Androgen receptor modulatory miR-1271-5p can promote hormone sensitive prostate cancer cell growth

In most patients with advanced prostate cancer treated with hormonal therapy, androgen independence eventually emerges, leading to death. Androgen receptor signalling remains an important prostate cancer driver, even in the advanced disease stage. MicroRNAs (miRs), non-coding RNAs that regulate gene expression by inhibiting translation and/or promoting degradation of target mRNAs, can act as tumour suppressors or “oncomiRs” and modulate tumour growth. Because of their stability in tissues and in circulation, and their specificity, microRNAs have emerged as potential biomarkers, as well as therapeutic targets in cancer. We identified miR-1271–5p as an androgen receptor modulatory microRNA and we show it can promote hormone sensitive prostate cancer cell growth. Inhibition or overexpression of miR-1271–5p levels affects prostate cancer cell growth, apoptosis and expression of both androgen receptor target genes and other genes that are likely direct targets, dependent on androgen receptor status, and tumour stage. We conclude that miR-1271–5p has the potential to drive progression of hormone-dependent disease and that the use of specific inhibitors of miR-1271–5p may have therapeutic potential in prostate cancer.

of the microwave.A container was also placed in the microwave, partially filled with water next to the container hosting the slides.Slides were microwaved at high (100%) power for 4mins (or until boiling) and the power was then reduced to a medium level (50%) for an additional period of 10mins.The containers were removed afterwards from the microwave and were allowed to cool down for 15-20min.The slides were then washed twice for 2-5min in PBS.Endogenous peroxidase was blocked, using 3% hydrogen peroxide in PBS for 5 min.
Slides were washed in PBS and then blocked with milk for 20min in a humid chamber.The primary antibody was placed on top of the slides, after being diluted, in order to reach the required concentration in the final blocking solution.The blocking solution was carefully placed drop-wise on top of the slides in a humid chamber and was incubated overnight at 4°C.
The following day, the slides were washed 3 times, 5min per wash, in PBS.The secondary antibody, provided by the kit (Immunostain kit, Invitrogen, Life Technologies, as per manufacturer), was then placed on top of the slides.The tissue was incubated at RT, for 1h.
The slides were washed again 3 times, 5min each, in PBS.Streptavidin was added drop-wise on top of the tissue slides with an incubation time of 10-15min in RT, as per manufacturer's instructions.Slides were further washed 3 times in PBS, 5min each.Antibodies were possible to be visualised using DAB+, as recommended by the manufacturer, after being stained for up to 10min, with constant monitoring, since excessive time of exposure to DAB+ could lead to overstaining.Slides were washed again three times, 5min each, in PBS.Further counterstaining was conducted with the use of Haematoxylin, for 10-30sec.Slides were rinsed with tap water and further dehydrated through graded dilutions of Ethanol: 50%, 70%, 95% twice and 100% twice as well, 5min per wash.Finally, the slides were immersed in Xylene/Histoclear to avoid any remaining water.Slides were covered and were left to set (refer to standard protocol)
RT reaction mix included the following reagents per sample: 2ul 5x Reaction Buffer, 1ul Enzyme mix, 0.5ul UniSp6 spike-in, 4.5ul water.The thermal cycle for the RT was set at 42°C for 60min (Polyadenylation-cDNA synthesis step) and 95°C for 5min (for Heat inactivation of the RTase).cDNAs were further diluted 80x in water + 1:20 ROX.The final concentration of ROX in each well was at 500nM (diluted 1:50).cDNAs were amplified using ExiLENT SYBR Green master mix (Exiquon) with the 7900HT RT-PCR System (Applied Biosystems, MA, USA).PCR parameters were set at 95°C for 10min, followed by 40 cycles at 95°C for 1min and 60°C for 1min.A melting curve analysis was also performed at the end of the 40 th cycle.Data were recorded using the Sequence Detection System.All data were analysed using the ΔΔCt method.Endogenous levels of miRs on the cell lines were normalised to the geomean of control primers: U6 and SNORD48.