MiR-543 Promotes Proliferation,Metastasis And Stem-like Traits Of Prostate Cancer Cells Through Targeting Numb

Background:More and more studies have shown that microRNAs(miRNAs,miRs)have become an important post-transcriptional regulators in various cancers,and it played an important role in the occurrence,development and metastasis of cancers.miR-543 has been reported to play an oncogenic role in prostate cancer by promoting the growth of prostate cancer.However,the regulation of proliferation,metastasis and stem-like traits of prostate cancer cells by miR-543 and its specific mechanisms have not been elucidated.Objective:To explore the mechanism of miR-543 promoting metastasis and maintaining the stem-like characteristics of prostate cancer cells.It is expected to reveal the molecular biological mechanism of tumor recurrence and metastasis,and to find a new method for the treatment of tumor metastasis and recurrence as soon as possible.Methods:The expression levels of miR-543 in CD44~+PC3,CD44~-PC3,CD44~+Du145and CD44~-Du145 cells were detected by fluorescence quantitative PCR.Mimic,mimic nc,inhibitor and inhibitor nc of miR-543 were transfected into DU145 and PC3 cell lines respectively.Real-time PCR was used to detect the expression levels of miR-543and CSC related markers.Transwell invasion assay was used to detect cell invasiveness.Colony formation assay was used to detect cell proliferation ability.Sphere formation assay and clonogenic assay were used to detect the stem-like characteristics of prostate cancer.Western Blot was used to detect the expression of Numb and cancer stem cell related proteins.After transfection of siRNA of Numb in CD44~-PC3 cells or transfection of Numb overexpression plasmid in CD44~+PC3 cells,clonal,sphere formation and transwell assays were used to detect self-renewal and invasiveness of prostate cancer cells.Luciferase reporter assays validated that Numb is the target gene of miR-543.At the same time,we constructed an over-expression plasmid containing all Numb cDNA sequences but without Numb 3'UTR sequences,and then co-transfected Numb and miR-543 mimic in PC3 cells.PC3 cells and Du145 cells transfected with miR-543 were inoculated subcutaneously into immunodeficient BALB/C mice to observe their tumorigenicity.Meanwhile,PC3 cells transfected with miR-543 were injected into caudal vein of mice to detect lung metastases.Results:The levels of miR-543 in CD44 positive PC3 and Du145 cells were significantly higher than those in CD44 negative PC3 and Du145 cells.The expression of miR-543 in prostate cancer cells after overexpression of miR-543 was significantly higher than that in the control group,while the expression of Numb was lower than that in the control group.The number of cell invasion,clones,sphere fromation in the miR-543 overexpression group was significantly higher than that in the control group.Prostate cancer cells transfected with inhibitors showed decreased expression of miR-543,but the expression of Numb increased.Moreover,the ability of cell invasion,3-D matrigel clone formation,sphere formation were significantly decreased.Western Blot results showed that the expression level of Numb was low when miR-543overexpression.When we knocked down Numb in CD44~-PC3 cells,the ability of cell invasion and colony formation increased.Luciferase reporter assays in 293T cells showed that miR-543 could inhibit the luciferase activity of wild-type Numb 3'UTR reporter gene vector,but had no effect on the luciferase activity of mutant Numb 3'UTR.This result was consistent with our prediction of the target gene of miR-543 by software.The results of co-transfection of PC3 cells with Numb and miR-543 showed that overexpression of Numb could reverse the carcinogenic effect of miR-543 in prostate cancer.Overexpression of miR-543 promoted subcutaneous tumorigenesis in BALB/C mice,while miR-543 knocked down in prostate cells could inhibite subcutaneous tumorigenesis in mice.The lung metastasis ability of mice increased after overexpression of miR-543.Conclusion:miR-543 could promote the occurrence and metastasis of prostate cancer by directly and negatively regulating Numb,thus positively regulating proliferation,metastasis and stem-like characteristics of prostate cancer cells.Targeting miR-543may become a potential target for the treatment of prostate cancer metastasis and relapse.