Multifaceted Function of MicroRNA-299-3p Fosters an Antitumor Environment Through Modulation of Androgen Receptor and VEGFA Signaling Pathways in Prostate Cancer

Prostate cancer (PCa) is one of the most common cancers to affect men worldwide. Androgen receptor (AR) signaling is central to PCa and PCa therapy. MicroRNAs (miRNAs) play crucial roles in the regulation of prostate cancer through modulation of signaling pathways. In the present study, we illustrate the functional significance and therapeutic benefit of miR-299-3p, an AR targeting microRNA, in PCa progression. We noted loss of expression of miR-299-3p in prostate tumors compared to noncancerous prostate tissues. Replenishment of miR-299-3p in C4-2B, 22Rv-1 and PC-3 cells contributed to cell cycle arrest, reduced proliferation, migration and increased expression of apoptotic markers. Additionally, overexpression of miR-299-3p induced a reduction of AR, PSA and VEGFA expression. AGO-RNA pulldown experiment showed enrichment of AR, VEGFA and miR-299-3p in C4-2B cells overexpressing miR-299-3p. miR-299-3p overexpression also inhibited epithelial mesenchymal transition, expression of Slug, TGF-β3, phospho-AKT and phospho-PRAS40, but increased expression of E-cadherin. Furthermore, miR-299 overexpression resulted in reduced tumor growth in xenograft models and increased drug sensitivity. Overall, this study has identified novel mechanisms of antitumor and antimigration function of miR-299-3p through modulation of AR and VEGFA signaling pathways which lead to improved drug sensitivity of PCa.


Preparation of inducible pLVX-TetOne-miR-299 construct:
For ectopic expression, a doxycycline-inducible construct encompassing human miR-299 precursor sequence obtained from OriGene (MI0000744) was prepared and cloned into the pLVX-TetOne-Puro vector (Clontech) at the BamH1 site, harboring a ZsGreen fluorescent marker between BamHI and EcoRI restriction sites.

Cell cycle and apoptosis analysis
C4-2B-299 and 22Rv-1-299 sublines and transfected PC-3 cells expressing miR-299 or scrambled RNA were induced with doxycycline and used for Propidium iodide staining. Cells were harvested at 48hr (PC-3) and 72hr (C4-2B-299 and 22Rv-1-299) post induction and fixed with cold 100% methanol. Cells were treated with 100μg/mL RNAse in 2% Bovine Serum Albumin (BSA) containing PBS. Cell were incubated at room temperature for 15 min and stained with 250μg/mL Propidium iodide in 2% BSA for 1hr at room temperature. Fluorescent cells were detected in CytoFlex S (Beckman Coulter) Flow Cytometer and analyzed using FlowJo software. For apoptosis assays, cells were harvested using 1X Trypsin EDTA-free (Fisher Scientific) 24h post induction and stained with 7-AAD and annexin V antibodies. Stained cells were analyzed in a CytoFlex S (Beckman Coulter) Flow Cytometer. Percentage of apoptotic cells were quantified using FlowJo software.

Scratch assay
Cells were induced with doxycycline and scratches were made with a pipet tip 24hr post induction. Cells were rinsed with PBS and incubated in media containing 5% FBS at 37°C. Migration of cells within the scratch was observed at the specified time 0 and 14 hrs for PC-3 cells and at 0 and 24hrs for C4-2B-299 and 22Rv-1-299 cells. Images were taken at 5X magnification using a Leica DMi8 inverted microscope and Leica LAS X software. ImageJ software was used to quantify the average width of each scratch.
Analysis of cell migration was determined by subtracting the width of the scratch at the 14 and 24hrs from the width of the scratch at 0hr followed by analysis of the ratio of distance migrated by each cell line samples.

Western blot analysis
Whole cell lysates from induced and uninduced C4-2B-299 and 22Rv-1-299 cells and PC-3 cells expressing miR-299 or scrambled RNA were prepared at 48hrs and 72hrs post induction with doxycycline. Similarly, PC-3 cells expressing miR-299 or scrambled RNA at 24 hours post induction with doxycycline were treated with 25nM DTX. After 48 hours of treatment whole cell lysates were prepared. Fifty g of extracted proteins was separated in a 10% Bis-Tris gel and transferred to activated PVDF membranes. Immunoblotting was performed with primary antibodies specific for AR (LSBio), VEGFA (Santa Cruz Biotechnology) and GAPDH (Sigma Aldrich). Antibodies for -tubulin, Slug, Vimentin, E-Cadherin, Cyclin D1, Cyclin B1, pan-Akt, phospho-Akt, PRAS40 and phospho-PRAS40 were obtained from Cell Signaling.

Immunohistochemistry
Briefly, 5M sections of tumor tissues were deparaffinized and rehydrated using xylene and ethanol.
Next, antigen retrieval was performed by boiling the slides immersed in 1X TE buffer for 13 minutes followed by cooling the slides for 20 minutes. Then Ki67 rabbit monoclonal antibodies conjugated with Alexa (R) 488 (Cell Signaling) and mouse monoclonal cytokeratin 8 antibodies (Santa Cruz Biotechnology) were used as primary antibodies. Anti-mouse antibodies conjugated with Texas Red Dye (Vector Laboratories) was used as the secondary antibodies and slides were mounted using DAPI Fluoromount-G (SouthernBiotech). All slides were imaged using the Leica SP5 confocal microscope Percentage of Ki67 positive cells were then quantified using ImageJ software.

E-cadherin
The blot was re-probed for E-cadherin  The blot was re-probed for E-cadherin