LNCap cells were obtained from The Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM (HyClone, United States) added with 100 U/ml penicillin/streptomycin and 15% fetal bovine serum (FBS; Gibco, United States) under a moisturized atmosphere with a 5% CO₂ level.

The CRISPR-dCas9-VPR and CRISPR-dCas9-KRAB plasmids were ordered from Addgene (Cambridge, MA, United States). The sgRNAs targeting ITGB5, TIMP1, and TMEM176B were designed by the online software CHOPCHOP, a CRISPR/Cas9, and TALEN web tool for genome editing.

LNCap cells were seeded in 6-well plates with a 3 × 10⁶ cells per well density in the environment of DMEM. Followed the manufacturer’s protocol of Lipofectamine 2000 Transfection Kit (Thermo Fisher Scientific, Waltham, MA, United States), transfection was conducted. After that, the cells were set for 48 h in order for future analysis.

At 48 h after gene transfection, TRIzol (Invitrogen) was used as a separating reagent for total RNA to get the first-strand cDNA using a FastKing RT Kit (With gDNase) (Tiangen) following the official instructions. The qRT-PCR analysis was carried out by SYBR Green (Tiangen). GAPDH was taken as endogenous controllers for the three mRNAs.

LNCap cells were seeded in a 96-well plate at a 1 × 10⁵/ml dose and incubated for 24 h. Subsequently, cells were starved for another 2 h. Culture medium was replaced with 20 μL of methyl thiazolyl tetrazolium (MTT) solution (5 g/L) (Sigma-Aldrich, St. Louis, MO, United States) and incubated at 37°C for 4 h. The supernatant was discarded and 100 μL of DMSO (dimethyl sulfoxide) (Sigma-Aldrich, St. Louis, MO, United States) was put on to each well. The absorbance value was tracked at the wavelength of 570 nm with a microplate reader.

At 48 h after cell transfection, the apoptosis of LNCap cells was determined by the Caspase-3 Assay Kit (Fluorometric) (ab39383). Caspase-3 activity was detected by spectrophotometry and each assay was performed in triple.

LNCap cells were treated with 1.8 mmol/L hydroxyurea for 12 h to inhibit cell proliferation and then a 100 μL pipette tip was used to make cell scratches in a vertical well plate. Then the cell culture medium was discarded and washed three times with PBS. Then the cells were culture and taken pictures. The 0 and 24 h pictures were recorded, and the imageproplus 6.0 software was used to analyze and calculate the cell migration distance.

After 48 h of transfection, cells from each group were taken, trypsinized, and then inoculated in a matrigel-Tran-swell chamber. The serum-free DMEM medium was used for culture in the chamber. 10% FBS culture medium was added to the lower chamber. After 24 h, the upper uninvaded cells was wipped with a cotton swab. After fixing with 4% polymethanol for 15 min, cells were stained with 1% crystal violet for 5 min. After washing with PBS for three times, five fields under the fluorescence microscope were randomly selected and observed, and the invaded cells were counted and recorded.

The data were expressed as means ± standard deviations (SD). The SPSS version 19.0 software (IBM Corporation, United States) was used for statistical analysis. A p-value < 0.05 was considered statistically significant.

In order to clarify the overall expression patterns of the three genes in prostate cancer, we tested the expression of ITGB5 (Figure 1A), TIMP1 (Figure 1B), and TMEM176B (Figure 1C) in the prostate cancer LNCap cell line. We found that ITGB5 was significantly higher in prostate cancer compared with BPH cells, and that TMP1 and TMEM176B were significantly low expressed in prostate cancer. This suggests that ITGB5 is a potential proto-oncogene in the prostate cancer LNCap cell line, while TMP1 and TMEM176B are potential tumor suppressor genes.

In order to further clarify whether ITGB5 is a proto-oncogene and whether TIMP1 and TMEM176B are tumor suppressor genes, we used dCas9-KRAB-mediated CRISPRi technology (Figure 2A) to knock down the expression of ITGB5 (Figure 2B), and used dCas9-VPR-mediated CRISPRa technology (Figure 2A) to activate TIMP1 and TMEM176B expression (Figure 2B). The experimental results of MTT show that inhibiting the expression of ITGB5 can reduce cell proliferation, while activating the expression of TIMP1 and TMEM176B can achieve the same effect (Figure 2C). In addition, the results of Caspase-3 ELISA assay show that inhibiting the expression of ITGB5 can increase cell apoptosis, while activating the expression of TIMP1 and TMEM176B can observe the same effect (Figure 2D).

To further clarify whether inhibition of ITGB5 and activation of TIMP1 and TMEM176B affect tumor cell migration, we also used dCas9-KRAB-mediated CRISPRi technology to knock down ITGB5 expression, and used dCas9-VPR-mediated CRISPRa technology to induce TIMP1 and TMEM176B activation. The results of the cell scratch experiment showed that inhibiting the expression of ITGB5 can reduce cell migration viability, while activating the expression of TIMP1 and TMEM176B can achieve similar effects (Figures 3A,B).

Cell migration is always directly related to invasion behavior. To further clarify whether knockdown of ITGB5 and activation of TIMP1 and TMEM176B expression affect tumor cell invasion, we used dCas9-KRAB-mediated CRISPRi to knock down ITGB5 expression, and used dCas9-VPR-mediated CRISPRa to activate TIMP1 and TMEM176B expression. The results of cell invasion experiments show that inhibiting the expression of ITGB5 can reduce the ability of cell invasion, while activating the expression of TIMP1 and TMEM176B can achieve similar effects (Figures 4A,B).