β-Catenin and transcriptional factor TCF-4 (human T-cell factor-4) genes comprise the Wnt signal. The Wnt signal pathway plays an important role in malignant transformation. We hypothesize that the β-catenin and TCF-4 gene and Wnt signal are important in the progression of renal cell carcinoma (RCC). To test this hypothesis, we investigated TCF-4 splicing isoforms, β-catenin, and Wnt signal pathway (cyclin D1, c-myc, c-jun, and MMP7) in three RCC cell lines (A498, Caki-1, and Caki-2), 38 primary RCCs, and 29 normal kidney samples. We also analyzed the relationship between TCF-4 gene splicing isoforms, proliferation (proliferating cell nuclear antigen labeling index), and apoptosis [antiapoptotic factors (Bcl-2 and Bcl-xL), proapoptotic factors (Bak and Bax), and caspase-3] in RCC samples. In 38 RCC samples, four splicing isoforms of the TCF-4 gene were present in the region between exon 12 and exon 17. Thirty (79%) of 38 RCCs and all (100%) of the normal kidney samples showed mixed isoforms with both long and short reading frames in the COOH-terminal region, whereas the remaining 8 RCC samples showed only the long-form reading frame. Two COOH-terminal-binding protein sites were present only in the long-form reading frame. The eight RCCs that demonstrated only the long reading frame isoform showed early disease progression and poor prognosis. In these 8 RCC samples, down-regulation of cyclin D1, c-myc, c-jun, and MMP7 expression was observed at the mRNA level. In addition, a marked reduction of caspase-3 expression was also found at both the mRNA and the protein level. However, the β-catenin gene was not overexpressed at the mRNA level and protein level, and mutation and deletion were not observed in exon 3. In these three renal cell lines, there was no significant difference in TCF-4 mRNA expression before and after 5-Aza-2′-deoxycytidine treatment, and there appeared to be no splicing isoforms in the region between exon 1 and exon 11. These findings suggest that alteration in β-catenin is an infrequent event in RCC. In samples in which β-catenin was not overexpressed, the target genes of Wnt signal were regulated through TCF-4 splicing isoforms. The imbalance between TCF-4 gene splicing isoforms with long and short reading frames is associated with RCC progression through the inhibition of the apoptotic pathway. We demonstrate for the first time that TCF-4 gene splicing isoforms and the Wnt signal pathway can induce progression of RCC by the inhibition of apoptosis and not by the induction of cell proliferation.

The Wnt signaling pathway, also called the APC/β-catenin/TCF pathway, is one of the pivotal mechanisms involved in the regulation of development and cell growth (1). The human TCF-43 gene is involved in the Wnt signaling pathway (2, 3). TCF-4 belongs to a family of transcriptional factors and shares sequence homology with the HMG box (4, 5). Because the consensus motif for the TCF-4 binding site (A/TA/TCAAAG) is present in the promoter region (6, 7, 8, 9, 10), c-myc, cyclin D1, c-jun, and MMP7 are considered targets for the Wnt signaling pathway. In the TCF-4 gene, three major domains are present; β-catenin-binding domain in exon 1, the DNA-binding HMG boxes in exons 10 and 11, and the COOH-terminal-binding domain in exon 17 (11, 12). Various splicing isoforms of TCF-4 mRNA are present at the COOH-terminal region (13). These splicing isoforms create long and short open reading frames in the COOH-terminal region. Only the long reading form of these splicing isoforms preserves the binding domain of CtBP. Therefore, splicing isoforms involved in the COOH-terminal region are related to the regulatory function of transcription (14, 15). In colorectal cancer with microsatellite instability, frequent frameshift mutations involved in exon 17 have been reported that may modulate transcriptional activity through the truncation at the COOH-terminal region (12). Whether or not splicing isoforms of TCF-4 involved in the COOH-terminal region modulate the target genes of the Wnt signaling pathway remains unclear in RCC.

The prognosis of RCC varies widely and depends on cell type, stage, and grade. The heterogeneity of RCC probably reflects differences in the loss of apoptosis. Recently, it has been reported that the activation of the Wnt-1 signaling provides not only a cell-growth advantage to cancer cells but also a protection against apoptosis (14). We hypothesize that downstream target genes of Wnt signaling pathways, cellular proliferation, and apoptosis are involved in RCC. To test these hypotheses, mRNA expression of the TCF-4 gene and its splicing isoforms, Wnt signaling pathways, cell proliferation, and apoptosis were analyzed in renal cancer cell lines (A498, Caki-1, and Caki-2), 38 human RCC samples, and 29 normal kidney samples.

Patients.

Thirty-eight RCC samples were collected from patients who underwent nephrectomy (Shimane Medical University, Izumo, Japan). Mean age at the time of surgery was 60.3 years (range, 26–87 years). Median follow-up was 28 months (range, 5–76 months). None received any treatment before surgery. Seventeen patients with primary RCC received a postoperative IFN-α (600 million IU) monthly injection for 6 months. During the postoperative course, the patients were examined every 4 months with chest X-ray, abdominal ultrasonography, computed tomography, bone scan, and/or magnetic resonance imaging. Disease progression was defined as local recurrence, new development of distant metastasis, or lymph node involvement. All of the tumor specimens were staged based on the TNM classification of malignant tumors by the Union International Contre Cancer (16). The staging was as follows: pT1-T3N0M0 in 33 cases, pT3N0M1 in 4 cases, and pT3N2M1 in 1 case; G1 in 18, G2 in 17, and G3 in 3 cases; and clear in 26, clear/granular in 7, chromophobe in 2, papillary in 2, and spindle in 1 case. In addition, the Robson staging system (17) was also used (stage I/II in 28 and III/IV in 10 cases).

Tissue Preparation.

All of the 38 specimens of primary RCCs and the 29 matched normal tissues obtained from the ipsilateral kidney were informative. One-half of each tissue sample was fixed in 10% buffered formalin (pH 7.0) and embedded in paraffin wax. Five-μm-thick sections were used for H&E staining for histological evaluation. The remaining one-half of each sample was immediately frozen and stored at −80°C until analyzed.

Cell Culture.

The human RCC cell lines A498, Cki-1, and Caki-2 were obtained from the American type of Culture Collection (Manassas, VA). A498 and Caki-1 cell lines were maintained in RPMI 1640 with l-glutamine and sodium pyruvate. The Caki-2 cell line was incubated in McCoy’s 5A medium with l-glutamine and 10% FCS was added to all of the media. The cells were maintained in a humidified atmosphere of 5% CO2/95% air at 37°C.

Nucleic Acid Extraction.

Genomic DNA was extracted using DNAzol Reagent (Life Technologies, Inc., Gaithersburg, MD). Total RNA was extracted by the Isogen reagent (Nippon Gene, Tokyo, Japan). The RNA pellet obtained after isopropanol and ethanol precipitation was dried, and resuspended in 50 μl of RNase-free water. RNA specimens were stored in aliquots at −80°C. The concentrations of DNA and RNA were determined by spectrophotometer, and their integrity was checked by each gel electrophoresis.

Mutational Analysis.

Genomic DNA from each sample was amplified using the primers described in Table 1. The location of the primers is shown in Fig. 1, a and b. A genomic DNA sample (100 ng) was diluted into 20 μl of solution containing 20 mm dNTP, 500 nm each primer, 0.5 units of Taq polymerase, and PCR reaction buffer provided by the manufacturer (TaKaRa, Kyoto, Japan). DNA samples were amplified through 35 cycles on a Thermal Cycler (MJ Research) at 94°C denaturation for 30 s, at 60°C annealing for exon 3 (B3S-B3AS) of β−catenin for 30 s, at 55°C annealing for exons 1, 10, 11, and 17 of TCF-4 for 30 s, and at 72°C extension for 45 s. In each set of PCR, a tube without template DNA was served as a negative control. Electrophoreses of the amplified PCR products were performed in 2% agarose gels containing ethidium bromide in Tris-Borate EDTA buffer. After electrophoresis, gels were examined using a UV transilluminator and were verified as a single band. A SSCP analysis was performed using a nonradioactive approach. Briefly, we mixed 5 μl of PCR product and 5 μl of SSCP loading dye, composed of 850 μl of formamide, 50 μl 10 mm EDTA (pH 8.0), 50 μl of 1.5% Ficoll in formamide, and 50 μl of 0.05% bromphenol blue and 0.05% xylene cyanol. The mixture was placed on ice immediately after denaturation at 95°C for 5 min. Then, one-half of the mixture volume was loaded onto 12% polyacrylamide gel. Electrophoresis was run using Tris-glycine buffer at both 10°C and 20°C for 5 h under the condition of 55 W. To improve the sensitivity of the detection of β-catenin mutation, we also performed TGGE. After amplifying the sample DNA using B3AS and sense primer with GC-clamp at 5′ end (BS3GC), we performed electrophoresis using Tris-acetate buffer in 12% polyacrylamide gel including 6 m urea with the temperatures ranging from 52°C to 68°C for 3 h at 150 V. After completing the electrophoresis, the gel was stained with a 1:10,000 solution of GelStar (BMA, Rockland, ME) in Tris-acetate buffer. In case of abnormal band shifts, the bands were cut and incubated with distilled water for 20 min at 95°C. The second PCR, with a supernatant of gel solution as a template, was carried out under the same conditions except for the PCR round cycles (30 cycles). Purified PCR products were used as templates for direct sequencing. After verification of the second PCR product as a single band on 2% agarose gel, the direct sequencing of the second PCR product by the dye terminator method was performed in both directions according to the manufacturer’s instructions (Applied BioSystem, Foster City, CA).

Deletion screening for the β-catenin gene was performed by PCR of genomic DNA with primers spanning exon 3 (including the consensus motif for the GSK-3β phosphorylation site). A DNA sample (100 ng) was amplified by 35 cycles on a Thermal Cycler (MJ Research): denaturation at 94°C for 50 s, annealing for the region encompassing exon 3 (Bi2S-B4eAS) at 56°C for 60 s, and extension at 72°C for 90 s. For PCR reaction, a tube without template DNA was served as a negative control. Each amplified PCR product was electrophoresed separately in series of 1.0, 1.5, and 2.0% agarose gels containing 2 mg/ml of ethidium bromide in Tris-Borate EDTA buffer.

cDNA Preparation and RT-PCR Analysis.

One μg of RNA was added to 0.5 μg of oligodeoxythymidilic acid primer (TaKaRa), and prepared to a final volume of 20 μl. The samples were placed at 55°C for 5 min and then cooled on ice. The primer RNA mixture was then combined with 0.25 unit of Avian myeloblastosis virus reverse transcriptase (TaKaRa) and 0.5 unit of RNase inhibitor. Finally, the mixture was added to 1 mm dNTP, 50 mm Tris-HCl, 75 mm KCl, and 5 mm MgCl2. The reverse transcriptase reaction was then carried out at 45°C for 45 min. The cDNA was then incubated at 95°C for 5 min to inactivate the reverse transcriptase. Samples were stored at −20°C until used.

The primer sets used in this study are shown in Table 1. The cDNA samples (2 μl) were diluted into 20 μl of solution containing 50 mm dNTP, 500 nm each primer, 0.5 units of Taq polymerase, and PCR reaction buffer provided by the manufacturer (TaKaRa). For a semiquantitative analysis of the amplified products, we prepared three other dilution sets of each sample (original sample, 1:2, 1:4, and 1:8 samples), and using these samples, we performed RT-PCR. Annealing temperature was 55°C for all of the RT-PCRs except MMP7 (62°C). The PCR products were electrophoresed on 2.0% agarose gel, and the expression level of these genes was evaluated by an AlphaImager 2200 (Alpha Innotech Corporation, San Leandro, CA) and expression of each gene relative to G3PDH expression was quantified and expressed as arbitrary units (AUs). We adjusted PCR cycle until 4 preparations in each sample were within exponential phase. We applied 32 cycles to Bcl-xL and Bak, and 30 cycles to β-catenin, cyclin D1, MMP7, Bcl-2, and G3PDH, and 26 cycles each to c-myc, c-jun, Bax, and caspase-3.

5-Aza-dC Treatment.

To screen whether an epigenetic alteration occurs in the TCF-4 gene in the A498, Caki-1, and Caki-2 cell lines, 5-Aza-dC was added to the fresh medium at the concentration of 5 μm in duplicate. The cultured cells were harvested after 4 days of 5-Aza-dC treatment. Using cDNA obtained from these renal cancer cell lines, we analyzed the difference in expression level of TCF-4 mRNA between before and after 5-Aza-dC treatments by RT-PCR using S1F and S1R primer sets (Table 1).

Determination of Splicing Isoform in TCF-4 mRNA.

Using cDNA samples of renal cancers as templates, splicing isoforms of TCF-4 mRNA encompassing the regions between exons 12 and 17 were determined (Fig. 1,c). In addition, cDNA samples of renal cancer cell lines were used to determine splicing isoforms in the region between exon 1 and exon 11, using the primer sets shown in Table 1. The sequence of forward primer P12F is located within exon 12, and that of forward primer P13F corresponds to the initial part of exon 13 and the last 2bp of exon 12. The sequence of reverse primer P17LR is present within exon 17. First PCR was performed using primer set P12F and P17LR or P13F and P17LR, and PCR products S2A and S2B, respectively. Then, nested second PCRs were performed using S2A and S2B PCR products as templates with primers (P14F and P17SR) spanning the region between exon 14 and 17. In some cases, the second round S2B PCRs were done using the S2A products as templates. Then, further PCR was performed using the second S2B PCR products as templates and primers P14F and P17SR. The location of primers P14F and P17SR were within exons 14 and 17, respectively. The precise locations of these primers are shown in Fig. 2. The PCR products were applied to 3% agarose gel electrophoresis using Nusieve CTG agarose (FMC BioProducts, Rockland, ME) or 12% polyacryl amide gel electrophoresis. Sequence of each splicing isoform was further confirmed using the dye-terminator sequencing method.

Immunostaining.

Immunostaining of β-catenin, caspase-3, and PCNA was performed on 5-μm-thick consecutive sections obtained from paraffin-embedded materials, by using mouse monoclonal antibody for β-catenin (1:500; Transduction Laboratories, Lexington, KY), rabbit polyclonal antibody for caspase-3 (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and mouse monoclonal antibody for PCNA (PC-10; Dako, Carpenteria, CA). Antigen retrieval was accomplished using citrate buffer (10 mm; pH 6.0) before the incubation of the primary antibody. In negative controls, the primary antibody was replaced with a nonimmune serum. 3,3′-diaminobenzidine (Sigma-Aldrich) was used as the chromogen, and counterstaining was performed using hematoxylin. To improve the sensitivity of caspase-3 detection, the technique of catalyzed signal amplification (CSA; Dako) was applied in addition to the usual immunostaining.

Statistical Analysis.

The relationship between TCF-4 splicing isoforms and mRNA expression was analyzed by the Mann-Whitney test. To evaluate the prognostic relevance of the variables, the probability was computed from the day of surgery until the occurrence of disease progression or until death or last follow-up. The curves were made using a Kaplan-Meier method (18), and the difference between curves was analyzed using a log-rank test. Multivariate survival analysis was carried out using a proportional hazard model. P of less than 0.05 was regarded as statistically significant.

TCF-4 Splicing Isoforms and Expression of Cyclin D1, c-myc, MMP7, and c-jun mRNA.

Four splicing isoforms of TCF-4 mRNA were detected in 38 RCC tissues: exons 12-13-14-15-17; exons 12-13-14-17; exons 12-14-15-17; and exons 12-14-17. The exon composition of these splicing isoforms was confirmed by DNA sequencing. In addition, there appeared to be no splicing isoforms containing exon 16 in RCC samples. Because individual RCC samples did not always exclusively exhibit a single isoform of TCF-4 mRNA, the combination patterns of splicing isoforms between exons 12 and 17 were evaluated in each RCC sample. Four patterns of splicing isoforms were detected as follows: type I, exon 12-13-14-17; type II, combination with exon 12-14-17 and exon 12-14-15-17; type III, combination with exon 12-13-14-17 and exon 12-13-14-15-17; and type IV, exon 12-14-17. (These types correspond to I, II, III, and IV in Fig. 3 a.) For example, in RCC with type I, both nested PCR1 and nested PCR2 showed only a single band corresponding to 75 bp, which confirmed the presence of exon 13 and the absence of exon 15. Therefore, the exons include 12, 13, 14, and 17. In RCC with type II, nested PCR1 showed two bands corresponding to 148 bp and 75 bp, whereas nested PCR2 showed no band. This indicates that exon 13 is absent. Thus, exons composition consisted of exons 12-14-15-17 or exons 12-14-17. In RCC with type III, both nested PCRs 1 and 2 showed two bands each, corresponding to 148 bp and 75 bp, indicating exon composition of 12-13-14-15-17 or 12-13-14-17. RCC with type IV showed only a single band in nested PCR1, whereas there was no band detectable in nested PCR2, suggesting the absence of exon 15. Thus, exon composition was considered exons 12-14-17.

In normal kidney samples, five patterns of electrophoresis data were obtained, and more complicated patterns of splicing isoforms were present than in RCC samples (Fig. 3,b). For example, in specimen A (Fig. 3,b, Lane A), the exon composition was exon 12-13-14-15-17 and exon 12-14-15-17. In specimens B, C, D, and E, exon 16-containing splicing isoforms are indicated by asterisks (∗) in Fig. 3 b. The exon compositions were: specimen B, exon 12-14-15-17, exon 12-14-17, exon 12-13-14-15-16-17, and exon 12-13-14-15-17; specimen C, exon 12-13-14-17, exon 12-(13)-14-16-17, and exon 12-(13)-14-15-17; specimen D, exon 12-13-14-15-16-17, exon 12-13-14-15-17, and exon 12-(13)-14-17, exon 12-13-14-15-16-17, and exon 12-13-14-15-17; and specimen E, exon 12-13-14-15-17, exon 12-14-15-17, and exon 12-13-14-16-17.

The reading frame of each splicing isoform is shown in Fig. 3 c. In RCC samples, irrespective of whether or not exon 13 is present, exon 15 skipping isoforms result in a long reading frame (L form). In L form, there are two binding sites (PLVL and PLVLS). On the other hand, isoforms with exon 15 result in a short reading frame (S form), in which the CtBP binding site is lacking. In normal kidney samples, the presence of exon 13 or exon 16 did not lead to a long reading frame. Thus, twenty-nine normal kidney samples showed L and S forms of the reading frame.

TCF-4 mRNA expression was detected in all three of the renal cancer cell lines. In exons 1–11, there was no splicing variant in A498, Caki-1, and Caki-2 renal cancer cell lines (Fig. 4,a). The expression level of TCF-4 mRNA was not increased after 5-Aza-dC treatment (Fig. 4 b).

The panel of mRNA expression of cyclin D1, c-myc, MMP7, and c-jun in relation to the patterns of TCF-4 splicing isoforms are shown in Fig. 5,a. C-myc, cyclin D1, c-jun, and MMP7 are considered targets for the Wnt signaling pathway. Only an L form of reading frame was present in types I and IV, whereas two reading frames (L and S forms) were present in the types II and III. As shown in Fig. 5 b, expression levels of cyclin D1 and c-myc mRNA are significantly lower in eight tumors exhibiting types I and IV isoforms and 30 tumors exhibiting types II and III isoforms. A similar tendency was observed for MMP7 mRNA expression (P = 0.07), but there was no significant difference in the levels of c-jun mRNA expression between the two groups.

TCF-4 Splicing Isoforms in Relation to Cell Proliferation and Apoptosis.

The PCNA labeling index was significantly lower in splicing isoforms types I, II, and IV [mean, 3.7 cell/high power field (×400)] than that in type IV isoforms (mean 15.9 cell/high power field; P < 0.05). Fig. 6,a demonstrates the relationship among types of TCF-4 splicing isoforms and reading frames with mRNA levels of antiapoptotic (Bcl-2 and Bcl-xL) and pro-apoptotic (Bak and Bax) factors and caspase-3. As shown in Fig. 6 b, the expression level of antiapoptotic factor (Bcl-2 and Bcl-xL) mRNA was significantly lower in the TCF-4 splicing isoform types I and IV than in the type II and III isoforms, respectively (P < 0.05). Interestingly, the expression level of proapoptotic factor Bak mRNA showed the parallel change as observed in the expression level of Bcl-2 and Bcl-xL mRNA (P < 0.05). On the other hand, expression of Bax mRNA was not significantly different among splicing isoform types.

There was a significant difference in the expression level of caspase-3 mRNA between isoform types I and IV and types II and III. RCC samples with isoform type I and IV showed significantly lower levels of expression, as compared with type II and III isoforms (P < 0.05). In addition, caspase-3 expression at protein level showed a parallel change in expression at mRNA level. As shown in Fig. 7, RCCs with the isoform types I and IV showed weak immunoreactivity to both caspase-3 and PCNA, whereas RCC with type III isoform showed high caspase-3 immunoreactivity and high PCNA labeling index.

SSCP Analysis of TCF-4 and Its Relation to Expression of Cyclin D1, c-myc, MMP7, and c-jun mRNA.

Thirteen samples showed band shifts in exon 17 of TCF-4 (Fig. 8,a), in which the COOH-terminal binding domain was present (Fig. 3,c). In these 13 RCC samples, sample A was substituted for sample C with the corresponding amino acid changing from proline (CCC) to threonine (ACC), indicating a SNP (Fig. 8,b). In A498, Caki-1, and Caki-2 renal cancer cell lines, there was no mutation in exon 17. The COOH-terminal-binding domain located in exon 17 of TCF-4 might modulate transcriptional activity on the target gene. There was no significant relationship between this SNP and Wnt signal target gene expression (cyclin D1, c-myc, MMP7, and c-jun) at the mRNA level (Fig. 8 c).

TCF-4 Splicing Isoforms and Prognosis.

Univariate analyses for disease progression and survival revealed that RCC patients with isoform types I and IV showed significantly lower progression-free probability (Fig. 9,a), as well as decreased survival probability (Fig. 9,b), than those with types II and III isoform, respectively (P < 0.01). As shown in Fig. 10, multivariate analysis also demonstrated that isoform types I and IV were significant and independent discriminators of Robson staging and nuclear grade.

Alteration of β-Catenin.

PCR-based SSCP analysis encompassing the region of exon 3, in which the GSK-3β phosphorylation motif is present, demonstrated no alteration in any of the 38 samples (Fig. 10,a). In addition, TGGE analysis to improve the sensitivity for detecting exon 3 mutations also demonstrated no alteration in these samples (Fig. 10,b). Deletion screening for exon 3 showed no abnormalities in any of the 38 samples (Fig. 10,c). RT-PCR analysis showed no overexpression of β-catenin at the mRNA level (Fig. 10,d). Only three RCC samples showed loss of β-catenin expression at mRNA level. These samples showed L and S forms of the reading frame. There was heterogeneity in β-catenin immunostaining with cells showing membranous staining and/or intracytoplasmic staining, but no nuclear accumulation (Fig. 11).

The Wnt signal pathway is composed of β-catenin and transcriptional factor TCF-4 (2, 3, 19, 20, 21). These factors activate the target genes that preserve the consensus motif (A/TA/TCAAAG) for TCF-4 binding in the promoter region (6). Recent studies have shown that cyclin D1, c-myc, MMP7, and c-jun could be target genes for the Wnt signal pathway (7, 8, 9, 10). Therefore, Wnt signal may induce cell proliferation through activation of these target genes (22, 23, 24, 25). To our knowledge, there is no report on RCCs regarding the significance of the Wnt signal factors in cell proliferation and/or apoptosis through regulation of downstream target genes. In this study, we evaluated the significance of the Wnt signal pathway in RCC through the analysis of cyclin D1, c-myc, MMP7, and c-jun in relation to β-catenin and TCF-4 alterations.

TCF-4 expression has been intensively investigated in the epithelium of the gastrointestinal tract (26, 27), and the presence of splicing isoforms in TCF-4 mRNA has been shown in colorectal cancer cell lines (13). There have been no reports regarding the mRNA expression of TCF-4 and its isoforms in RCC. In this study, we demonstrated that TCF-4 mRNA is expressed in normal and cancerous kidney tissue. We identified various splicing isoforms in the region spanning exon 12 to 17. In renal cancer tissues, however, there were only four splicing isoforms identified. The open reading frame of the TCF-4 gene appears to be determined by the presence of exon 15. Because each individual RCC did not always show an exclusive dominant splicing isoform, combination types of TCF-4 isoforms were analyzed. As shown in Fig. 3, the corresponding reading frames of each type of isoforms were different from each other: type I, L form only; type II, L and S forms; type III, L and S forms; and type IV, L form only. In normal kidney samples, although there were various splicing isoforms, all of the samples showed the L and S forms of the reading frame. It is important to note that two CtBP binding sites (PLSL and PLSLV in Fig. 4 b) are present only in the L form. Thus, RCCs with the L form could suppress the transcriptional activity through binding with CtBP, whereas those RCCs with the S form might escape from the negative regulation of transcription. In the present study, we have shown that isoforms type I and IV down-regulate expression of the target genes (cyclin D1, c-myc, MMP7, and c-jun) as compared with types II and III isoforms. In types II and III isoforms, it is possible that the balance between L and S forms affects the expression level of target genes.

Previous studies have shown that cyclin D1, c-myc, and c-jun genes affect the cell cycle during cell proliferation (28, 29, 30). In the present study, the TCF-4 type I and IV isoforms (L form only) have worse progression-free and survival probabilities despite the marked reduction of Wnt target gene expression. Because c-myc and cyclin D1 are involved in the pathway of apoptosis as well as in proliferation (31, 32, 33), antiapoptosis factors and proapoptosis factors were analyzed in relation to splicing types of Tcf4 isoforms. Both Bcl-2 and Bcl-xL represent intrinsic apoptotic inhibitors (34, 35). If Bcl-2 and Bcl-xL proteins are present, Bak and Bax can promote apoptosis by blocking the antiapoptotic function of Bcl-2 and Bcl-xL through their ability to form homodimers and/or heterodimers with Bcl-2 and Bcl-xL proteins (36, 37). However, it has also been reported that Bcl-xL can inhibit cell death by a mechanism independent of Bak and Bax (38). Likewise, Bak and Bax may have a proapoptotic function that is independent of their ability to heterodimerize with Bcl-2 and Bcl-xL proteins (39). In comparison with types II and III isoforms, the expression levels of antiapoptotic factors Bcl-2 and Bcl-xL as well as proapoptotic Bak were significantly reduced in type I and IV isoforms. In addition, a significant reduction of caspase-3 expression at both mRNA and protein levels was observed in type I and IV isoforms. Considering that caspase-3 comprises a final and critical step of the apoptotic pathway, irrespective of an intrinsic or extrinsic apoptotic stimulus (40), these findings appear to suggest that (a) TCF-4 relating apoptosis is not regulated by the involvement of antiapoptotic factors but by proapoptotic factor Bak, and (b) RCC tissues harboring splicing types I and IV isoforms of TCF-4 showed poor prognosis through down-regulation of apoptosis inhibition. Recently, using a peptide nucleic acid model, an antisense c-myc treatment has been reported to strongly inhibit both cell proliferation and apoptosis (41). In addition, caspase-3 has been involved in the c-myc-induced apoptosis pathway (32). In this study, types I and IV showed also a low level of PCNA labeling index in addition to the low levels of caspase-3 expression. The majority (76%) of RCCs exhibited the type III isoform, in which L and S forms of the reading frames are concomitantly present. In contrast to type I and IV isoforms, in which only the L form reading frame is present, the type III isoform was not related to prognosis. Again, in RCC, the presence of exon 15 determines the L and S forms of reading frame (Fig. 4). RCCs with the L form reading frame result in the inhibition of apoptosis through down-regulation of Wnt target gene expression.

We further investigated the alteration of β-catenin in RCC at the genomic DNA, mRNA, and protein levels. Because β-catenin is degraded through the ubiquitin proteasome by GSK-3β (25, 42), the genomic changes in exon 3 of the β-catenin, in which the consensus motif for the phosphorylation site of GSK-3β is located, were extensively analyzed. SSCP, combined with the more sensitive TGGE method, could not detect any mutations in exon 3 of the β-catenin gene. Although large deletions were reported to be present in the regions spanning exon 3 in melanoma and hepatocellular carcinoma (43, 44), there were no deletions found in our RCC samples. Therefore, at the genomic level, β-catenin alteration appeared to be a less frequent event in RCC. In addition, the lack of β-catenin mRNA overexpression or intracellular accumulation strongly suggested that activation of the Wnt signal through β-catenin alteration was not a significant event in RCC.

The genomic structure of TCF-4 contains three major domains, the β-catenin binding domain (exon 1), the DNA-binding HMG Boxes (exons 10 and 11), and the CtBP binding sites (exon 17; Refs. 6, 11, and 13). In the present study, the SSCP analysis demonstrated no mutations in exons 1, 10, or 12, but did detect one SNP (proline to threonine) in exon 17. Exon 17 of the TCF-4 appeared to be important because the binding sites for the CtBP are located in the COOH-terminal region. The CtBP has been reported to repress the transcriptional activity of the target genes (15). However, we could not verify any relationship between the SNP in exon 17 of TCF-4 and the expression levels of the Wnt signal target genes. An increased number of renal tumors should be investigated to determine differences in target gene expression between the presence and absence of this SNP.

It has been reported that the activated β-catenin/TCF transcription could inhibit apoptosis (14). However, in this study of renal cancer, there does not appear to be overexpression of β-catenin leading to activation of β-catenin/TCF transcription. On the other hand, recent investigations have shown that overexpression of β-catenin induces apoptosis independent of the transcriptional factor LEF1 overexpression (45). In summary, the lack of overexpression of β-catenin appears to be insufficient to induce apoptosis, and renal cancer of the L form splicing isoform with the CtBP site could suppress transcriptional activity of TCF4. Furthermore, the L form reading frame results in down-regulation of MMP7. Initially known as an enzyme that degrades tissue matrix components (9), MMP7 has also been implicated in angiogenesis by generating angiostatin, a potent inhibitor for tumor vascularization, from circulating plasminogen (46). In the present study, type I and IV isoforms, exhibiting the L form reading frame, correlate with the progression of RCC through the inhibition of both apoptosis and antiangiogenesis. In conclusion, the present study demonstrates for the first time that TCF-4 splicing isoforms and the Wnt signal pathway are involved in the progression of RCC through the inhibition of apoptosis.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by NIH Grants RO1DK55040, RO1DK511-1, RO1DK47517, and RO1AG16870 and Veterans Affairs Merit review and Veterans Affairs Research Enhancement Award Program grants.

3

The abbreviations used are: TCF-4, T-cell factor-4; PCNA, proliferating cell nuclear antigen; CtBP, COOH-terminal-binding protein; SSCP, single-strand conformational polymorphism; TGGE, temperature gradient gel electrophoresis; RT-PCR, reverse transcription-PCR; 5-Aza-dC, 5-Aza-2′-deoxycytidine; RCC, renal cell carcinoma; SNP, single nucleotide polymorphism; HMG, high mobility group; GSK-3β, glycogen synthase kinase 3 beta; G3PDH, glyceraldehyde 3 phosphate dehydronase.

Fig. 1.

Schematic presentation of the β-catenin and TCF-4 genes. a, relationship between the β-catenin gene at the 5′ end and primer location are shown. b, location of the primer sets used for PCR-SSCP analysis of TCF-4 gene, β-catenin-binding domain (Exon 1), DNA-binding HMG Box (Exon 10 and Exon 11), and COOH-terminal binding domain (Exon 17). c, locations of the primer sets used for the determination of TCF-4 splicing isoforms between the regions encompassing exon 12 to exon 17.

Fig. 1.

Schematic presentation of the β-catenin and TCF-4 genes. a, relationship between the β-catenin gene at the 5′ end and primer location are shown. b, location of the primer sets used for PCR-SSCP analysis of TCF-4 gene, β-catenin-binding domain (Exon 1), DNA-binding HMG Box (Exon 10 and Exon 11), and COOH-terminal binding domain (Exon 17). c, locations of the primer sets used for the determination of TCF-4 splicing isoforms between the regions encompassing exon 12 to exon 17.

Close modal
Fig. 2.

cDNA sequence of TCF-4 region between exon 12 and exon 17, and its relationship to primer sequences are shown. Underlined, the primer sequences.

Fig. 2.

cDNA sequence of TCF-4 region between exon 12 and exon 17, and its relationship to primer sequences are shown. Underlined, the primer sequences.

Close modal
Fig. 3.

Splicing isoforms of TCF-4 in the regions between exon 12 and 17, and the corresponding reading frames. a, exon composition was determined by the location of the primers shown in Fig. 2,a. There were four splicing types (I, II, III, and IV) in renal cancer on 3% agarose gel electrophoresis. The lateral lanes show 100-bp DNA ladder. b, exon compositions were determined by the location of the primers shown in Fig. 2 a. There were five splicing types (A, B, C, D, and E) in normal kidney on 12% PAGE. ∗, the splicing isoforms, which includes exon 16. The lateral lanes, show 25-bp DNA ladder. c, exon compositions and the corresponding reading frames were shown. Splicing isoform with exons 12-13-14-15-17 or exons 12-14-15-17 showed a short reading frame at COOH-terminal region (S form). On the other hand, splicing isoforms with exons 12-13-14-17 or exons 12-14-17 showed a long reading frame (L form), where two CtBP binding sites (PLSL or PLSLV) were preserved. ∗, the insertion of stop codon.

Fig. 3.

Splicing isoforms of TCF-4 in the regions between exon 12 and 17, and the corresponding reading frames. a, exon composition was determined by the location of the primers shown in Fig. 2,a. There were four splicing types (I, II, III, and IV) in renal cancer on 3% agarose gel electrophoresis. The lateral lanes show 100-bp DNA ladder. b, exon compositions were determined by the location of the primers shown in Fig. 2 a. There were five splicing types (A, B, C, D, and E) in normal kidney on 12% PAGE. ∗, the splicing isoforms, which includes exon 16. The lateral lanes, show 25-bp DNA ladder. c, exon compositions and the corresponding reading frames were shown. Splicing isoform with exons 12-13-14-15-17 or exons 12-14-15-17 showed a short reading frame at COOH-terminal region (S form). On the other hand, splicing isoforms with exons 12-13-14-17 or exons 12-14-17 showed a long reading frame (L form), where two CtBP binding sites (PLSL or PLSLV) were preserved. ∗, the insertion of stop codon.

Close modal
Fig. 4.

Splicing isoforms between exon 1 and exon 11 and the effects of 5-Aza-dC treatment on TCF-4 expression in RCC cell lines. a, there were no splicing isoforms in the region encompassing between exon 1 and exon 11. A, B, and C correspond to Caki-1, Caki-2 and A498 cell lines, respectively. b, no significant difference was observed between in TCF-4 mRNA expression in Caki-1, Caki-2, and A498 RCC cell lines before 5-Aza-dC treatment and in TCF-4 mRNA expression in Caki-1, Caki-2, and A498 RCC cell lines after 5-Aza-dC treatment. (+), 4 days after 5-Aza-dc treatment; (−), before 5-Aza-dC treatment.

Fig. 4.

Splicing isoforms between exon 1 and exon 11 and the effects of 5-Aza-dC treatment on TCF-4 expression in RCC cell lines. a, there were no splicing isoforms in the region encompassing between exon 1 and exon 11. A, B, and C correspond to Caki-1, Caki-2 and A498 cell lines, respectively. b, no significant difference was observed between in TCF-4 mRNA expression in Caki-1, Caki-2, and A498 RCC cell lines before 5-Aza-dC treatment and in TCF-4 mRNA expression in Caki-1, Caki-2, and A498 RCC cell lines after 5-Aza-dC treatment. (+), 4 days after 5-Aza-dc treatment; (−), before 5-Aza-dC treatment.

Close modal
Fig. 5.

Splicing types of TCF-4 isoforms and expression of c-myc, cyclin D1, MMP7, and c-jun at mRNA level are shown. a, representative types of TCF-4 isoforms and expression levels of the mRNA. Typically, in types I and IV isoforms, the expression levels of c-myc and cyclin D1 mRNA were markedly decreased, whereas in type III isoform, the expression level varied from one RCC to another. In type II isoform, the expression level was somewhat decreased. MMP7 and c-jun expression at mRNA level was also decreased in comparison with that in the remaining types. At the bottom, corresponding types of reading frame. b, expression of c-myc and cyclin D1 at the mRNA level was significantly lower in the types I and IV isoforms than in the types II and III isoforms. Matrix metalloproteinase 7 expression showed the same tendency, however, the difference did not reach statistical significance (P = 0.07). No significant difference in c-jun expression was found between types I and IV isoforms and types II and III isoforms. A.U., arbitrary unit(s); L, long reading frame; S, short reading frame.

Fig. 5.

Splicing types of TCF-4 isoforms and expression of c-myc, cyclin D1, MMP7, and c-jun at mRNA level are shown. a, representative types of TCF-4 isoforms and expression levels of the mRNA. Typically, in types I and IV isoforms, the expression levels of c-myc and cyclin D1 mRNA were markedly decreased, whereas in type III isoform, the expression level varied from one RCC to another. In type II isoform, the expression level was somewhat decreased. MMP7 and c-jun expression at mRNA level was also decreased in comparison with that in the remaining types. At the bottom, corresponding types of reading frame. b, expression of c-myc and cyclin D1 at the mRNA level was significantly lower in the types I and IV isoforms than in the types II and III isoforms. Matrix metalloproteinase 7 expression showed the same tendency, however, the difference did not reach statistical significance (P = 0.07). No significant difference in c-jun expression was found between types I and IV isoforms and types II and III isoforms. A.U., arbitrary unit(s); L, long reading frame; S, short reading frame.

Close modal
Fig. 6.

Splicing types of TCF-4 and expression of anti- and proapoptotic factors at mRNA level. a, representative types of isoforms and expression levels of the mRNA. Typically, the type I and IV isoforms exhibited lower levels of Bcl-2, Bcl-xL, Bak, and caspase-3 mRNA in comparison with type III isoform. As for Bax expression, no significant difference was observed among isoform types. b, expression of Bcl-2, Bcl-xL, Bak, and caspase-3 at mRNA level was significantly lower in types I and IV isoforms than in types II and III isoforms. On the other hand, no significant difference in Bax expression was found between types I and IV isoforms and types II and III isoforms. A.U., arbitrary unit(s); L, long reading frame; S, short reading frame.

Fig. 6.

Splicing types of TCF-4 and expression of anti- and proapoptotic factors at mRNA level. a, representative types of isoforms and expression levels of the mRNA. Typically, the type I and IV isoforms exhibited lower levels of Bcl-2, Bcl-xL, Bak, and caspase-3 mRNA in comparison with type III isoform. As for Bax expression, no significant difference was observed among isoform types. b, expression of Bcl-2, Bcl-xL, Bak, and caspase-3 at mRNA level was significantly lower in types I and IV isoforms than in types II and III isoforms. On the other hand, no significant difference in Bax expression was found between types I and IV isoforms and types II and III isoforms. A.U., arbitrary unit(s); L, long reading frame; S, short reading frame.

Close modal
Fig. 7.

Splicing types of TCF-4 in relation to immunostaining of caspase-3 (a, c, and e) and PCNA (b, d, and f). In type I isoform (a and b, pT2N0M0, G2) and type IV isoform (e and f, pT3N0M0, G3), immunoreactivity of both caspase-3 and PCNA was markedly reduced, whereas in type III isoform (c and d, pT3N0M0, G3), both immunoreactivities were strong. These alterations in caspase-3 immunoreactivity were parallel with the change in caspase-3 expression at mRNA level. L, long reading frame; S, short reading frame.

Fig. 7.

Splicing types of TCF-4 in relation to immunostaining of caspase-3 (a, c, and e) and PCNA (b, d, and f). In type I isoform (a and b, pT2N0M0, G2) and type IV isoform (e and f, pT3N0M0, G3), immunoreactivity of both caspase-3 and PCNA was markedly reduced, whereas in type III isoform (c and d, pT3N0M0, G3), both immunoreactivities were strong. These alterations in caspase-3 immunoreactivity were parallel with the change in caspase-3 expression at mRNA level. L, long reading frame; S, short reading frame.

Close modal
Fig. 8.

SNP in exon 17 of TCF-4 and expression levels of c-myc, cyclin D1, MMP7, and c-jun mRNA. a, PCR-SSCP analysis in exon 17. Lanes A–J are representative RCC samples. Lanes A, I, and J, heterozygotes (hetero.) for SNP (+) allele and SNP (−) allele. Lanes B, C, D, F, G, and B, homozygotes (homo.) for SNP (−) alleles; Lanes E, homozygotes for SNP (+) alleles. b, direct sequencing of SNP negative allele (left) and positive allele (right). SNP allele resulted in amino acid change from proline (Pro) to threonine (Thr). c, relationship of expression levels of c-myc, cyclin D1, MMP7, and c-jun mRNA with SNP. No significant difference (N.S.) in the expression levels of the mRNA was found between SNP (−) and SNP (+) RCCs. A.U., arbitrary unit(s).

Fig. 8.

SNP in exon 17 of TCF-4 and expression levels of c-myc, cyclin D1, MMP7, and c-jun mRNA. a, PCR-SSCP analysis in exon 17. Lanes A–J are representative RCC samples. Lanes A, I, and J, heterozygotes (hetero.) for SNP (+) allele and SNP (−) allele. Lanes B, C, D, F, G, and B, homozygotes (homo.) for SNP (−) alleles; Lanes E, homozygotes for SNP (+) alleles. b, direct sequencing of SNP negative allele (left) and positive allele (right). SNP allele resulted in amino acid change from proline (Pro) to threonine (Thr). c, relationship of expression levels of c-myc, cyclin D1, MMP7, and c-jun mRNA with SNP. No significant difference (N.S.) in the expression levels of the mRNA was found between SNP (−) and SNP (+) RCCs. A.U., arbitrary unit(s).

Close modal
Fig. 9.

Splicing types of TCF-4 in relation to prognosis. a, progression-free probability in types I and IV isoforms is significantly lower than in types II and III. Multivariate analysis revealed splicing types as an independent powerful discriminator over Robson staging and grade. b, survival probability in the type I and IV isoforms was significantly lower than in the types II and III. Multivariate analysis revealed splicing types as an independent powerful discriminator over Robson staging and grade.

Fig. 9.

Splicing types of TCF-4 in relation to prognosis. a, progression-free probability in types I and IV isoforms is significantly lower than in types II and III. Multivariate analysis revealed splicing types as an independent powerful discriminator over Robson staging and grade. b, survival probability in the type I and IV isoforms was significantly lower than in the types II and III. Multivariate analysis revealed splicing types as an independent powerful discriminator over Robson staging and grade.

Close modal
Fig. 10.

Alteration of β-catenin at genomic DNA and mRNA levels. a, SSCP analysis for exon 3 of β-catenin gene. Lanes A–I are representative RCC samples. Left lane, normal genomic DNA was applied. No abnormal bands were detected. b, TGGE analysis for exon 3 of β-catenin gene. Lanes A–I are representative RCC samples. Left lane, positive control (mutation sample) was applied. No abnormal bands were detected. c, deletion screening for exon 3 of β-catenin gene. Lanes A–J are representative RCC samples. No large deletions were detected. d,. RT-PCR for β-catenin gene. Lanes A–J are representative RCC samples. ∗, 100-bp DNA ladder. Although samples of C, D, and J showed loss of β-catenin expression, at least overexpression of β-catenin was not found.

Fig. 10.

Alteration of β-catenin at genomic DNA and mRNA levels. a, SSCP analysis for exon 3 of β-catenin gene. Lanes A–I are representative RCC samples. Left lane, normal genomic DNA was applied. No abnormal bands were detected. b, TGGE analysis for exon 3 of β-catenin gene. Lanes A–I are representative RCC samples. Left lane, positive control (mutation sample) was applied. No abnormal bands were detected. c, deletion screening for exon 3 of β-catenin gene. Lanes A–J are representative RCC samples. No large deletions were detected. d,. RT-PCR for β-catenin gene. Lanes A–J are representative RCC samples. ∗, 100-bp DNA ladder. Although samples of C, D, and J showed loss of β-catenin expression, at least overexpression of β-catenin was not found.

Close modal
Fig. 11.

β-catenin staining of pT2N0M0 G2 RCC sample. Immunohistochemistry, tumor cells showed mainly cytoplasmic and membranous expression. There was no nuclear accumulation of β-catenin in tumor cells. a, ×200; b, ×400.

Fig. 11.

β-catenin staining of pT2N0M0 G2 RCC sample. Immunohistochemistry, tumor cells showed mainly cytoplasmic and membranous expression. There was no nuclear accumulation of β-catenin in tumor cells. a, ×200; b, ×400.

Close modal
Table 1

Primer sequence

PrimerSequence
β-catenin  
 Bi2S 5′-CCTCCTAATGGCTTGGTGAA-3′ 
 B2S 5′-GAAAATCCAGCGTGGACAAT-3′ 
 B3S 5′-ATTGATGGAGTTGGACATGGC-3′ 
 B3AS 5′-CCAGCTACTTGTTCTTGAGTGAAGG-3′ 
 B3SGC 5′-CCCCGCCCGGCCCGCCCCGCCCCCCGCCCCCCCTCCCGGCCCGCCCCCATTGATGGAGTTGGACATGGC-3′ 
 B4AS 5′-TGCATGTTTCAGCATCTGTG-3′ 
 B4eAS 5′-CTGAGCTCGAGTCATTGCAT-3′ 
TCF-4  
 T1F 5′-AATTGCTGCTGGTGGGTGA-3′ 
 T1R 5′-CCCGAGGGGCTTTTCCTA-3′ 
 T10AF 5′-TTTATTGGGTTGCGTCTGTT-3′ 
 T10AR 5′-ACCTTTGCTCTCATTTCCTT-3′ 
 T10BF 5′-CCTCTTAATGCATTCATGTTG-3′ 
 T10BR 5′-ACTCTGGTCCCTCACCAC-3′ 
 T11F 5′-TCTCTTTGTTCATGTCTTTCT-3′ 
 T11R 5′-TCTACAGACTCCAATCCTAA-3′ 
 T17F 5′-GCCTCTATTCACAGATAACTC-3′ 
 T17R 5′-GTTCACCTTGTATGTAGCGAA-3′ 
 P13F 5′-TGAACACAGCGAATGTTTCC-3′ 
 P12F 5′-CAAGCAGCCGGGAGAGAC-3′ 
 P17LR 5′-TCAGCGAGCAGGAGGC-3′ 
 P14F 5′-AACTGGTGCGGCCCTTGCAG-3′ 
 P17SR 5′-TGGGCTGAGGCAGCTGCCTT-3′ 
 S1F 5′-CTTCCAAAATTGCTGCTGGTG-3′ 
 S1R 5′-TCTCTGGACAGTGCATGCC-3′ 
RT-PCR  
c-myc S 5′-AGACTCCAGCGCCTTCTCTCCG-3′ 
c-myc AS 5′-CTGTGAGGAGGTTTGCTGTGGCC-3′ 
cyclin D1-S 5′-TCTAAGATGAAGGAGACCATC-3′ 
cyclin D1-AS 5′-GCGGTAGTAGGACAGGAAGTTGTT-3′ 
MMP7 S 5′-AACTCCCGCGTCATAGAAATAATG-3′ 
MMP7 AS 5′-ACCCAAAGAATGGCCAAGTTCATG-3′ 
c-jun S 5′-GGATCAAGGCGGAGAGGAAG-3′ 
c-jun AS 5′-GCGTTAGCATGAGTTGGCAC-3′ 
G3PDH S 5′-TCCCATCACCATCTTCCA-3′ 
G3PDH AS 5′-CATCACGCCACAGTTTCC-3′ 
Bcl-2 S 5′-GGTGCCACCTGTGGTCCACCT-3′ 
Bcl-2 AS 5′-CTTCACTTGTGGCCCAGATAGG-3′ 
Bcl-X S 5′-TTGGACAATGGACTGGTTG-3′ 
Bcl-X AS 5′-GTAGAGTGGATGGTCAGTG-3′ 
Bax S 5′-CTGACATGTTTTCTGACGGC-3′ 
Bax AS 5′-TCAGCCCATCTTCTTCCAGA-3′ 
Bak S 5′-TGAAAAATGGCTTCGGGGCAAGGC-3′ 
Bak AS 5′-TCATGATTTGAAGAATCTTCGTACC-3′ 
Caspase-3 S 5′-CAAACTTTTTCAGAGGGGATCG-3′ 
Caspase-3 AS 5′-GCATACTGTTTCAGCATGGCA-3′ 
PrimerSequence
β-catenin  
 Bi2S 5′-CCTCCTAATGGCTTGGTGAA-3′ 
 B2S 5′-GAAAATCCAGCGTGGACAAT-3′ 
 B3S 5′-ATTGATGGAGTTGGACATGGC-3′ 
 B3AS 5′-CCAGCTACTTGTTCTTGAGTGAAGG-3′ 
 B3SGC 5′-CCCCGCCCGGCCCGCCCCGCCCCCCGCCCCCCCTCCCGGCCCGCCCCCATTGATGGAGTTGGACATGGC-3′ 
 B4AS 5′-TGCATGTTTCAGCATCTGTG-3′ 
 B4eAS 5′-CTGAGCTCGAGTCATTGCAT-3′ 
TCF-4  
 T1F 5′-AATTGCTGCTGGTGGGTGA-3′ 
 T1R 5′-CCCGAGGGGCTTTTCCTA-3′ 
 T10AF 5′-TTTATTGGGTTGCGTCTGTT-3′ 
 T10AR 5′-ACCTTTGCTCTCATTTCCTT-3′ 
 T10BF 5′-CCTCTTAATGCATTCATGTTG-3′ 
 T10BR 5′-ACTCTGGTCCCTCACCAC-3′ 
 T11F 5′-TCTCTTTGTTCATGTCTTTCT-3′ 
 T11R 5′-TCTACAGACTCCAATCCTAA-3′ 
 T17F 5′-GCCTCTATTCACAGATAACTC-3′ 
 T17R 5′-GTTCACCTTGTATGTAGCGAA-3′ 
 P13F 5′-TGAACACAGCGAATGTTTCC-3′ 
 P12F 5′-CAAGCAGCCGGGAGAGAC-3′ 
 P17LR 5′-TCAGCGAGCAGGAGGC-3′ 
 P14F 5′-AACTGGTGCGGCCCTTGCAG-3′ 
 P17SR 5′-TGGGCTGAGGCAGCTGCCTT-3′ 
 S1F 5′-CTTCCAAAATTGCTGCTGGTG-3′ 
 S1R 5′-TCTCTGGACAGTGCATGCC-3′ 
RT-PCR  
c-myc S 5′-AGACTCCAGCGCCTTCTCTCCG-3′ 
c-myc AS 5′-CTGTGAGGAGGTTTGCTGTGGCC-3′ 
cyclin D1-S 5′-TCTAAGATGAAGGAGACCATC-3′ 
cyclin D1-AS 5′-GCGGTAGTAGGACAGGAAGTTGTT-3′ 
MMP7 S 5′-AACTCCCGCGTCATAGAAATAATG-3′ 
MMP7 AS 5′-ACCCAAAGAATGGCCAAGTTCATG-3′ 
c-jun S 5′-GGATCAAGGCGGAGAGGAAG-3′ 
c-jun AS 5′-GCGTTAGCATGAGTTGGCAC-3′ 
G3PDH S 5′-TCCCATCACCATCTTCCA-3′ 
G3PDH AS 5′-CATCACGCCACAGTTTCC-3′ 
Bcl-2 S 5′-GGTGCCACCTGTGGTCCACCT-3′ 
Bcl-2 AS 5′-CTTCACTTGTGGCCCAGATAGG-3′ 
Bcl-X S 5′-TTGGACAATGGACTGGTTG-3′ 
Bcl-X AS 5′-GTAGAGTGGATGGTCAGTG-3′ 
Bax S 5′-CTGACATGTTTTCTGACGGC-3′ 
Bax AS 5′-TCAGCCCATCTTCTTCCAGA-3′ 
Bak S 5′-TGAAAAATGGCTTCGGGGCAAGGC-3′ 
Bak AS 5′-TCATGATTTGAAGAATCTTCGTACC-3′ 
Caspase-3 S 5′-CAAACTTTTTCAGAGGGGATCG-3′ 
Caspase-3 AS 5′-GCATACTGTTTCAGCATGGCA-3′ 

a S, sense; AS, antisense; F, forward; R, reverse.

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