J Gastrointest Surg (2015) 19:1484–1496 DOI 10.1007/s11605-015-2767-6
ORIGINAL ARTICLE
Properties and Clinical Relevance of Speckle-Type POZ Protein in Human Colorectal Cancer Junfei Xu & Feiran Wang & Haiyan Jiang & Yasu Jiang & Jinpeng Chen & Jun Qin
Received: 5 November 2014 / Accepted: 30 January 2015 / Published online: 29 May 2015 # 2015 The Society for Surgery of the Alimentary Tract
Abstract Background The aims of this study are to evaluate the effect of Speckle-type POZ protein (SPOP) in colorectal cancer (CRC) patients and explore its significance in the prognosis. Methods We used immunohistochemistry to detect the expression of SPOP in CRC. Moreover, this result was further confirmed at the protein and messenger RNA (mRNA) level in paired CRC specimens and matched adjacent noncancerous colon tissues by Western blotting and real-time quantitative PCR (qRT-PCR), respectively. Furthermore, we evaluate the effects of SPOP on CRC cell proliferation and migration in vitro. The Kaplan–Meier method and log-rank test were employed to compare the overall survival between SPOP low expression group and SPOP high expression group. Correlation of survival with clinicopathologic parameters, including SPOP level, was investigated with multivariate analyses. Results We confirmed frequent SPOP downregulation in both mRNA (P=0.0286) and protein (P=0.004) levels in CRC tissues as compared to matched adjacent nontumorous tissues. Besides, the downregulated SPOP expression in CRC tissues was significantly correlated to poor differentiation (P=0.013), distant metastasis (P=0.003), gross type (P75 % as 4. Moreover, staining intensity is rated on a scale of 0 to 3, with 0, negative; 1, weak; 2, moderate; and 3, strong. We created an immunoreactive score by multiplying the score for the percent of positive cells and the score for staining intensity. Theoretically, the ISS could range from 0 to 12. An ISS of 9~12 was considered strong immunoreactivity (+++), 5~8 was considered moderate (++), 1~4 was considered weak (+), and 0 was scored as negative (−). Sections were considered positive for SPOP when more than 25 % of tumor cells were stained in the cell cytoplasm.
J Gastrointest Surg (2015) 19:1484–1496
polyvinylidene fluoride (PVDF) membrane. Nonspecific binding was blocked for 2 h with 5 % nonfat milk in Trisbuffered saline containing 0.1 % Tween 20 (TBST). After incubation with anti-SPOP antibody (sc-66649, Santa Cruz Biotechnology, USA), anti-E-cadherin antibody (ab1416, Abcam, Cambridge, MA, USA), anti-vimentin antibody (ab92547, Abcam), anti-N-cadherin antibody (ab18203, Abcam), anti-Snail antibody (ab180714), anti-Slug antibody (ab27568, Abcam), and anti-Twist antibody (ab49254, Abcam) overnight at 4 °C, membranes were washed three times in TBST for 5 min and subsequently incubated secondary antibody with conjugated IRDye800 (1:5000, Rockland Gilbertsville, CA) at room temperature for 2 h, followed by scanning with an Odyssey infrared imaging system (LI-COR, Lincoln, NE), and analyzed with PDQuest 7.2.0 software (Bio-Rad). Real-Time RT-PCR The messenger RNA (mRNA) expression of SPOP was analyzed by quantitative real-time PCR. Total RNAs were extracted using the Trizol (Gibco). Quantitative real-time PCR was performed using HotStart-IT SYBR Green QPCR Master Mix (2×; USB Corporation, Cleveland, OH 75762, USA). According to the HotStart-IT protocol, 25-μl reactions were run of with 2 μl cDNA. RT-PCR experiments were performed in a LightCycler 480 system (Roche Applied Sciences). Cycling parameters were as follows: hot start at 95 °C for 10 min; 40 cycles of amplification/quantification at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s during which time fluorescence was measured. Melting curve analysis was performed using continuous fluorescence acquisition from 65 to 97 °C. These cycling parameters generated single amplicons for both primer sets used according to the presence of a single melt peak. The β-actin was selected as the internal reference. All quantitative real-time PCRs were repeated three times for each gene, and each sample was done in triplicate. Primer sequences were as follows: SPOP forward 5′-TGACCACCAG GTAGACAGCG-3′, SPOP reverse 5′-CCCGTTTCCCCC AAGTTA-3′. The β-actin gene served as an internal control. The sequences of the primers for β-actin were as follows: βactin forward 5′-AACTTCCGTTGCTGCCAT-3′, β-actin reverse 5′-TTTCTTCCACAGGGCTTTG-3′. Relative expression level for each target gene was normalized by the Ct value of β-actin (internal control) using a 2ΔΔCt relative quantification method.
Western Blotting Cell Proliferation Assay Coupled tumor and their adjacent nontumor tissues were taken for Western blot analysis. Total protein was extracted by lysis buffer containing protease inhibitors (Promega, Madison, WI). Equal amounts of protein separated by 10 % sulfate polyacrylamide gel electrophoresis and then transferred to a
Cell viability and proliferation was assessed using an MTT assay. Following transfection, HCT116 and LoVo cells were plated in 96-well plates at 1×103 cells/well and incubated for 24, 48, and 72 h. A total of 20 μl MTT (5 mg/ml, Sigma-
J Gastrointest Surg (2015) 19:1484–1496
Aldrich) was added to each corresponding test well and incubated for 4 h at 37 °C. The supernatant was then discarded, and 200 μl dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) was added to each well to dissolve the formazan. Optical density was assessed by measuring the absorbance of each well at 490 nm using a spectrophotometer (SpectraMax Plus384; Molecular Devices, Sunnyvale, CA, USA). All experiments were performed in triplicate. Colony Formation Assay After transfection of SPOP-siRNA/p-SPOP or control vector, HCT116 and LoVo cells (5×104 cells/well) were separately plated in a 24-well plate. After 24 h, the cells collected and seeded (1000–1500/well) in a fresh six-well plate for 14 days. Surviving colonies (>50 cells per colony) were counted after fixed with methanol/acetone (1:1) and stained with 5 % Gentian Violet (ICM Pharma, Singapore, Singapore), and then rinsed three times with PBS to remove excess dye, photographed, and counted. The experiment was carried out in triplicate wells for three times. Cell Migration Assay The cell migratory capacity was determined using transwell chambers with 8-mm diameters (Corning, Corning, NY, USA). Briefly, HCT116 and LoVo cells (1×105/well) were transfected and suspended in 100-μl serum-free medium and then added to the upper chamber of the inserts; DMEM medium (Gibco) containing 10 % FBS (500 μl) was added to the lower chamber as a chemoattractant. After culture for 24 h at 37 °C, nonmigrated cells on the upper surface were removed gently with a cotton swab and cells that migrated to the lower side of the department were fixed with methanol and dyed with 0.1 % crystal violet. Then, cells that migrated through the upper chamber were counted in five random fields under a light microscope, and the average value of five fields was expressed. All assays were performed in triplicate. Wound Healing Assay Cells were plated in six-well plates and incubated overnight; when the cells had grown by 90–100 %, they were scraped with a pipette tip to generate straight wounds. To eliminate dislodged cells, culture medium was removed and wells were washed with PBS. We measured the closure or filling of the wounds at 0, 24, 48, and 72 h using the phase-contrast microscope with a magnification×100. To ensure documentation of the same region, the wells were marked across the wounded area. The untreated cells served as controls. Each experiment was repeated in triplicate.
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Statistical Analysis The SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied for statistical analysis. The association between SPOP expression and clinicopathologic features was analyzed using χ2 test. Spearman rank correlation analysis was used to analyze the relationship between SPOP expression in CRC and TNM stage. Survival was calculated by the Kaplan–Meier method, and differences in survival were determined by the log-rank analysis. A multivariable analysis of several independent prognostic factors was carried out using Cox’s proportional hazards regression model. The results are expressed as the mean±SD of at least three independent experiments. P < 0.05 was considered statistically significant.
Results Downregulation of SPOP in CRC Tissue Samples To investigate SPOP gene expression profile, a total of 126 paraffin-embedded colorectal tissue blocks were evaluated by IHC analysis. We found that SPOP was expressed at various levels in the CRC tissues and the adjacent noncancerous tissue samples (Fig. 1). Overall, 78 of 126 (61.90 %) cases showed low SPOP expression (SPOP − or SPOP +) in CRC tissues, whereas the remaining 48 (38.10 %) cases displayed high SPOP expression (SPOP ++ or SPOP +++) (Fig. 1, Table 1). The adjacent noncancerous colorectal tissues showed the strongest SPOPpositive staining (Fig. 1a). The protein expression of SPOP in CRC patients was forward examined by Western blotting analysis in 24 randomly selected pairs of CRC and their matched nontumor colorectal tissues. Five representative cases of the Western blotting result are shown in Fig. 2a. The relative quantity of SPOP protein expression was normalized to the β-actin of the same samples. SPOP protein was downregulated in the CRC tissues compared with their adjacent nontumor colorectal tissues in most of CRC patients (75 %, 18 of 24 patients), and the average SPOP protein level in 24 CRC tissues was significantly lower than that in their adjacent nontumor colorectal tissues (P=0.004) (Fig. 2b). To check SPOP mRNA level in CRC tissues, we performed real-time RT-PCR analysis in the 42 colorectal tumor tissues and their corresponding nontumor mucosal samples. As shown in Fig. 2c, the SPOP expression was significantly reduced in colorectal cancer samples as compared with that in their corresponding nontumor mucosal samples (P=0.0286).
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J Gastrointest Surg (2015) 19:1484–1496
Fig. 1 SPOP protein expression in CRC and surrounding nontumorous tissues detected by immunohistochemistry. a Normal CRC tissues distant from the tumor were scored as SPOP (+++); b well-differentiated CRC was scored as SPOP (++); c moderately differentiated CRC was scored as SPOP (+); and d Poorly differentiated CRC was scored as SPOP (−). Original magnification×200
Correlation Between SPOP Relative Expression and Clinicopathologic Factors in CRC The degree of SPOP immunohistochemical staining correlates with clinicopathologic characteristics. Clinicopathologic factors were compared between two SPOP expression groups (Table 1). We used χ2 test to analyze the correlation between immunohistochemical SPOP expression and various clinicopathologic characteristics of CRC patients. In colorectal tumor tissues from 126 CRC patients, decreased SPOP expression was found to be significantly correlated to poor differentiation (P = 0.013), distant metastasis (P = 0.003), gross type (P0.05; Table 1). To further clarify the relationship between SPOP expression and TNM stage, Spearman rank correlation analysis was employed. The result showed that SPOP expression in CRC was negative correlation with TNM stage, which suggested the more advanced clinical TNM stage corresponding to the lower expression level of SPOP in CRC (rs =0.306, P5 Location Colon Rectum Gross type Massive Ulcerative Infiltrating type Differentiation Well/moderate Poor Lymph node metastasis Negative Positive Invasive depth T1/T2 T3/T4 Distant metastasis M0 M1 TNM stage Stage I/II Stage III/IV
Total (n=126)
SPOP expression Low level (n=78)
High level (n=48)
70 56
48 30
22 26
45 81
32 46
13 35
P value
0.085
0.113
0.061 68 58
37 41
31 17
59 67
38 40
21 27
0.587
ORIGINAL ARTICLE
Properties and Clinical Relevance of Speckle-Type POZ Protein in Human Colorectal Cancer Junfei Xu & Feiran Wang & Haiyan Jiang & Yasu Jiang & Jinpeng Chen & Jun Qin
Received: 5 November 2014 / Accepted: 30 January 2015 / Published online: 29 May 2015 # 2015 The Society for Surgery of the Alimentary Tract
Abstract Background The aims of this study are to evaluate the effect of Speckle-type POZ protein (SPOP) in colorectal cancer (CRC) patients and explore its significance in the prognosis. Methods We used immunohistochemistry to detect the expression of SPOP in CRC. Moreover, this result was further confirmed at the protein and messenger RNA (mRNA) level in paired CRC specimens and matched adjacent noncancerous colon tissues by Western blotting and real-time quantitative PCR (qRT-PCR), respectively. Furthermore, we evaluate the effects of SPOP on CRC cell proliferation and migration in vitro. The Kaplan–Meier method and log-rank test were employed to compare the overall survival between SPOP low expression group and SPOP high expression group. Correlation of survival with clinicopathologic parameters, including SPOP level, was investigated with multivariate analyses. Results We confirmed frequent SPOP downregulation in both mRNA (P=0.0286) and protein (P=0.004) levels in CRC tissues as compared to matched adjacent nontumorous tissues. Besides, the downregulated SPOP expression in CRC tissues was significantly correlated to poor differentiation (P=0.013), distant metastasis (P=0.003), gross type (P75 % as 4. Moreover, staining intensity is rated on a scale of 0 to 3, with 0, negative; 1, weak; 2, moderate; and 3, strong. We created an immunoreactive score by multiplying the score for the percent of positive cells and the score for staining intensity. Theoretically, the ISS could range from 0 to 12. An ISS of 9~12 was considered strong immunoreactivity (+++), 5~8 was considered moderate (++), 1~4 was considered weak (+), and 0 was scored as negative (−). Sections were considered positive for SPOP when more than 25 % of tumor cells were stained in the cell cytoplasm.
J Gastrointest Surg (2015) 19:1484–1496
polyvinylidene fluoride (PVDF) membrane. Nonspecific binding was blocked for 2 h with 5 % nonfat milk in Trisbuffered saline containing 0.1 % Tween 20 (TBST). After incubation with anti-SPOP antibody (sc-66649, Santa Cruz Biotechnology, USA), anti-E-cadherin antibody (ab1416, Abcam, Cambridge, MA, USA), anti-vimentin antibody (ab92547, Abcam), anti-N-cadherin antibody (ab18203, Abcam), anti-Snail antibody (ab180714), anti-Slug antibody (ab27568, Abcam), and anti-Twist antibody (ab49254, Abcam) overnight at 4 °C, membranes were washed three times in TBST for 5 min and subsequently incubated secondary antibody with conjugated IRDye800 (1:5000, Rockland Gilbertsville, CA) at room temperature for 2 h, followed by scanning with an Odyssey infrared imaging system (LI-COR, Lincoln, NE), and analyzed with PDQuest 7.2.0 software (Bio-Rad). Real-Time RT-PCR The messenger RNA (mRNA) expression of SPOP was analyzed by quantitative real-time PCR. Total RNAs were extracted using the Trizol (Gibco). Quantitative real-time PCR was performed using HotStart-IT SYBR Green QPCR Master Mix (2×; USB Corporation, Cleveland, OH 75762, USA). According to the HotStart-IT protocol, 25-μl reactions were run of with 2 μl cDNA. RT-PCR experiments were performed in a LightCycler 480 system (Roche Applied Sciences). Cycling parameters were as follows: hot start at 95 °C for 10 min; 40 cycles of amplification/quantification at 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 30 s during which time fluorescence was measured. Melting curve analysis was performed using continuous fluorescence acquisition from 65 to 97 °C. These cycling parameters generated single amplicons for both primer sets used according to the presence of a single melt peak. The β-actin was selected as the internal reference. All quantitative real-time PCRs were repeated three times for each gene, and each sample was done in triplicate. Primer sequences were as follows: SPOP forward 5′-TGACCACCAG GTAGACAGCG-3′, SPOP reverse 5′-CCCGTTTCCCCC AAGTTA-3′. The β-actin gene served as an internal control. The sequences of the primers for β-actin were as follows: βactin forward 5′-AACTTCCGTTGCTGCCAT-3′, β-actin reverse 5′-TTTCTTCCACAGGGCTTTG-3′. Relative expression level for each target gene was normalized by the Ct value of β-actin (internal control) using a 2ΔΔCt relative quantification method.
Western Blotting Cell Proliferation Assay Coupled tumor and their adjacent nontumor tissues were taken for Western blot analysis. Total protein was extracted by lysis buffer containing protease inhibitors (Promega, Madison, WI). Equal amounts of protein separated by 10 % sulfate polyacrylamide gel electrophoresis and then transferred to a
Cell viability and proliferation was assessed using an MTT assay. Following transfection, HCT116 and LoVo cells were plated in 96-well plates at 1×103 cells/well and incubated for 24, 48, and 72 h. A total of 20 μl MTT (5 mg/ml, Sigma-
J Gastrointest Surg (2015) 19:1484–1496
Aldrich) was added to each corresponding test well and incubated for 4 h at 37 °C. The supernatant was then discarded, and 200 μl dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) was added to each well to dissolve the formazan. Optical density was assessed by measuring the absorbance of each well at 490 nm using a spectrophotometer (SpectraMax Plus384; Molecular Devices, Sunnyvale, CA, USA). All experiments were performed in triplicate. Colony Formation Assay After transfection of SPOP-siRNA/p-SPOP or control vector, HCT116 and LoVo cells (5×104 cells/well) were separately plated in a 24-well plate. After 24 h, the cells collected and seeded (1000–1500/well) in a fresh six-well plate for 14 days. Surviving colonies (>50 cells per colony) were counted after fixed with methanol/acetone (1:1) and stained with 5 % Gentian Violet (ICM Pharma, Singapore, Singapore), and then rinsed three times with PBS to remove excess dye, photographed, and counted. The experiment was carried out in triplicate wells for three times. Cell Migration Assay The cell migratory capacity was determined using transwell chambers with 8-mm diameters (Corning, Corning, NY, USA). Briefly, HCT116 and LoVo cells (1×105/well) were transfected and suspended in 100-μl serum-free medium and then added to the upper chamber of the inserts; DMEM medium (Gibco) containing 10 % FBS (500 μl) was added to the lower chamber as a chemoattractant. After culture for 24 h at 37 °C, nonmigrated cells on the upper surface were removed gently with a cotton swab and cells that migrated to the lower side of the department were fixed with methanol and dyed with 0.1 % crystal violet. Then, cells that migrated through the upper chamber were counted in five random fields under a light microscope, and the average value of five fields was expressed. All assays were performed in triplicate. Wound Healing Assay Cells were plated in six-well plates and incubated overnight; when the cells had grown by 90–100 %, they were scraped with a pipette tip to generate straight wounds. To eliminate dislodged cells, culture medium was removed and wells were washed with PBS. We measured the closure or filling of the wounds at 0, 24, 48, and 72 h using the phase-contrast microscope with a magnification×100. To ensure documentation of the same region, the wells were marked across the wounded area. The untreated cells served as controls. Each experiment was repeated in triplicate.
1487
Statistical Analysis The SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) was applied for statistical analysis. The association between SPOP expression and clinicopathologic features was analyzed using χ2 test. Spearman rank correlation analysis was used to analyze the relationship between SPOP expression in CRC and TNM stage. Survival was calculated by the Kaplan–Meier method, and differences in survival were determined by the log-rank analysis. A multivariable analysis of several independent prognostic factors was carried out using Cox’s proportional hazards regression model. The results are expressed as the mean±SD of at least three independent experiments. P < 0.05 was considered statistically significant.
Results Downregulation of SPOP in CRC Tissue Samples To investigate SPOP gene expression profile, a total of 126 paraffin-embedded colorectal tissue blocks were evaluated by IHC analysis. We found that SPOP was expressed at various levels in the CRC tissues and the adjacent noncancerous tissue samples (Fig. 1). Overall, 78 of 126 (61.90 %) cases showed low SPOP expression (SPOP − or SPOP +) in CRC tissues, whereas the remaining 48 (38.10 %) cases displayed high SPOP expression (SPOP ++ or SPOP +++) (Fig. 1, Table 1). The adjacent noncancerous colorectal tissues showed the strongest SPOPpositive staining (Fig. 1a). The protein expression of SPOP in CRC patients was forward examined by Western blotting analysis in 24 randomly selected pairs of CRC and their matched nontumor colorectal tissues. Five representative cases of the Western blotting result are shown in Fig. 2a. The relative quantity of SPOP protein expression was normalized to the β-actin of the same samples. SPOP protein was downregulated in the CRC tissues compared with their adjacent nontumor colorectal tissues in most of CRC patients (75 %, 18 of 24 patients), and the average SPOP protein level in 24 CRC tissues was significantly lower than that in their adjacent nontumor colorectal tissues (P=0.004) (Fig. 2b). To check SPOP mRNA level in CRC tissues, we performed real-time RT-PCR analysis in the 42 colorectal tumor tissues and their corresponding nontumor mucosal samples. As shown in Fig. 2c, the SPOP expression was significantly reduced in colorectal cancer samples as compared with that in their corresponding nontumor mucosal samples (P=0.0286).
1488
J Gastrointest Surg (2015) 19:1484–1496
Fig. 1 SPOP protein expression in CRC and surrounding nontumorous tissues detected by immunohistochemistry. a Normal CRC tissues distant from the tumor were scored as SPOP (+++); b well-differentiated CRC was scored as SPOP (++); c moderately differentiated CRC was scored as SPOP (+); and d Poorly differentiated CRC was scored as SPOP (−). Original magnification×200
Correlation Between SPOP Relative Expression and Clinicopathologic Factors in CRC The degree of SPOP immunohistochemical staining correlates with clinicopathologic characteristics. Clinicopathologic factors were compared between two SPOP expression groups (Table 1). We used χ2 test to analyze the correlation between immunohistochemical SPOP expression and various clinicopathologic characteristics of CRC patients. In colorectal tumor tissues from 126 CRC patients, decreased SPOP expression was found to be significantly correlated to poor differentiation (P = 0.013), distant metastasis (P = 0.003), gross type (P0.05; Table 1). To further clarify the relationship between SPOP expression and TNM stage, Spearman rank correlation analysis was employed. The result showed that SPOP expression in CRC was negative correlation with TNM stage, which suggested the more advanced clinical TNM stage corresponding to the lower expression level of SPOP in CRC (rs =0.306, P5 Location Colon Rectum Gross type Massive Ulcerative Infiltrating type Differentiation Well/moderate Poor Lymph node metastasis Negative Positive Invasive depth T1/T2 T3/T4 Distant metastasis M0 M1 TNM stage Stage I/II Stage III/IV
Total (n=126)
SPOP expression Low level (n=78)
High level (n=48)
70 56
48 30
22 26
45 81
32 46
13 35
P value
0.085
0.113
0.061 68 58
37 41
31 17
59 67
38 40
21 27
0.587
