LNCaP cell culturing and drug screening
Human LNCaP clone FGC cell lines were purchased from the Shanghai (Zhong Qiao Xin Zhou Biotechnology Co., Ltd, Lot#ZQ0039). LNCaP cell was cultured in 1640 medium supplemented with 10% FBS and 1% Glutamax (USA, Invitrogen, Lot#35050061). Cells were grown at 37℃ in a humidified atmosphere containing 5%CO2. Enzalutamide (MDV3100, Lot# PHB00235-1G) was obtained from Merk (USA, https://www.sigmaaldrich.com/). Transplant LNCaP cells into a 96-well plate (8000 cells/well) and then divided into 6 groups. One group was treated DMSO, and the other five groups were treated with enzalutamide at final concentrations of 5, 10, 20, 50 and 100 μM for 7 days, with a drug change on the 4th day. After the processing of each group is completed, Cell Counting Kit-8 (CCK-8) assay was performed for testing cell proliferation. Knockout interfering RNA (shRNA) were synthesized (Sangon Biotech Shanghai Co., Ltd. China) and transfected into LNCaP cells by using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer’s protocol. For TP53 knockout gene assessment, primers were as follows:
shRNA-1: 5’- UUGCGGAGAUUCUCUUACU -3’,
shRNA-2: 5’- UUCUCUUACUCCACACGCA -3’
shRNA-3: 5’- AUUCUCUUACUCCACACGC -3’.
CRISPR Cas9 sgRNA library construction and sequencing.
sgRNA library plasmid (20,000 genes, 6 sgRNA targets for each gene) was obtained from Addgene (http://www.addgene.org). sgRNA library plasmid was directly packed with lentivirus and transfected with 293T cells with the aid of Lipofectamine 2000 reagent (ThermoFisher, Lot#11668027). After 48 and 72 hours of transfection, the virus was collected, filtered with a 0.45uM membrane, and then concentrated to 6ml. The concentrated virus was aliquoted and stored at -80℃. Current drug screening experiment was divided into Enzalutamide treated group and DMSO group, and each group contains 1x107 LNCaP cells (two 15cm dishes). The enzalutamide group was treated with the pre-experimentally determined drug (20μM) for 6 days, and the solution was changed once every 3 days. The DMSO group was added with the same volume of DMSO as the enzalutamide group. The surviving cells will continue to expand and culture after 6 days, and half of them will be frozen and stored, and the remaining half of the cells will be added to the respective dose of drugs. This cycle is repeated for 3 times. After that, cell genomic DNA was extracted with kit of DP304 (TIANGEN, Lot#DP304-02). Then, PCR and sequencing were done by using illumina hiseq 3000. We use the RNAi Gene Enrichment Ranking (RIGER) algorithm to analyze the enrichment score of each gene. There is a total of 10048 enriched genes which at least 3 different sgRNA targets are enriched.
Western blot and RT-PCR
Total protein was extracted by using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) following the instruction. After measuring protein concentrations, 12% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels were conducted and transferred to polyvinylidene fluoride (PVDF) membranes (MerckMillipore, Darmstadt, Germany). Antibody applicated in current investigation list as following: p53 (1C12) Mouse mAb #2524, dilution ratio 1:1000 in western blot and 1:2000 in immunofluorescence. Androgen Receptor (D6F11) XP® Rabbit mAb #5153, dilution ratio 1:2000 in western blot and 1:600 in immunofluorescence. Total RNA was extracted using the RNAiso plus reagent (Takara Bio, Dalian, China). Quantitative real‐time PCR was performed using SYBR Green mix (Toyobo, Osaka, Japan) in a StepOnePlus Real‐Time PCR System (Life Technologies, Carlsbad, CA). For RT-PCR, primers were as follows:
AR fwd: 5’-ATGGTGAGCAGAGTGCCCTATC-3’,
AR rev: 5’-ATGGTCCCTGGCAGTCTCCAAA-3’,
GAPDH fwd: 5’-GAAGGTGAAGGTCGGAGTC-3’,
GAPDH rev: 5’-GAAGATGGTGATGGGATTTC-3’,
TP53 fwd: 5’-GAGCTGAATGAGGCCTTGGA-3’,
TP53 rev: 5’- CTGAGTCAGGCC CTTCTGTCTT-3’.
Cell proliferation, apoptosis and cell cycle
Cell proliferation was assessed with Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. The optical density (OD) values at the wavelength of 450nm was measured on an enzyme-linked immunosorbent assay plate reader (Varioskan Flash, Thermo Scientific, Waltham, MA). Apoptosis and cell cycle detection was analyzed by a flow cytometer (MoFlo, Beckman, CA, USA). Cells were harvested 48 h after lentivirus transfection and incubated with annexin V-FITC and PI according to the manufacturer’s instructions (BD, San Diego, CA, USA).
Database sources and statistical
PPI network analysis was done by using STRING (https://string-db.org/). RNA-seq data of LNCaP cell treated with enzalutamide and DMSO were obtained from GEO publications (GSE137833). Pathway analyses was conducted by using methods of GSEA (https://www.gsea-msigdb.org/gsea/index.jsp). PRAD data was download form publica database of The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/). All statistical or figures was dealt with software of GraphPad Prism v5.0 or R platform (https://www.r-project.org/).