Role Of Transcriptional Factor KLF4 In Regulation Of Prostate Cancer Proliferation And Migration

Objective: Prostate cancer(PCa)is one of the most common malignancies in the world and is a serious threat to male's life and health.In our previous work,we demonstrated that miR-7 targets KLF4 and inhibits the KLF4/PI3K/p21 pathway,leading to an attenuation of stemness in prostate cancer stem-like cells to inhibit the proliferation of PCa cells.As a pivotal stemness-associated transcription factor,KLF4 has been found to activate or repress the transcription of multiple genes including microRNAs.Considering our previous work together with other groups' work,in this study we attempt to explore whether and how KLF4 modulates the transcription of miR-7 and in turn regulates the proliferation of PCa cells.Methods: KLF4 stable knockdown or control subclone cell line was constructed in PCa cell line LNCaP and named LNCaP-shKLF4 and LNCaP-con respectively.Alang with PC3-shKLF4 vs PC3-con cells constructed in our previous work,real-time fluorescence quantitative PCR,chromatin immunoprecipitation(ChIP)and double luciferase reporter assay were used to detect the effect of KLF4 knockdown on the expression of miR-7 and epithelial-mesenchymal transition(EMT)associated genes.The effect of KLF4 knockdown or overexpression of mature miR-7 mimics on the proliferation and migration of PCa was verified by the cell proliferation assay,plate cloning assay,3D sphere formation assay and cell migration assay respectively.The co-immunoprecipitation(co-IP)was used to detect the interaction of nuclear translocated YAP with p72 and to determine whether this interation impaired p72 binding-dependent miR-7 processing.Differences between groups were analyzed using the student's t test.Statistical analysis and graphes were done using Prism GraphPad5 software.P < 0.05 is considered to be statistically significant.Results: In this study we identified that KLF4 promotes the transcription of miR-7 primary precursors in prostate cancer(PCa)cells.Considering together with our previous finding that miR-7 inhibits KLF4 expression at post-transcriptional level,we demonstrated a KLF4-miR-7 auto-regulatory feedback loop for mutual regulation of their expression.Interestingly,this homeostatic feedback loop is unbalanced due to impaired miR-7-processing in PCa cell lines,leading to decreased mature miR-7 production and KLF4 overexpression.Mechanistically,enhanced oncogenic YAP nuclear translocation mediates sequestration of p72,a co-factor of the Drosha/DGCR8 complex for pri-microRNA processing,leading to an attenuation of p72 binding-dependent miR-7 processing.The production of mature miR-7 can be recovered to inhibit tumor proliferation in PCa through exogenous overexpression of p72 to restore Drosha/p72/DGCR8 complex or transfection with mature miR-7 mimics to bypass its impaired processing.On the other hand,stable knockdown of KLF4 has a similar effect on inhibition of proliferation,EMT and cell migration in PCa cells in vitro.Conclusion: Unbalanced KLF4-miR-7 auto-regulatory feedback loop leads to the overexpression of KLF4 and the surppression of miR-7 in PCa cells.Stable knockdown of KLF4 or restoration of miR-7 can efficiently inhibit cell proliferation and migration in PCa cells in vitro.