HOXB2 increases the proliferation and invasiveness of colon cancer cells through the upregulation of CCT6A

Abstract

Colon cancer has a high mortality rate, thus there is an urgent need to develop novel therapeutic options for clinical management of the disease. Studies have revealed that chaperonin containing TCP1 subunit 6A (CCT6A) promoted the development of multiple types of cancer, and dataset analysis revealed that homeobox B2 (HOXB2) has the potential to modulate the expression of CCT6A. However, whether HOXB2 affects the proliferation, migration and invasion of colon cancer cells remains to be determined. A CCT6A knockdown colon cancer cell line was established and colony formation, wound healing and Transwell invasion assays were performed to assess proliferation, migration and invasion of the altered colon cancer cells. Subsequently, luciferase reporter gene assays and chromatin immunoprecipitation assays were performed to detect the relationship between HOXB2 and CCT6A. A HOXB2 overexpression colon cancer cell line was established and the proliferation, migration and invasion of these cells was determined using the same methods. Knockdown of CCT6A reduced the proliferation, migration and invasion of colon cancer cells. HOXB2 enhanced the expression of CCT6A in colon cancer cells by binding to the promoter of CCT6A. Overexpression of HOXB2 abolished the inhibitory effect of CCT6A knockdown on the proliferation, migration and invasion of colon cancer cells. HOXB2 increased the proliferation and invasiveness of colon cancer cells by increasing the expression of CCT6A.

Keywords: chaperonin containing TCP1 subunit 6A; colon cancer; homeobox B2; invasiveness.

Figure 1.

Expression of CCT6A is enhanced in the colon cancer cells. (A) The data of expression of CCT6A in colorectal cancer cells was obtained from GEPIA. (B) The data of survival rates of colon cancer patients and expression of CCT6A was obtained from GEPIA. (C) The data of the expression of CCT6A in colon cancer cells was obtained from the CCLE database. The expression of CCT6A in colon cancer cell was detected with (D) reverse transcription-quantitative PCR and (E) western blotting. *P<0.05, **P<0.01 and ***P<0.001. CCT6A, chaperonin containing TCP1 subunit 6A.

Figure 2.

Knockdown of CCT6A suppresses the proliferation of colon cancer cells. (A) Reverse transcription-quantitative PCR and (B) western blotting were performed to detect the expression of CCT6A in colon cancer cells. (C) The viability of colon cancer cells was detected with the Cell Counting Kit-8 assay. (D) The proliferation of colon cancer cells was detected with the colony formation assay. (E) Immunofluorescence was performed to detect the expression of Ki-67 in colon cancer cells. **P<0.01 and ***P<0.001. CCT6A, chaperonin containing TCP1 subunit 6A; shRNA, short hairpin RNA; NC, negative control.

Figure 3.

Inhibition of CCT6A restricts the migration and invasion of colon cancer cells. (A) Wound healing and (B) Transwell assays were performed to detect the migration and invasion of colon cancer cells. (C) Statistical analysis of cell invasion and migration. (D) Western blotting was performed to determine the expression of epithelial-mesenchymal transition-related proteins in colon cancer cells. **P<0.01 and ***P<0.001. CCT6A, chaperonin containing TCP1 subunit 6A; shRNA, short hairpin RNA; NC, negative control.

Figure 4.

HOXB2 activates the expression of CCT6A in colon cancer cells. (A and B) The binding sites between HOXB2 and CCT6A. The expression of HOXB2 in colon cancer cells was determined with (C) western blotting and (D) RT-qPCR. The expression of HOXB2 in colon cancer cells of the overexpression group was determined with (E) RT-qPCR and (F) western blotting. (G) A luciferase reporter assay was performed to detect the relationship between CCT6A and HOXB2. (H) Chromatin immunoprecipitation assays were performed to demonstrate the interaction between CCT6A and HOXB2. The expression of CCT6A in colon cancer cells was detected with (I) RT-qPCR and (J) western blotting. ***P<0.001. CCT6A, chaperonin containing TCP1 subunit 6A; RT-qPCR, reverse transcription-quantitative PCR; Ov, overexpressing; NC, negative control; WT, wild-type; MUT, mutant; shRNA, short hairpin RNA.

Figure 5.

Overexpression of HOXB2 rescues the proliferation of colon cancer cells. (A) Cell Counting Kit-8 was performed to detect the viability of colon cancer cells. (B) The proliferation of colon cancer cells was determined with the colony formation assay. (C) Immunofluorescence was performed to detect the expression of Ki-67 in colon cancer cells. *P<0.05, **P<0.01 and ***P<0.001. ^##P<0.01 vs. shRNA-CCT6A + Ov-NC. CCT6A, chaperonin containing TCP1 subunit 6A; shRNA, short hairpin RNA; Ov, overexpressing; NC, negative control.

Figure 6.

Overexpression of HOXB2 rescues the invasion of colon cancer cells. (A) Wound healing and (B) Transwell assays were performed to determine the migration and invasion of colon cancer cells. (C) Western blotting was used for the detection of the expression of epithelial-mesenchymal transition-related proteins in colon cancer cells. **P<0.01 and ***P<0.001. CCT6A, chaperonin containing TCP1 subunit 6A; shRNA, short hairpin RNA; Ov, overexpressing; NC, negative control.