Seven pancreatic cancer cell lines SW1990, PATU8988, AsPC-1, BxPC-3, CFPAC-1, Capan-2, PANC-1 were cultured in DMEM medium supplemented with 10% FBS and incubated in a 5% CO₂ humidified incubator at 37°C. Total RNA was extracted from cells using RNA iso Plus (Takara, Japan) according to the manufacturer’s instructions. The yield of RNA was determined using a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA) and the integrity was evaluated using agarose gel electrophoresis stained with ethidium bromide.

Two reference sequence transcript variants of SUFU gene are published on NCBI. They are transcript variant 1 of SUFU (SUFUv1, NM_016169) and transcript variant 2 of SUFU (SUFUv2, NM_001178133). 3′RACE was made to obtain the 3′ cDNA of SUFU transcripts.

3′-Full RACE Core Set Ver.2.0 (product code, D314, Takara) kit contains: 3′RACE adaptor (a reverse transcription primer adaptor which can addict to mature mRNA poly A tail by its poly T sequences), M-MLV reverse transcriptase, 3′RACE outer primer (reverse primer of the first nested PCR), 3′RACE inner primer (reverse primer of the second nested PCR), etc. The steps are shown in Fig. 1A.

We performed reverse transcription. The reaction consisted of 1 μl total RNA (500 ng/μl), 1 μl 3′RACE adaptor, 0.25 μl M-MLV reverse transcriptase, 0.25 μl RNase inhibitor, 2 μl 5× M-MLV buffer, 1 μl dNTP mixture and 4.5 μl RNase-free dH₂O, in a total volume of 10 μl. Reaction was performed in a GeneAmp^® PCR System 9700 (Applied Biosystems, USA) for 60 min at 42°C, followed by heat inactivation of RT for 15 min at 70°C.

The forward primer of the first nested PCR was designed by us according to the kit instructions (Table I). We call this forward primer P1. It is situated in the 9th exon of SUFUv1. The first base pair of P1 is situated in the 1,218 bp of the whole RNA sequences. The reverse primer of the first nested PCR is in the kit called 3′RACE outer primer. The reaction consisted of 3 μl reverse transcription product, 7 μl 1× cDNA dilution buffer, 2 μl P1 (10 μmol/l), 2 μl 3′RACE outer primer, 5 μl 10× Ex Taq buffer (including Mg₂), 0.25 μl Ex Taq enzyme and 30.75 μl dH₂O. Reactions were performed in a GeneAmp PCR System 9700 for 3 min at 94°C, followed by 25 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 5 min, and a last step of 72°C for 10 min.

The forward primer of the second nested PCR was also designed by us according to the kit instruction (Table I). We call this forward primer P2. It is situated in the 10th exon of SUFUv1. The first base pair of P2 is situated in the 1,357 bp of the whole RNA sequences. The reverse primer of the second nested PCR is in the kit called 3′RACE inner primer. The reaction consisted of 1 μl the first nested PCR product, 8 μl dNTP mixture, 5 μl 10× Ex Taq buffer (including Mg₂), 0.5 μl Ex Taq enzyme, 2 μl P2 (10 μmol/l), 2 μl 3′RACE inner primer and 31.5 μl dH₂O. Reactions were performed in a GeneAmp PCR System 9700 for 3 min at 94°C, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 5 min, and a last step of 72°C for 10 min.

The product of second nested PCR was ligated with pMD-18T vector, and infected DH5α. Then, we selected 10 positive clones, and made sequence detection.

We performed reverse transcription by SUFU ORF reverse transcription primer (Table I), then, by PCR. The forward primer of PCR was designed by us called P3 (Table I). It is situated in the first exon of SUFUv1. Reverse primer of PCR was also designed by us called P4 (Table I). It contains partial 11th exon of SUFUv1 and partial new exon. Product of PCR was used for sequence detection. The steps are shown in Fig. 1B.

The forward primer terminal is added with protection base pairs, BamHI enzyme sites and Kozak sequences. The reverse primer terminal is added with protection base pairs, AgeI enzyme sites. The forwad primer and reverse primer were designed to clone open reading frame (ORF) of SUFU. We performed reverse transcription by SUFU ORF reverse transcription primer (Table I), then by PCR. The sequence of forward primer is: 5′-cgggatccGGGATGGC GGAGCTGCGGCCTA-3′. The sequence of reverse primer is: 5′-gcgcaccggtCTAGTGTAG CGGACTGTCGAACA-3′. BamHI and AgeI double enzyme digestion was made in both PCR product and pcDNA3.1mychisA (+) expression vector. After ligation of PCR product with pcDNA3.1mychisA (+) expression vector and DH5α infection we selected positive clones and sequence detection was performed.

SW1990 cells were transiently transfected with empty vector, SUFUv1 and SUFUvN expression vector, respectively, by use of Lipofectamine 2000 kit (Takara) according to the manufacturer’s instructions. The cells were cultured for 24 h after transfection. Then the cells were harvested for further studies.

The empty vector, SUFUv1 and SUFUvN expression vector transfected SW1990 cells were collected. Cells were dissolved in cell lysis buffer. Protein of pancreatic cancer tissue was extracted by RIPA lysis buffer. The lysates were cooled with ice for 30 min, and then centrifuged at 13,000 × g for 30 min. Proteins (10 μg) in the collected supernatant were separated by SDS-PAGE on 12% gels and then transferred to PVDF membranes. The membrane, after a block with 5% skim milk, was incubated with primary antibodies to SUFU (rabbit anti-human monoclonal antibody, catalog no. NBP1-40515, Novus, USA) and GAPDH (ab97626, Abcam, Cambridge, UK). This SUFU antibody is made by N terminal of SUFU antigen, so it can bind the N terminal of protein encoded by SUFUv1, SUFUv2 and SUFUvN (SUFU isoform 1, isoform 2 and isoform N). Primary antibody is 1:200 diluted to detect SUFU isoforms. The secondary antibody is HRP labeled goat anti-rabbit polyclonal IgG antibody. GAPDH is used as inner control. ECL kit (product code, 34096, Thermo Fisher Scientific, USA) and the camera are used to capture the results.

Tumors were obtained from 40 adult patients diagnosed with pancreatic ductal adenocarcinoma who undergwent surgery at The Second Military Medical University affiliated Changhai Hospital between 2013 and 2014. This study was approved by the Ethics Committee of Changhai Hospital. The total RNA of the pancreatic cancer tissue was extracted by mirVana™ miRNA isolation kit (am1561, Takara). DNA contamination was removed by DNase in this kit. Total RNA was stored at −70°C.

The 18s control (Hs99999901_s1) MGB probe and primers kit were purchased from Invitrogen Co., USA. The primer pairs and the TaqMan MGB probe sequences of SUFU different variants were designed by Primer Express 3.0 software and were synthesized by Invitrogen Co. The sequences are shown in Table II, and the probes are shown in Fig. 1C.

Quantification was performed with a two-step reaction process: reverse transcription (RT) and PCR. Each RT reaction consisted of 1 μg RNA, 2 μl of PrimerScript buffer, 0.5 μl of oligodT, 2 μl of random 6 mers and 0.5 μl of PrimerScript RT Enzyme Mix I (Takara), in a total volume of 10 μl. Reactions were performed in a GeneAmp PCR System 9700 (Applied Biosystems) for 15 min at 37°C, followed by heat inactivation of RT for 5 sec at 85°C. Real-time PCR was performed using LightCycler^® 480 II Real-time PCR Instrument (Roche, Swiss) with 20 μl PCR reaction mixture that included 10 μl of 2× TaqMan mix (Takara), 1 μl of 20× probe and primers mixture (Invitrogen), 2 μl of cDNA, and 7 μl of nuclease-free water. Reactions were incubated in a 384-well optical plate (Roche) at 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 30 sec. Each sample was run in triplicate for analysis. At the end of the PCR cycles, melting curve analysis was performed to validate the specific generation of the expected PCR product. The expression levels of mRNAs were normalized to 18s rRNA and were calculated using the 2^−ΔΔCt method.

The relation of SUFU variant mRNA RQ values (−ΔΔCt) with clinical characteristics were assessed by Mann-Whitney U test. Statistical analysis was processed by the SPSS 18.0 software package. P-values <0.05 were considered statistically significant.

Following the strategy described in Fig. 1A, the 3′RACE amplification for 3′ cDNA of SUFU in pancreatic cancer cells was performed. The products of first cycle (using P1 and 3′RACE outer primer) and second cycle of nested PCR (using P2 and 3′RACE inner primer) are shown in Fig. 2A. A cDNA fragment of ~1,000 bp products from SW1990 cells was cloned into pMD18-T and sequenced. As shown in Figs. 2B and 3, a novel cDNA fragment sequence was found at 3′ termination of SUFU gene compared with cDNA of transcript variant 1 of SUFU (SUFUv1, NM_016169) and transcript variant 2 of SUFU (SUFUv2, NM_001178133) published in NCBI database. This fragment sequence contains a new protein-coding exon (126 bp) the same as a fragment of the intron sequence (102,625,811–102,625,936 of the human chromosome 10, hg38 version) between exon 10 and 11 of SUFUv1. We hypothesized that there exists a novel alternative splicing transcript variant of SUFU (SUFUvN) which contains an additional new exon compared with SUFUv1.

In order to identify the full length of SUFUvN cDNA squence, the 5′ cDNA fragment of SUFUvN was amplified by RT-PCR following the strategy described in Fig. 1B, in which a specific primer 4 (P4) covering the new exon and exon 11 of SUFUv1 pairing with a specific primer 3 (P3) siting in exon 1 of SUFUv1 were used. As shown in Fig. 4A, a cDNA fragment of 1,400 bp products from SW1990 cells was obtained by RT-PCR and its sequence was the same as the exon 2-exon 10 of SUFUv1 cDNA. The combination of exon 10 and new exon was also been detectable in 1,400 bp product (data not shown). Thus, the new exon is combined with exon 1–10 in 5′-terminal and with exon 11–12 in 3′ terminal.

RT-PCR product of SUFU transcript was double enzyme digested by BamHI and AgeI and was ligated with expression vector pcDNA3.1mychisA (+). After infection to DH5α, positive clones were selected and sequenced. From the sequencing and alignment result (data not shown), we identified two kinds of SUFU cDNA clones. They are SUFUv1 and full length of SUFUvN as described above. Figure of BLAT in UCSC web site show us SUFUvN, 5′cDNA of SUFUvN, 3′cDNA of SUFUvN, and other two SUFU variants (Fig. 4B).

There were three bands present in the western blotting figure of PDAC tissue (Fig. 4C). Compared with lanes with SUFUv1 and SUFUvN expression vector transfected SW1990 serum, two bands were identified to be protein encoded by SUFUv1 (isoform 1) and isoform N in PDAC tissues. Isoform 1 is ~54 kDa, while isoform N is ~60 kDa. The additional band ~47 kDa may be protein encoded by SUFUv2 (isoform 2).

We designed TaqMan MGB probes to identify different transcript variants of SUFU. Each probe is described in the figure, so each probe can uniquely detect the corresponding SUFU variant (Fig. 1C). After detecting transcription levels of SUFU variants in PDAC tissues of 40 patients, we found that SUFUvN transcription levels are significantly higher in PDAC tissues of N1 stage patients than in N0 stage patients, while there is no relationship between other variants transcription levels and lymph node metastasis (Fig. 5). As shown in Table III, we found that the elevated transcription of SUFUvN was related with high N stage, while no correlation was found with other clinical features, including T stage, age of onset, gender, tumor size and tumor differentiation.