CDKL1 promotes tumor proliferation and invasion in colorectal cancer

Figures & data

Figure 1 Representative micrographs of the staining patterns of CDKL1 in (A–D) CRC and (E–G) normal mucosa.

Notes: Original magnification: upper rows ×200, lower rows ×400. CDKL1 expression was noted mainly in the nucleus of tumor cells. Positive cells were stained brown. (A) High-intensity CDKL1 expression was scored as “+++”. (B) Moderate-intensity CDKL1 expression was scored as “++”. (C) Low-intensity CDKL1 expression was scored as “+”. (D) The near absence of CDKL1 staining was scored as “−”. (E–G) Moderate, low, and negative expressions of CDKL1 in normal mucosa.
Abbreviations: CDKL1, cyclin-dependent kinase-like 1; CRC, colorectal cancer.

Figure 2 Infection of CRC cells by CDKL1-shRNA lentivirus.

Notes: (A) Analysis of CDKL1 protein in 8 CRC cell lines by Western blot assay. (B) The CDKL1-shRNA or nonsense sequence was constructed and packaged within lentivirus. HCT116 and Caco2 cells were infected with CDKL1-shRNA lentivirus or control-shRNA lentivirus for 3 days and observed under fluorescence microscope in fluorescent field (left panel), bright field (middle panel), and merged picture (right panel) with ×200 magnification. (C) CDKL1 expression was detected by Western blot in the shRNA-transduced cells. CDKL1 protein was significantly downregulated in CDKL1-shRNA lentivirus-infected cells. Error bar denotes mean ± SD. ^*P<0.05.
Abbreviations: CDKL1, cyclin-dependent kinase-like 1; CRC, colorectal cancer; shRNA, small hairpin RNA; SD, standard deviation.

Table 1 Expression of CDKL1 in 100 cases of colorectal cancer patients

Characteristics	CDKL1 immunostaining				Positive rate (%)	P-value*
	−	+	++	+++
Normal mucosa	36	42	22	0	64.0
Cancer	18	33	42	7	82.0	0.000

Notes:

* The Wilcoxon test was used to determine statistical significance between the 2 groups. −, negative; +, slightly positive; ++, moderately positive; +++, strongly positive.

Abbreviation: CDKL1, cyclin-dependent kinase-like 1.

Figure 3 The effect of CDKL1 knockdown on the proliferation, colony formation, and invasion of colorectal cancer cells in vitro.

Notes: (A, B) Analysis of cell viability of CDKL1-shRNA-transfected cells for 2 days by the CCK-8 assay. The proliferation of both (A) HCT116 and (B) Caco2 cells was significantly inhibited after CDKL1 knockdown. (C, D) Colony formation in lentivirus-infected cells was assayed 2 weeks after culture. The statistical results of colony number showed that the colony-forming ability was impaired in CDKL1-shRNA lentivirus-infected (C) HCT116 and (D) Caco2 cells. (E, F) Representative images are shown for CDKL1-shRNA-transfected HCT116 and Caco2 cells, respectively, in a transwell assay. Photomicrographs of stained migrating cells were captured at ×200 magnification. The migration was quantified by counting the cells in 4 random microscopic fields. (G, H) The effect of CDKL1 knockdown on the cell cycle profile of HCT116 and Caco2 cells by FACS. The percentages of cells in the G1, G2, and S phases are shown. (I, J) Lentivirus-infected HCT116 and Caco2 cells were cytometrically analyzed for positive Annexin-V-APC and PI staining. The dual parameter dot plot of APC vs PI is shown in logarithmic fluorescence intensity. Q2 and Q3 quadrants are regarded as late and early apoptotic cells, respectively. ^*P<0.05; ^**P<0.01.
Abbreviations: CDKL1, cyclin-dependent kinase-like 1; CRC, colorectal cancer; FACS, fluorescence-activated cell sorting; OD, optical density; PI, propidium iodide; shRNA, small hairpin RNA.

Figure 4 CDKL1 knockdown inhibits colon tumorigenesis in vivo.

Notes: (A, B) Representative images of the tumors (arrows) formed in BALB/c mice 4 weeks after inoculation of control-shRNA- and CDKL1-shRNA-transfected HCT116 and Caco2 cells. (C, D) The average tumor volume assessed weekly after the inoculation of cells. ^**P<0.01.
Abbreviations: CDKL1, cyclin-dependent kinase-like 1; CRC, colorectal cancer; shRNA, small hairpin RNA.

Figure 5 Microarray analysis between control-shRNA and CDKL1-shRNA-transduced CRC cells in the parameter of |FC|>2 and P-value <0.01 and confirmation of microarray results using Western blotting.

Notes: (A) Volcano plot. (B) Scatter illustration. (C) Cluster figure. (D) Representative Western blot images of P15, CDK4, CDK6, cyclinD1, tubulin, Rb, Rbser807/811, and Rbser780 in HCT116 and Caco2 cells transduced with control-shRNA and CDKL1-shRNA. Tubulin and LaminB1 were used as loading controls.
Abbreviations: CDKL1, cyclin-dependent kinase-like 1; CRC, colorectal cancer; shRNA, small hairpin RNA; |FC|, fold change.

Table 2 Pathway and GO analysis of potential gene sets or pathways involved in the pathogenic mechanisms in CDKL1-knockdown CRC cells

Gene set name	Number	P-value	Genes
GO analysis
Biological process
Regulation of TGF-β receptor signaling	2	3.90E-03	CDKN2B, EID2
Cellular defense response	2	5.87E-02	VEZF1, LY96
Enzyme linked receptor protein signaling	3	6.89E-02	CDKN2B, EID2, FIBP
Secretory pathway	2	1.11E-01	LMAN1, CADPS
Regulation of signal transduction	3	1.91E-01	CDKN2B, EID2, GOLT1B
Molecular function
RNA polymerase II transcription factor activity	3	1.73E-01	JUN, VEZF1, TEF
Enzyme inhibitor activity	2	2.42E-01	APP, CDKN2B
Cellular component
Chromosome	2	2.60E-01	TMPO, JUN
Membrane fraction	4	2.84E-01	FIBP, SLC16A3, SLC39A1, SSR3
Pathway analysis
TOLL pathway	2	3.14E-02	JUN, LY96
MAPK pathway	2	7.07E-02	JUN, DUSP1
Pathogenic Escherichia coli infection	2	7.28E-02	LY96, CLDN1
Spliceosome	2	2.52E-01	PRPF19, RBM22

Abbreviations: CDKL1, Cyclin-dependent kinase-like 1; CRC, colorectal cancer; GO, gene ontology; TGF, transforming growth factor.