From October 2010 to December 2012, 212 primary CRC tissues and 47 matching adjacent noncancerous tissues were obtained from patients who had the operation at the Hunan Provincial People's Hospital. Patients with preoperative chemotherapy and/or radiotherapy, and palliative surgery were excluded. Survival analysis excluded patients who died within 30 days after surgery, since their death could be attributed to surgical complications. All patients have signed an informed consent. The Ethical Review Board of Hunan Provincial People's Hospital have approved the study.
According to previously described methods, we performed the following procedures by the classic biotin-streptavidin-peroxidase IHC staining protocols.15 We obtained sections from the Hunan Provincial People's Hospital Pathology Department, and incubated overnight at 48℃ with polyclonal primary antibody against PARP1 (1:100; Abcam, Cambridge, UK). Following incubated with diaminobenzidine and horseradish peroxidase-conjugated sheep anti-rabbit secondary antibody (Beyotime; Guangzhou, China), used Mayer’s hematoxylin to counterstain the slides. Positive control was primary CRC tissue slides. In negative control staining, used phosphate-buffered saline (PBS) buffer to replace the primary antibody. The immunostaining results were scored according to the methods previously described below.16
Culture and treatment of Cell
We obtained human CRC cell lines FHC, SW480, SW620, LoVo, SW403, HT-29, COLO205 and COLO320DM from the American Type Culture Collection (Manassas, VA, USA). Incubated cell lines in DMEM/RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) complemented with penicillin (100U/mL), streptomycin (100μg/ml), and 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2.
Vectors and retroviral infection
Through subcloning the PCR-amplified human PARP1 coding sequence into a pBABE-puro vector, we generated pBABE/PARP1-overexpressing human PARP1. Cloned two RNA interference (RNAi) oligonucleotides into pSuper-retro-puro vectors to produce the pSuper-retro-PARP1-RNAi respectively, thereby silencing endogenous PARP1. As previously described, the generation and infection of retroviruses were carried out.17 After infection 48 hours, cell line stably expressing PARP1(pBABE-puro-PARP1 or SW480/SW620-PARP1; control, pBABE-puro or SW480/SW620-Vector, respectively) or PARP1 RNAi (pSuper- retro-puro-si PARP1 or SW480/SW620-PARP1/RNAi; control, pSuperretro-puro or SW480/SW620-Scramble, respectively) were selected using puromycin (0.5mg/ml) over 10 days. SDS-PAGE was used to segregate SW480 and SW620 cell lysates to detect PARP1 protein levels.
Extraction and reverse transcription of RNA, real-time quantitative PCR
In line with manufacturer’s illustrations, applied Trizol reagent (Invitrogen, Carlsbad, CA, USA) to accomplish total RNA extraction from cultured cells or tissues. Equipped with the ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, USA), SYBR Green I (Invitrogen) was applied for real-time quantitative PCR. Selected housekeeping gene GAPDH as an internal control. Used the primers below:
PARP1 forward, 5′-ACAGTGTGCAGGCCAAGGTG -3′, and reverse 5′-CTCGGC TTCTTCAGAATCTCTGTC-3′; XRCC2 forward: 5′-TCACCTGTGCATGGTG ATATT-3′, and reverse: 5′-TTCCAGGCCACCTTCTGATT-3′; GAPDH forward: 5′-GACTCATGACCACAGTCCATGC-3′, and reverse: 5′-AGAGGCAGGGATGATG
TTCTG-3′; p21 forward: 5′-CGATGCCAACCTCCTCAACGA-3′, and reverse: 5′-TCGCAGACCTCCAGCATCCA-3′; cyclin D1 forward: 5′-AACTACCTGGA CCGCTTCCT-3′, and reverse: 5′-CCACTTGAGCTTGTTCACCA-3′.
According to manufacturer’s instructions，proteins were prepared from cell lysates, isolated on SDS-PAGE, and transferred to PVDF membranes. To detect specific proteins, primary antibodies that were used included α-Tubulin mouse monoclonal antibody (1:1000; Sigma-Aldrich, St. Louis, MO, USA), anti-human XRCC2 mouse monoclonal antibody (1:1500; Abcam), anti-human PARP1 mouse monoclonal antibody (1:1500; Abcam), anti-human cyclin D1 rabbit monoclonal antibody (1:1500; Abcam), and anti-human P21 rabbit monoclonal antibody (1:1500; Abcam). The secondary antibody was goat anti-mouse antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After the membrane exposed to electrochemiluminescence reagent (GE Healthcare, Buckinghamshire, UK), storm imaging system (Amersham Biosciences, Piscataway, NJ, USA) was used for visualization to achieve signal amplification and detection.
Cell proliferation detection
In accordance with the manufacturer’s illustrations, used the Cell Counting Kit-8 (CCK-8) cell proliferation kit (Dojindo Laboratories, Kumamoto, Japan) to assess cell proliferation. Concisely, seeded the cells into 96-well plates (2×103 cells/well), and cultured under regular circumstances with 100μL complete medium. At the specified time, incubated cells with RPMI-1640 medium (100μL) plus CCK8 reagent (10μL) for 2 hours at 37°C. After that, measured the absorbance at 450nm wavelength on a microplate reader (Bio-Rad, La Jolla, CA, USA). Conducted three repetition experiments independently.
Colony formation assay
In brief, plated exponential growth cells into 6-well plates at 1000 cells/well and cultured for 10-14 days at 37°C with 5% CO2. For visualization and counting, used 75% ethanol to fix the colonies for 30mins and stained with 0.5% crystal violet (Beyotime, Nanjing, China) afterwards. When colonies with more than 50 cells would be manually calculated. Every group of cells comprised three wells, and three independent repeat experiments were conducted.
SPSS 20.0 (SPSS Inc, Chicago, IL, USA) was used for statistic analysis. Employed the Chi-square test to evaluate the association between PARP1 expression and clinicopathological characteristics. The significant differences between two groups of data were analyzed with the Student’s t test. The log-rank test and the Kaplan-Meier method were employed for survival curves analysis. The time from surgery to last follow-up date or patient’s death was 5-year overall survival (OS). The time from radical operation to recurrence, last follow-up date, or death was defined as Relapse-free survival (RFS). Recurrences were defined as local and distant relapses. Statistically significant was set at p <0.05.