Patient and specimens
The tissue chip (TMA) is composed of 226 colorectal cancer tissues and matched adjacent tissues collected by the Department of Pathology, Affiliated Hospital of Xuzhou Medical University, all of which are paraffin embedded blocks. All patients underwent radical surgery in the Affiliated Hospital of Xuzhou Medical University from April 2010 to March 2015. Clinicopathological characteristics such as the patient's age, sex, lymph node metastasis, tumour-node-metastasis (TNM) stage, degree of differentiation, tumour diameter, distant metastasis and infiltration depth were obtained from the Department of Pathology, Affiliated Hospital of Xuzhou Medical University. A total of 8 pairs of fresh colorectal cancer tissues and adjacent tissues were taken directly from the operating room of the Affiliated Hospital of Xuzhou Medical University, and postoperative pathology confirmed colorectal adenocarcinoma. The clinical pathological information of the patients was obtained from the hospital medical records, and informed consent was obtained from all patients. The patient study was conducted in accordance with the Declaration of Helsinki. The use of these specimens and data for research purposes was approved by the hospital ethics committee. Clinicopathological characteristics such as the patient's age, sex, lymph node metastasis, tumour-node-metastasis (TNM) stage, degree of differentiation, tumour diameter, distant metastasis and infiltration depth were obtained from the Department of Pathology, Affiliated Hospital of Xuzhou Medical University. A total of 8 pairs of fresh colorectal cancer tissues and adjacent tissues were taken directly from the operating room of the Affiliated Hospital of Xuzhou Medical University.
Immunohistochemistry (IHC)
The paraffin-embedded CRC tissues and corresponding paracancerous tissues were punched to obtain 1.5 mm diameter cores. The standard protocol for immunostaining of the TMAs was described previously. Immunohistochemistry was performed according to the streptavidin–peroxidase (Sp) method using a standard Sp Kit (Zhongshan Biotech, Beijing, China). The TMA slide was incubated with monoclonal rabbit anti-LMNB2 (1:100, ab151735, Abcam, Cambridge, MA, USA) overnight at 4 ℃, anti-p21 antibodies were applied at 1:100 dilutions (#2947, Cell Signaling Technology, USA), and anti-Ki67 antibodies were applied at 1:200 dilutions (ab16667, Abcam, USA). Diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate. The immunoreactivity was assessed blindly by two independent observers using light microscopy (Olympus BX-51), and the image was collected by a Camedia Master C-3040 digital camera (Beijing, China).
Assessment of IHC
Two pathologists separately assessed the TMAs under blinded experimental conditions, and all differences that arose were resolved by discussion. The staining scores of LMNB2 were evaluated by combining the percentage of cells with the staining intensity and were dependent on the IRS. The intensity of LMNB2 immunostaining was scored as 0–3 (0, negative; 1, weak; 2, moderate; 3, strong); the percentage of immunoreactive cells was graded as 1 (0–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). Based on the IRS, the level of LMNB2 expression was categorized as low (IRS: 0–6) and high (IRS: 8–12) expression.
Cell lines and cell culture
The colorectal cancer cell lines FHC, HCT116, DLD1, LOVO, HCT29, SW480, and SW620 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai China). HCT116, SW480, and SW620 cells were cultured in DMEM, while DLD1 and LoVo cells were cultured in RPMI 1640 medium supplemented with 10% foetal bovine serum and 100 μg/ml streptomycin and incubated in a 37 °C humidified incubator with 5% CO2.
Small interfering RNA and transient transfections
Small interfering RNA (siRNA) specific for LMNB2 (siLMNB2) and nonspecific control siRNA (siCtrl) were purchased from GenePharma (Shanghai, China) and transfected with siLentFect Lipid Reagent (Bio-Rad Laboratories, Inc.) according to the manufacturer’s protocol when CRC cells were grown to ~50% confluency. Six hours after transfection, the medium containing transfection reagents was replaced by fresh medium. The siRNA sequences are as follows:
SiLMNB2#1 sense: GCGAGGUGAGUGGCAUCAATT;
SiLMNB2#2 sense: GGAAGUGGCCAUGAGGACUTT;
sip21# sense: CCUCUGCAUUAGAAUUAUTT;
siCtrl sense: UUCUCCGAACGUGUCACGUTT.
Stable cell line generation
For stable suppression of LMNB2 expression, LMNB2 short hairpin RNA (shRNA) and control lentivirus were obtained from GenePharma. HCT116 and DLD1 cells were infected with lentivirus for 48 h and then selected with 2 ng/ml puromycin for 2 weeks, with the medium refreshed every 3 days. The shRNA target sequences were as follows: shLMNB2 sense: GCGAGGUUGGCAUCAATT;
shCtrl sense: TTCTCCGAACGTGTCACGT.
Cell proliferation and colony formation assays
CCK-8 assays were carried out to determine the function of LMNB2 in cell proliferation. HCT116 and DLD1 cells were transfected with siRNA targeting LMNB2 or negative control siRNA using siLentFect™ Lipid Reagent for RNAi (Bio-Rad Laboratories, Inc.), respectively. Forty-eight hours after transfection, for CCK-8 analysis, 4000 cells were seeded in each well of 96-well plates, and CCK-8 solution was added 24, 48, 72, and 96 h after transfection. Cells were incubated at 37 °C for 1 h after 10 μl of CCK-8 solution was added. The absorbance at 450 nm was measured. Five millilitres of cell suspension containing 7x102 cells was inoculated into a 60 mm dish for continuous culture until visible clones appeared. Then, the cells were fixed with methanol and stained with 0.05% crystal violet solution. After two washes with PBS, the plates were photographed using a digital camera. Positive colony formation, defined as colonies with more than 50 cells, was confirmed by manual counting.
EdU assay
The effect of LMNB2 on the proliferation of HCT116 and DLD1 cells was measured by a 5‐ethynyl‐2’‐deoxyuridine (EdU) incorporation assay using an EdU assay kit (RiboBio, Guangzhou, China) according to the manufacturer's protocol. Briefly, the cells were cultivated in 96‐well plates at 4 × 103 cells/well. Twenty hours after culture, the cells were treated with 50 μmol/L 5‐ethynyl‐2'‐deoxyuridine (EdU; RiboBio, Guangzhou, China) and incubated for 2 h at 37 °C. The cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X‐100 for another 20 min. Afterwards, the cells were washed five times with PBS and incubated with 100 μL of 1×Apollo® reaction cocktail for 30 min at room temperature. Finally, the nuclei of the cells were dyed with 100 μL of Hoechst 33342 (5 μg/mL) for 20 min and visualized with fluorescence microscopy (IX71; Olympus, Tokyo, Japan).
Cell cycle analysis
Forty-eight hours after transfection, the cells were synchronized by serum starvation overnight and induced to re-enter the cell cycle by incubating in medium containing 10% foetal bovine serum for 4 h. Then, the cells were collected, washed with PBS, and fixed in precooled 70% ethanol at 4 °C overnight. The day after, the cells were washed twice with PBS and resuspended in RNase A at 37 °C for 30 min, and then, propidium iodide (PI) was added to the cells in the dark at 4 °C for 30 min. Finally, all samples were analysed by flow cytometry (BD, FACSCantoTM II).
Apoptosis
Apoptosis assays were carried out using the Annexin V-FITC/PI apoptosis detection kit (Nanjing KeyGen Biotech, Inc.) according to the manufacturer's protocol. Briefly, the cells were collected after transfection with siRNA for 48 h, washed twice with PBS, and resuspended in binding buffer. Sequentially, the cells were stained with Annexin V-FITC and PI at room temperature for 15 min and then analysed by flow cytometry (BD, FACSCantoTM II).
Western blot analysis
The cells were lysed in RIPA buffer and centrifuged at 12 000 g for 10 min at 4 °C. The supernatants were collected for Western blotting assays. Equal amounts of proteins were subjected to 10% SDS‐PAGE and then transferred to 0.45‐μm pore size PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 2 h with 5% skim milk in Tris buffered saline containing 0.1% Tween 20 and incubated overnight at 4 °C with anti-mouse GAPDH (Cell Signaling Technology, USA), anti-rabbit LMNB2 (Abcam, USA), and anti-rabbit P21 (Cell Signaling Technology, USA). The membranes were washed 10 min three times with Tris-buffered saline containing 0.1% Tween 20 and incubated for 2h with appropriate secondary antibodies. Specific primary antibodies against LMNB2 (ab151735) and Ki67 (ab16667) were purchased from Abcam. Antibodies against p21 (#2947), p27 (#3686), cyclin D1 (#2978), cyclin E2 (#4132), and Cdk2 (#2546) were obtained from Cell Signaling Technology. Antibodies against GAPDH (60004-1-Ig) were purchased from Proteintech.
RNA extraction and quantitative real-time PCR
TRIzol reagent was used for total RNA extraction (Invitrogen) following the manufacturer's instructions. After RNA purity was measured, a reverse transcription reaction was performed. The PrimeScript™ RT kit (1 µg RNA) and gDNA Eraser (Vazyme) were used. The primers for the p21 promoter fragment were reported(26). The primer sequences are as folloewed:
5′-TGGAGATCAACGCCTACCG -3′ (forward) and
5′-AGCCGCTTCCGCTTACTG -3′ (reverse) for LMNB2;
5′-AAGGTCGGAGTCAACGGATTTG-3′ (forward) and
5′-CCATGGGTGGAATCATATTGGAA-3′ (reverse) for GAPDH.
Real-time PCR was performed using SYBR Green PCR Master Mix with an ABI-7500 qRT-PCR thermal cycler (Vazyme Biotech, Nanjing, China). The relative mRNA level of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control.
Dual-luciferase reporter assays
HCT116 cells were transiently transfected with siRNA specific for LMNB2 and nonspecific control siRNA using siLentFect™ Lipid Reagent for RNAi (Bio-Rad Laboratories, Inc.), p21 promoter plasmid, and Renilla luciferase plasmid. Forty-eight hours post transfection, we collected the cells and measured the activities of both firefly luciferase and Renilla luciferase according to the dual-luciferase reporter assay system (Promega, Madison, WI, USA). The internal standard for transfection efficiency was normalized to Renilla luciferase activity. The p21-luc plasmid (−2400/+11) was a gift from Dr. Baiqu Huang (The Institute of Genetics and Cytology, Northeast Normal University).
Chromatin immunoprecipitation assay
ChIP assays were performed according to the protocol of the ChIP assay kit (Upstate Biotechnology, Lake Placid, NY). HCT116 cells cultured in 100 mm dishes (approximately 1 × 107 cells) were crosslinked by adding formaldehyde to a final concentration of 1% and incubated at room temperature for 10 min, washed twice with cold PBS containing protease inhibitors, lysed by ChIP lysis buffer, and sonicated to shear DNA at 4 °C to reduce the average length. Sonicated lysates were then diluted 10-fold with ChIP dilution buffer, and nonspecific binding was reduced with protein A-agarose for 1 h at 4 °C. In this step, 20 μl of lysate was removed as an input control, followed by incubation with anti-LMNB2 or anti-IgG (as a negative control) at 4 °C overnight with rotation. After several washes with a series of buffers, qRT-PCR was performed to amplify the genomic region of p21 flanking the potential LMNB2-binding sites.
In vivo tumour xenograft model
The animal experiments were approved by the Animal Care Committee of Xuzhou Medical University, Xuzhou, China. Female BALB/c nude mice (6–8 weeks old) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd., and maintained under specific pathogen-free conditions. HCT116 cells (5 × 106 cells) with knockdown of LMNB2 and control cells were injected subcutaneously into the flanks of the mice. Tumour volume (V) was monitored every 2 days by measuring the long axis (L) and the short axis (W) with a Vernier caliper and calculated with the following formula: V=(L× W2)/2. Twenty days later, the mice were killed, and the tumours were weighed and processed to detect the expression of LMNB2, p21, and Ki67 by IHC.
Statistical analysis
SPSS 23.0 was used for all statistical analyses. The difference between LMNB2 staining of CRC tumours and its corresponding adjacent noncancerous tissues was evaluated by a paired Wilcoxon test. The χ2-test was used to evaluate the relationship between the expression of LMNB2 and clinicopathological features. The Kaplan–Meier method and log-rank test were chosen to investigate the correlation between LMNB2 expression and 5-year overall survival and disease-free survival. Univariate and multivariate Cox proportional hazards regression analyses were performed.