The promoter region (from −632 to +12 nucleotides) of the PSA gene was amplified from the genome of 293 cells by PCR and inserted into the dCas9-KRAB plasmid, which was a gift from Dr. George Church’s lab. The sgRNA targeting and inhibiting the open reading frame (ORF) region of the human PSA gene was designed by CRISPR-ERA online software, and its sequence is: 5′-GCTCCCAGCTGCTTTACTAA-3′.

Prostate cancer cell lines (LNCap, PC3, AT3B-1, and DU145) and the normal prostate tissue cell line (RWPE1) were purchased from the cell bank of Chinese academy of science, Shanghai. Cells were all cultured in medium RPMI-1640 supplemented with 20% fetal bovine serum (FBS).

The synthesized luciferase reporter plamid was transiently transfected into cells (LNCap, PC3, AT3B-1, DU145, and RWPE1). The whole cell lysate was collected 24 h after transfection and the luciferase activities were measured by the luciferase reporter assay kit (Invitrogen). For data normalization, one reporter generally functions as an experimental reporter and the second as an internal control to account for non-specific experimental variations.

Total RNA was isolated from cells using Trizol agent (Invitrogen) and then reversely transcribed to synthesize cDNA template. Next, quantitative reverse transcription PCR (qRT–PCR) was performed by using a cDNA template and specific PCR primers. The change of the fluorescent signal was used to detect the change in the amount of the amplification product in each cycle of the PCR amplification reaction in real time, and finally the starting template was accurately quantitatively analyzed.

The MTT assay was used for determining cell proliferation 48 h after transfection according to the supplier’s instructions. Prostate cancer cells in the logarithmic growth phase were cultured in 96-well plates, and 5000 cells were seeded per well. The light absorption value was measured with an enzyme-linked immunosorbent assay (ELISA) at 570 nm wavelength, which indirectly reflects the number of living cells.

For colony formation assay, cells were seeded into each well of a six-well plate and incubated for 2 weeks. Cell colonies were fixed with 4% PFA and then stained with 1% crystal violet for 15 min. Finally, the plate was rinsed gently with distilled water. A BX51 microscope (Olympus, Tokyo, Japan) was used to count stained colonies.

Migration of human prostate cancer cell lines was assessed using a cell scratch assay. A marker was used to draw two parallel lines on the back of the six-well plate before cell seeding, and cells are seeded into the six-well plate after digestion. When the cells cover the bottom of the plate, a 10 μl pipette tip was used to gently draw lines on the plate, and the width of each scratch should be as close to the same as possible. After rinsing the plate three times with phosphate-buffered saline (PBS) buffer to remove cell debris from scratches, the cells were photographed. Finally, pictures were collected for analysis, and migration rate was quantified by dividing the change in wound width by the time spent in migration.

Transwell migration assay was performed by using uncoated membranes of Transwell chambers. Briefly, cells were suspended in serum-free medium. A total of 200 µl of cell suspension was added to the upper chamber; 700 µl of culture medium (10% FBS) was added to the lower chamber. Next, cells were grown in a 37 °C incubator. After 24 h, cells that passed through the membrane were fixed with 4% PFA and then stained with 1% crystal violet to remove cells on the other side of the membrane. Stained cells were counted in five random fields under a BX51 microscope.

Cell apoptosis was determined by ELISA, which is based on a fluorescent immunosorbent enzyme reaction, using the Caspase-3 Activity Assay kit (Beyotime Shanghai, China). Briefly, whole cell lysates were added to 96-well plates and incubated with 2 mM caspase-3 substrate for 4 h at 37 °C. Then, the absorbance was determined at 405 nm using a microplate reader. Apoptosis was also detected using Annexin V-PI Apoptosis Detection Kit (A211, Vazyme, Nanjing, China). Transfected prostate cancer cells were harvested and washed with PBS, then cells were resuspended in 100 µl of Annevix Binding Buffer and incubated with 5 µl of Annevix FITC and 5 µl of propidium iodide (PI) for 15 min. Finally, apoptosis was detected after adding 150 µl of Annevix Binding Buffer.

Data are shown as the mean ± one standard deviation (SD) and analyzed by a t-test. A P < 0.05 was considered statistically significant. All the processes of statistical analysis were performed using IBM SPSS Statistics Version 20 software.