance of Spheroid-Derived Colon Cancer Stem Cells. Int. J. Mol. Med. 34 (1), 35–42. 10.3892/ijmm.2014.1752 - DOI - PMC - PubMed
    1. De S., Cipriano R., Jackson M. W., Stark G. R. (2009). Overexpression of Kinesins Mediates Docetaxel Resistance in Breast Cancer Cells. Cancer Res. 69 (20), 8035–8042. 10.1158/0008-5472.Can-09-1224 - DOI - PubMed
    1. Dekker E., Tanis P. J., Vleugels J. L. A., Kasi P. M., Wallace M. B. (2019). Colorectal Cancer. Lancet 394 (10207), 1467–1480. 10.1016/s0140-6736(19)32319-0 - DOI - PubMed