Tumor-suppressive MicroRNA-497 Targets IKKβ To Regulate NF-κB Signaling Pathway In Human Prostate Cancer Cells

Objective: Prostate cancer(PCa)is one of the most prevalent malignant tumors,PCa-related death is mainly due to the high probability of metastasis.MicroRNAs(miRNAs)play an important role in cancer initiation,progression and metastasis by regulating their target genes.The aim of this research is to explore the possible mechanisms of tumor-suppressive microRNA-497 targeting inhibitors of NF-κB(IκBs)kinase β(IKKβ)to regulate NF-κB signaling pathway in human prostate cancer cells,in order to verify that targeting this miRNA may have significant therapeutic potential.Methods: Real-time PCR was used to detect the expression of microRNA-497 in 40 pairs of serum samples collected from patients with PCa and healthy control subjects.The molecular biological function was investigated by using cell proliferation assays,cell cycle assay,and migration and invasion assay after transfected with miR-497 and anti-miR-497,knockdown of IKKβ,IMD-0354(a novel IKK-β inhibitor).We used several Algorithms and confirmed that IKKβ is directly regulated by miR-497 using luciferase reporter assay and western blot analysis.Western blot analysis was used to detect the expression of CDK8、MMP9 and PSA after transfected with miR-497 and anti-miR-497,knockdown of IKKβ,IMD-0354.Results: Here,qRT-PCR analysis showed that the expression level of miR-497 was downregulated in PCa serum samples,compared with samples from healthy control subjects.The CCK-8 assay showed that miR-497 mimics significantly inhibited the proliferation of PC3-AR cells at 48 and 72 h,respectively.Conversely,anti-miR-497 transfection in PC3-AR cells could promote cell proliferation.Cell cycle analysis found that overexpression of miR-497 resulted in S and G0/G1 phase cell cycle arrest in PC3-AR cells after 24 and 48 h of exposure,respectively.On the contrary,anti-miR-497 transfection suppressed the effect.Transwell assays illustrated that the forced expression of miR-497 resulted in a decrease in the migratory and invasion ability of PC3-AR cells when compared to the control cells.Subsequently,IKKβ is confirmed as a target of miR-497.Furthermore,knockdown of IKKβ expression resulted in decreased proliferation,migration and invasion activity.Finally,similar results were found after treatment with a novel IKK-β inhibitor(IMD-0354)in PC3-AR cells.CDK8,MMP9,and PSA were involved in all these process.Conclusions: The consistent decrease in miR-497 in the human prostate tumor tissues and blood samples decreases the feasibility of using them as a signature for the progression of PCa.Furthermore,considering the inhibitory effect of cell proliferation,migration,and invasion with miR-497,siR-IKKβ,and IMD-0354,it can be concluded that miR-497 participates in the development and progression of human PCa by targeting the IKKβ-mediated NF-κB/MMP9/PSA signaling pathway.In addition,miR-497 is likely to be a new diagnostic marker to partly replace or complement PSA in PCa.Taken together,all these results suggest that targeting the miR-497/IKKβ/NF-κB interaction or perturbing the expression of miR-497 may prove to be a new insight into prevention,diagnosis,and treatment of patients with PCa.