2.1 Patients and clinical samples
Confirmed by the Department of Pathology for Harbin Medical University Cancer Hospital, gathered of 96 samples of patients with cervical squamous cell carcinoma who accepted cervical cancer staging surgery at the Harbin Medical University Cancer Hospital from January 2010 to December 2012. (not accepted any treatment before surgery, including immunotherapy, chemotherapy and radiotherapy). Clinical samples were processed by experienced pathologists through fixed, dehydrated, transparent, wax-transparent, embedded, sliced, patched, stained, transparent, sealed made into Paraffin embedded samples. Clinical pathological features including age, lymph node metastasis, tissue differentiation, and clinical stage (according to cervical cancer 2009 FIGO staging) were found by Hospital medical record system. The follow-up period was from the day of surgery to November 2018 (the follow-up date was 11–105 months, average 81 months). The study was ratified by the Harbin Municipal Ethics Committee and supported by the Harbin Medical University in China. All patients submit Informed consents.
2.2 Immunohistochemistry
According to the manufacturer's instructions, the purchased RRBP1 antibody (1: 1000, RRBP1 antibody, Abcam, Ab95983, UK) was diluted. In short, 98 paraffin sections fixed from formalin of the Tumor Hospital of Harbin Medical University were dewaxed. In order to eliminate the effect of endogenous peroxidase, pretreatment with 3% H2O2 solution for 10 minutes at room temperature. Antigen retrieval was achieved by microwave in citrate buffer pH 6.0 for 30 minutes. Block with the same source for serum of the secondary antibody for 15–30 minutes, add all the samples to the primary antibody diluted with anti-RRBP1 antibody in a ratio of (1:1000), and incubate at 4 °C for overnight, and rewarmed, and rinse with phosphate buffered saline (PBS) 3 minutes × 5 times. Next the biotin-labeled secondary antibody (goat anti-rabbit IgG-HRP, wanleibio, WLA023, China) was further added dropwise and incubated at 37 ° C for 30 minutes, and the cells were further washed with PBS for 3 minutes × 5 times. Then Immunoperoxidase staining was developed for 5 minutes using DAB chromogen and incubated at room temperature for 30 minutes to 1 hour. Finally, it was washed with PBS for 3 minutes x 5 times and counterstained with hematoxylin. We used a diluent instead of a primary antibody as a negative control. Immunohistochemistry results were analyzed by two experienced pathologists without understand of clinical information, and RRBP1 staining was analyzed by semi-quantitative methods. The staining intensity score was colorless (0), light yellow (1), brownish yellow (2), tan (3); Total percentage of positive cells scored 0 means < 5%; 1 means 5–25%; 2 means: 26–50%; 3 means 51–75%; 4 means > 75%. The score for each sample is calculated by the product of two values. The score (≥ 4) was evaluated as high expression and the score (< 4) was evaluated as low expression.
2.3 Western blot analysis
All fresh surgical specimen tissues (n = 36) were frozen in a -80 °C ultra-low temperature freezer, and the experimental reagents and polyacrylamide gels were prepared according to the manufacturer's instructions. Firstly, in order to extract the protein, aliquoted the lysate (containing 1% PMSF) according to the needed of the experiment, and added each sample to the lysate, and left it on ice for 5 minutes. The lysate was transferred to a centrifuge tube and centrifuged at 12000 rpm for 5 minutes at 4 °C,and took the supernatant and stored at -20 °C. Then total protein was quantified used the purchased BCA protein concentration determination kit (wanleibio, WLA004, China). The complex protein mixture was separated by SDS-PAGE (wanleibio, WLA013, China) and transferred to a PVDF membrane (Millipore, IPVH00010, USA) in a conventional manner. So as to incubate the primary antibody, the purchased RRBP1 antibody (1: 1000, RRBP1 antibody, Abcam, Ab95983, UK) was incubated in a buffer at 4 ° C overnight. Then incubated the secondary antibody through goat anti-rabbit IgG-HRP (1:5000, Goat anti-rabbit IgG-HRP༌wanleibio, WLA023, China) was incubated for 45 minutes at 37° C. Finally, the configured chemiluminescent reagent was mixed with the membrane, shaken at room temperature for 5 minutes, the residual liquid was placed in the X-ray film at a dark room before final development, and the exposure time was appropriately adjusted according to the strength of the signal, and the image processing system (Gel-Pro-Analyzer software) analyzed the optical density value of the target band. The β-actin antibody (wanleibio, WL01845, China) was used as an internal reference antibody.
2.4 Real-time PCR analysis
First mRNA was extracted from fresh surgical samples (n = 36) according to the manufacturer's instructions and the concentration of RNA in each sample was determined used a UV spectrophotometer NANO 2000 (UV spectrophotometer NANO 2000, Thermo, USA). The cDNA was then synthesized in a PCR instrument (Real-Time PCR, Exicycler 96, BIONEER, Korea) used Super M-MLV reverse transcriptase (BioTeke, PR6502, Beijing), and the products were subjected to quantitative fluorescence analysis. This experimental analysis method used the 2-△△ CT method. PCR amplification conditions: Incubated at 94.00 ℃, for 5 minutes, incubated at 94.00 ℃, for 10 seconds, incubated at 60.00 ℃, for 20 seconds, incubated at 72.00 ℃, for 30 seconds. Scanned, went to line 2, Cycle 40, incubated at 72.00 ℃, for 2 minutes 30 seconds, incubated at 40.00 ℃, for 1 minutes 30 seconds, melted 60 ℃ to 94 ℃, Every 1.0 ℃, 1 seconds, incubated at 25.00 ℃, for 1–2 minutes. The primer sequence of RRBP1-F is 5'-TCCATCCAGAGTCTCACTTC-3 ', and the primer sequence of RRBP1-R is 5'-GCCCTCGTTGAACACCAT-3'. The primer sequence of GAPDH-F is 5'-GGCACCCAGCACAATGAA-3 ', and the primer sequence of GAPDH-R is 5'-TAGAAGCATTTGCGGTGG-3'. The β-actin antibody (wanleibio, WL01845, China) was used as an internal reference antibody.
2.5 Statistical analysis
We utilized chi-square test to analyze the results of immunohistochemistry, and applied the Kaplan-Meier method to estimate the total survival (OS) and disease-free survival (DFS) of cervical cancer, and applied log rank test. Finally, multivariate analysis was assessed according to Cox regression (proportional risk model). A P < 0.05 was deemed statistically implication. All the above statistical analysis applied Windows SPSS software V25.0 (IBM SPSS. Inc., Chicago, IL, USA).