microRNA-372 Suppresses Migration and Invasion by Targeting p65 in Human Prostate Cancer Cells

Abstract

Prostate cancer (PCa) is one of the most prevalent malignant tumors. microRNAs (miRNAs) play an important role in cancer initiation, progression, and metastasis, and their roles in PCa are becoming more apparent. In this study, we found that microRNA-372 (miR-372) is downregulated in human PCa and inhibits the proliferation activity, migration, and invasion of DU145 cells. Subsequently, p65 is confirmed as a target of miR-372, and knockdown of p65 expression similarly resulted in decreased proliferation activity, migration, and invasion. CDK8, MMP-9, and prostate-specific antigen were involved in both these processes. Taken together, our results show evidence that miR-372 may function as a tumor suppressor gene by regulating p65 in PCa and may provide a strategy for blocking PCa metastasis.

Keywords: PCa; invasion; miR-372; migration; p65.

FIG. 1.

The expression of miR-372 in PCa and the proliferation assay in DU145 cells. (A) The relative expression of miR-372 in 20 pairs of serum samples from patients with PCa and healthy control subjects was analyzed by qRT-PCR. (B) The expression level of miR-372 in a panel of human prostate cancer cells LNCaP, PC-3, DU145 cell, and normal prostate epithelial cells RWPE-1 cell. (C) CCK-8 assay of DU145 cell transfection with miR-372, miR-NC, anti-miR-372, siR-P65, siR-NC, and control. (*p < 0.05). PCa, prostate cancer; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

FIG. 2.

(A) Cell cycle analysis showed that there was significant cell cycle transition in DU145 cells after 24 and 48 h of treatment with miR-372, NC, anti-miR-372, and siR-p65. (B) The colony formation assay was conducted to determine the colony-forming growth of cells, and the colonies were captured on the 14th day after seeding (*p < 0.05).

FIG. 3.

miR-372 suppresses the migration and invasion of DU145 cells. (A) The migratory activity of DU145 cells was detected using a transwell migration assay treated with miR-372, anti-miR-372, control, and siR-p65. Representative images are shown on the right; the values shown are expressed as mean ± standard deviation. (B) The invasive activity of DU145 cells was detected using a transwell invasion assay. (*p < 0.05).

FIG. 4.

p65 is a target gene of miR-372 in DU145 cells. (A) Western blot analysis was used to detect the expression of p65, CDK8, PSA, and MMP9 in DU145 cells after treatment with miR-497, siR-p65, and control. The expression of p65 and IKKβ was also detected after transfection with siR-p65 and siR-IKKβ to clear the relationship between P65 and IKKβ in PCa. β-actin served as an internal control. (B) Relative luciferase activity of the p65 WT-UTR and the DEL-UTR luciferase constructs in DU145 cells transfected with miR-372 or control (*p < 0.05). (C) p65 was predicted to be a candidate target of miR-372 using the bioinformatics algorithm TargetScan. PSA, prostate-specific antigen.