Patients information and tissues specimen
We collected clinical information and tissue samples of patients undergoing radical prostatectomy in our institution from 2016 to 2019. Clinical information includes age, PSA level at initial diagnosis, clinical stage, Gleason score, and whether the seminal vesicles and lymph nodes are invaded. None of these patients received androgen deprivation therapy, chemotherapy or radiation before surgery. All patients signed informed consent. This study was approved by the Ethics Committee of Tangshan Gongren Hospital. The clinical trial registry number is GRYY-LL-2019-40.
The human PCa cell lines PC-3, C4-2, and Lncap was used in this study, which was purchased from the ATCC (American Type Culture Collection, USA). The human prostatic hyperplasia cell line BPH-1 was purchased from the HonSun company (Shanghai). The PC-3, C4-2, Lncap and BPH-1 cell line was cultured in RPMI 1640 (Biological Industries, 01-100-1A, Israel) medium containing 10% fetal bovine serum (Biological Industries, 04-400-1A, Israel) in a 37 °C incubator containing 5% carbon dioxide.
Knockdown KIF20A by small hairpin RNAs
The lentiviral vector which contained short hairpin RNA(shRNA) targeting KIF15 was purchased from Genechem Technology (Shanghai, China). The most efficient shRNA sequence to knock down KIF15 was: 5’-TGAAGTGAAGAGGCTCAAA-3’ and the control shRNA sequence was: 5’-TTCTCCGAACGTG TCACGT-3’. According to the instructions, a mixture of 100 nM shRNA and lipofectamine 3000 Transfection Reagent (Invitrogen,L3000015,USA) was added to the serum-free RPMI 1640 medium. Detection of KIF15 knockdown efficiency by Western blot after 48 hours
Quantitative real-time PCR
Total RNA was extracted from tumor tissues and normal tissues by using Trizol reagent (Invitrogen,15596018,USA).According to the manufacturer’s protocol, Reverse Transcription system, which included DEPC water, primer-mix, dNTP-mix, DTT and 5×PrimeScript buffer, was used to reverse-transcribed total RNA to generate cDNA. SYBR Premix Ex Taq was performed to quantified the expression of KIF15 at mRNA level and the expression of GAPDH was used as internal reference. The primers of KIF15 and GAPDH used for qPCR were as follows: KIF15: Forwards (5′-3′) : CTCTCACAGTTGAATGTCCTTG, Reverse(5′-3′): CTCCTTGTCAGCAGAATGAAG; GAPDH: Forwards(5′-3′): TGACTTCAACAGCGACACCCA, Reverse(5′-3′): CACCCTGTTGCTGTAGCCAAA. 2 –ΔΔCT analysis program was used to analyze the expression of KIF15.
RIPA buffer (Invitrogen,89900,USA) was used to lyse the cell lines to obtain protein. The concentration of extracted protein was measured by using BCA kit (Solarbio,PC0020,Beijing). 25ug protein samples was separated by SDS-PAGE and then transferred to the PVDF membrane. Subsequently, the membrane was incubated with the KIF15 antibody (Abcam,ab272615,UK), GAPDH antibody (Abcam, ab181602,UK), Ki67 antibody (Abcam, ab16667,UK), or MMP9 antibody (Abcam, ab219372,UK) at 4ºC overnight. At last, the membrane was incubated with secondary antibodies for 1 hour and the bands was displayed by using ECL kit (Solarbio,PE0020,Beijing). ImageJ was performed for the semi-quantitative analysis of bands.
The cells in the logarithmic growth phase, which transfected with shCON or shKIF15, were collected and seeded on 96-well plates with 800 cells/well. After 6 days, MTT reagent (Solarbio,M1020,Beijing) was added into every well and cultured with cells for 2 hours. Then remove the MTT reagent, incubate with the cells for half an hour after adding DMSO (Solarbio,D8371,Beijing), and detect the absorbance by using a microplate reader at 570 nm.
The matrigel (Corning,354234,USA) and the pre-chilled serum-free 1640 medium were mixed on ice at a ratio of 1 to 8. 60 ul diluted matrigel was added to the upper chamber (Corning,3496,USA) of transwell and 1640 medium containing 20% serum was added below the chamber. Add 9,000 cells to each chamber in a 24-well plate and incubate in the incubator for 30 hours. Discard the supernatant and stain the cells with crystal violet. Remove the remaining cells in the upper chamber at the same time. Take a photo under the microscope and Perform a cell count.
The tissue-embedded paraffin block was cut into sections with a thickness of 4 um. The sections were subjected to dewaxing, hydration, and antigen retrieval in order. A peroxidase blocker was added to the sections to block the effect of peroxidase. After washing with PBS, the sections were incubated with the primary antibody (KIF15, Abcam,ab272615,1:50,UK) at 4ºC overnight. Sections were then incubated with secondary antibodies for 1 hour at 37 ºC. DAB kit (Solarbio,DA1010,Beijing)was used for coloration and then hematoxylin staining was added. Finally, the slices were dehydrated and transparently treated, and neutral gum was used for sealing. Observe and take pictures under the microscope.
GEPIA(http://gepia.cancer-pku.cn/) was used to analyze the PCa patients’ data from TCGA(The Cancer Genome Atlas) dataset. Using the median value of KIF15 mRNA expression level in PCa patients as the cutoff value, the patients were divided into a KIF15 high expression group and a KIF15 low expression group. Kaplan-Meier method was performed to analyze the overall survival and disease-free survival in the two groups of patients. P <0.05 was considered statistically significant.
Statistical analysis was performed by using SPSS 22.0 software. The correlation between KIF15 expression and the clinical feature of PCa patient was analyzed by using Chi-square test. Paired t test and rank sum test were used to analyze the differences between two groups. P < 0.05 is considered statistically significant. * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, and n.s. indicates no significant difference.