Cell culture and transfection
Human cervical cancer cell lines Ca Ski, MS751, ME-180, SiHa, HT-3, HeLa, C4-I and SW756 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Natocor) supplemented with 10% fetal bovine serum (FBS) (GIBCO). Ectocervical Ect1/E6E7 and endocervical End1/E6E7 cells were cultured in Keratinocyte-Serum Free medium (GIBCO-BRL, 17005-042, USA) supplemented with 0.1 ng/mL human recombinant EGF, 0.05 mg/mL bovine pituitary extract (BPE, Sigma) and 0.4 mM CaCl2 (Natocor). The cells were maintained in 5% CO2 at 37 °C.
The full-length BASP1 cDNA was cloned into vector pMSCV-puro; pMSCV-empty vector was used as the negative control. pMSCV-BASP1 and pMSCV-empty vector were cotransfected with the IK packaging plasmid into 293T cells using the calcium phosphate transfection method. Supernatants were collected at 48 h after transfection, and infected with cervical cells for 12 h in the presence of polybrene (2.5 μg/mL). Puromycin was used to select the transfected cell lines.
For BASP1 knockdown experiments, two siRNAs for BASP1 and one scrambled siRNA were synthesized by Guangzhou RiboBio Co (Guangzhou, China). The sequences used to downregulate BASP1 were: siBASP1#1: 5′CGGGATCCATGGGA3′, siBASP1#2: 5′CGGAATTCTCACTCT3′. 50 nM of the siRNA was transfected into ME-180 and HT-3 cells using the Lipofectamine RNAiMAX Transfection Reagent (Life Technologies).
Patients and tissue samples
Six cervical cancer tissues and matched adjacent normal cervical epithelium tissues were obtained from the First Affiliated Hospital of Sun Yat-sen University. Guangzhou. These samples were snap-frozen immediately and stored in liquid nitrogen until use. To further analyze the relationship between BASP1 expression and the clinicopathological parameters, a cohort of 136 paraffin-embedded cervical cancer tissues was used. These tissues were diagnosed histopathologically and clinically at the First Affiliated Hospital of Sun Yat-sen University. For the use of above clinical samples for research purposes, prior patient’s consent and approval from the Institute Research Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University were obtained. The detailed clinicopathological parameters are shown in Additional file 5: Table S1.
Western blotting and immunohistochemistry (IHC)
Western blotting was performed according to standard methods, as described previously [19], using anti-BASP1 (ab101855, Abcam), anti-Ki67 (sc-7846, Santa Cruz) antibodies. The membranes were stripped and reprobed with anti-β-actin antibodies as a loading control. The band intensity was analyzed using Image J software.
IHC was performed as described previously [20] using the anti-BASP1 antibody (ab101855, Abcam). The results of staining were scored independently by two pathologists blinded to the clinical outcome, and were based on both the proportion of positively stained tumors cells and the intensity of staining. The proportion of stained tumor cells was scored as follows: score 0, no positive cells; score 1, up to 25% positive cells; score 2, 26–50% positive cells; score 3, 51–75% positive cells; score 4, over 75% positive cells. The intensity of staining was determined as: 0 (no staining), 1 (light yellow = weak staining), 2 (yellow brown = moderate staining), and 3 (brown = strong staining). The staining index (SI) was determined as the product of staining intensity × percentage of positive tumor cells. Cutoff values for high and low expression of BASP1 were chosen based on a measurement of heterogeneity using the log-rank test with respect to overall survival. An SI score of greater than or equal to 6 was considered to be high expression, and an SI score of less than 6 was considered low expression.
Cell proliferation and cell cycle assay
To examine the role of BASP1 in the proliferation of cervical cancer cells, an MTT assay and colony formation assay were performed using previous described methods [21].
Cell cycle analysis was performed using a previously described method [21]. Briefly, cells were harvested and washed in cold PBS followed by fixation in 70% alcohol overnight at 4 °C. After washing with cold PBS three times, the cells were resuspended in PBS solution with 20 μg/mL propidium iodide (Sigma) and μg/mL RNase A for 30 min at 37 °C. Samples were then analyzed using a FACSCalibur cytometer (Becton–Dickinson).
Anchorage-independent growth assay
An anchorage-independent growth assay was performed according to a previously described method [21, 22]. Briefly, 500 cells were suspended in 2 mL of complete medium containing 0.3% agar (Sigma). The agar–cell mixture was plated on top of a solid bottom layer containing 1% complete medium agar mixture. After 10 days, colonies that contained more than 50 cells or were larger than 0.1 mm in diameter were counted. The experiment was performed in triplicate.
Growth of tumor xenografts in nude mice
Animal studies were performed according to institutional guidelines. BALB/c nude mice (4–5 weeks old) were used to make a xenograft model using ME-180 cervical cancer cell lines. Five mice were assigned randomly to each group and injected with ME-180 transfected with pMSCV-BASP1, pMSCV-empty, scramble siRNA, or BASP1 siRNA, and used to determine the role of BASP1 in tumor progression. 1 × 106 cells were injected into the subcutaneous sites of nude mice. The tumor volume was calculated every 2 days for 1 month. The tumors were excised and subjected to protein extraction to determine Ki67 levels using western blotting.
Statistical analysis
All statistical analyses were performed using the SPSS 19.0 statistical software package. Results are presented as the mean ± standard deviation (SD) for at last three repeated individual experiments for each group. Chi square and Fisher’s exact tests were used to analyze the relationship between BASP1 levels and clinicopathological parameters. Bivariate correlations between variables were calculated using Spearman’s rank correlation coefficients. The survival curve was plotted using Kaplan–Meier survival analysis and compared using a log-rank test. Univariate and multivariate Cox regression analyses were used to estimate the significance of various variables for survival. A value of p < 0.05 was considered significant in all cases.
