HOXD13 knockdown attenuates apoptosis in prostate cancer via the upregulation of MDM2/p53 signaling

Abstract

Background: Prostate cancer (PCa) is a common leading cause of death worldwide and is recognized as the second most frequently diagnosed cancer in the male population, making it a significant focus of long-term oncological research. The HOXD13 gene, belonging to the HOX gene family, has been associated with various malignancies, including breast and colon cancer, and is associated with prognostic outcomes. However, its specific role in prostate cancer remains to be elucidated.

Methods: The correlation of HOXD13 expression in PCa was analyzed by UALCAN database. qRT-PCR and western blot assays were employed to assess the expression levels of HOXD13 in several prostate cancer cell lines. Construct siRNA and overexpression plasmids for HOXD13 and transfect them into cells. Western blot was used to assess the knockdown efficiency of HOXD13 in the cells. The influence of HOXD13 expression on cell proliferation in PC-3 M and C4-2 cells was assessed by EDU staining. The role of HOXD13 in cell migration and invasion of prostate cancer cells was detected by wound healing and transwell assay. Western blot analysis was performed to examine apoptosis-related proteins (C-caspase3, Bax, and Bcl-2) in PC-3 M cells. The co-immunoprecipitation (Co-IP) assay evaluates the interaction between MDM2 and p53.

Results: An examination of the data obtained from the UALCAN database reveals that HOXD13 was significantly reduced in prostate cancer tissues and correlated with patient survival. In prostate cancer cell lines, notably PC-3M, HOXD13 expression was markedly reduced. Knockdown of HOXD13 promotes PC-3 M and C4-2 cells proliferation, migration, and invasion. However, the elevated expression of HOXD13 hampers PC-3 M and C4-2 cells proliferation, migration, and invasion. Furthermore, knockdown of HOXD13 in PC-3 M cells resulted in decreased levels of apoptosis markers, including C-caspase-3 and Bax. Co-immunoprecipitation assays demonstrated that HOXD13 depletion enhanced the association between MDM2 and p53.

Conclusion: HOXD13 was a critical regulator in the pathogenesis of prostate cancer, modulating the MDM2/p53 signaling pathway to promote apoptosis.

Keywords: HOXD13; Apoptosis; MDM2; Prostate cancer; p53.

Fig. 1

HOXD13 is low expression in prostate cancer and is related to the outcome of the disease. A Analysis of data from UALCAN revealed that the expression of HOXD13 was markedly reduced in tumor tissues in comparison to normal control tissues. B The survival analysis of prostate cancer patients was conducted in relation to the expression levels of HOXD13

Fig. 2

HOXD13 represses cell proliferation in PC-3 M and C4-2 cells. AHOXD13 expression in normal prostate epithelial cells RWPE-1 and different prostate cancer cell lines DU-145, PC-3 M, and C4-2. B, C Western blot was conducted to assess the HOXD13 overexpression or knockdown efficiency in PC-3 M and C4-2 cells. D, E Knockdown or overexpression of HOXD13 in PC-3 M and C4-2 cells, followed by EDU staining to assess cell proliferation. DAPI stains the nucleus. *P < 0.05 indicated a statistically significant difference; oe, overexpression; si, small interfering RNA

Fig. 3

HOXD13 suppresses cell migration and invasion in PC-3 M cells. AHOXD13 silencing promoted the rate of cell migration. B Overexpression of HOXD13 inhibited the velocity of cell migration. C Knockdown of HOXD13 promoted the rate of cell invasion. D Overexpression of HOXD13 inhibited the rate of cell invasion. *P < 0.05 indicated a statistically significant difference. oe, overexpression; si, small interfering RNA

Fig. 4

HOXD13 suppresses cell migration and invasion in C4-2 cells. AHOXD13 silencing promoted the rate of cell migration. B Overexpression of HOXD13 inhibited the velocity of cell migration. C Knockdown of HOXD13 promoted the rate of cell invasion. D Overexpression of HOXD13 inhibited the rate of cell invasion. *P < 0.05 indicated a statistically significant difference. oe, overexpression; si, small interfering RNA

Fig. 5

HOXD13 accelerated the apoptosis in PC-3 M cells. A, B Following the knockdown or overexpression of HOXD13, a TUNEL assay was conducted to assess apoptotic cell death. C, D Western blot analysis was employed to investigate the expression levels of apoptosis-related proteins, specifically cleaved caspase-3, Bax, and Bcl-2 after knockdown or overexpression of HOXD13. *P < 0.05 indicated a statistically significant difference; oe, overexpression; si, small interfering RNA

Fig. 6

HOXD13 promotes its tumor inhibition function by destroying the interaction between p53 and MDM2. A, B After knockdown or overexpression of HOXD13, the expression and distribution of p53 in the cells. C Co-immunoprecipitation was employed to identify the interaction between MDM2 and p53 after HOXD13 knockdown in PC-3M. D quantitative analysis of p53. E After the addition of the proteasome inhibitor MG-132, Western blot analysis was conducted to assess alterations in p53 protein levels within the cells. *P < 0.05 indicated a statistically significant difference; si, small interfering RNA