Structure of the Candidate Gene.

To establish the full structure of CB987890, we performed 5′ and 3′ RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE). No additional exons or isoforms were detected. Exons 1 (at the 5′ end) and 4 (at the 3′ end) were expanded by 183 bp and 192 bp, respectively. The total length of the gene is 1154 bp. We named this unique gene Papillary Thyroid Carcinoma Susceptibility Candidate 3 (PTCSC3). PTCSC3 has a potential open reading frame (ORF) predicting 95 amino acids starting in exon 1 and ending in exon 4. A single polyA signal sequence (AATAAA) was detected 18 bp before a polyA tail (Fig. 1). To confirm the size of PTCSC3 obtained by RACE, we performed a Northern blot using RNA from 293T cells (no endogenous expression of PTCSC3; negative control), 293T cells transfected with PTCSC3 expression construct (positive control) and from thyroid tissue. As shown in Fig. 1 a band around 1.2 kb was observed in the thyroid samples supporting our findings from RACE.

PTCSC3 Is Likely a Noncoding RNA Gene.

To define the putative function of PTCSC3 we performed computational analysis by integrating with the current genome annotations available at UCSC Genome Browser (16). We found that although the sequence of exon 1 is unique in the genome, exons 2, 3, and 4 are located within repetitive elements. Exons 2 and 3 completely overlap with LINEs (Family L1) and SINEs (Family Alu), respectively, ~75% of exon 4 overlaps with LTR repetitive elements (Family ERVL), suggesting that these exons were produced by retrotransposable elements. The genes that contain mobile genetic elements such as transposons or retrotransposons are usually noncoding RNA genes despite having sometimes short ORFs (17, 18).

PTCSC3 Expression Is Highly Thyroid Specific.

To test whether the PTCSC3 gene is expressed in other tissues than thyroid, we performed RT-PCR using RNA obtained from multiple tissues. Importantly, the PTCSC3 is almost uniquely expressed in thyroid tissue with very low expression additionally in kidney tissue (Fig. 1).

PTCSC3 Expression Is Down-Regulated in Tumor Tissue.

We analyzed PTCSC3 expression in paired, adjacent, unaffected (PTC-N) and tumor (PTC-T) thyroid tissue samples from 46 patients by real-time PCR (Fig. 2). The PTCSC3 RNA level was strongly or moderately down-regulated in tumor tissue compared with unaffected thyroid tissue in all of the tested pairs (P = 2.84 × 10⁻¹⁴). We also analyzed the expression of PTCSC3 in six thyroid cancer cell lines (C643, FTC133, SW1736, TPC-1, BCPAP, KTC-1) as shown in Fig. S3 and no expression was detected in any of them.

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Fig. 2.

Quantitative RT-PCR of PTCSC3 expression in paired samples of unaffected tissue and PTC tumor (n = 46). N, nonaffected tissue; T, tumor tissue. Shown are values from tumor normalized against values from the corresponding unaffected tissue. Samples arranged by SNP rs944289 genotypes (CC, n = 6; CT, n = 19; TT, n = 21; CC vs. CT, P = 0.687; CC vs. TT, P = 0.110; CT vs. TT, P = 0.004).

SNP Risk Allele [T] Is Associated with Dysregulation of PTCSC3 Expression.

To assess the impact of the risk allele on suppression of PTCSC3, we analyzed fold changes in expression between tumor and unaffected thyroid tissue. The rs944289[T] allele was associated with stronger down-regulation of PTCSC3 when [TT] homozygotes (n = 21) were compared with heterozygous [CT] patients (n = 19) (P = 0.004), but in the comparison with the few available homozygous [CC] subjects (n = 6) the difference did not reach statistical significance (P = 0.110; Fig. 2 and Fig. S4). Interestingly, in adjacent unaffected thyroid tissue PTCSC3 expression was slightly higher in [TT] homozygotes than in [CT] heterozygotes (P = 0.034) (Fig. S4).

C/EBPα and C/EBPβ Regulate Transcriptional Activity of the PTCSC3 Promoter.

Computational analysis revealed the presence of a binding site for CCAAT/enhancer binding protein (C/EBP) α and β overlapping with rs944289. The SNP is located within the conserved part of the binding site (Fig. 1). We used an electrophoresis mobility shift assay (EMSA) to determine whether the binding of the C/EBP proteins to the position of rs944289 occurs in vitro. A [α-P³²]-dCTP radiolabeled (by Klenow) probe containing the rs944289[C] allele was used, together with a nuclear extract from 293T cells transfected 24 h before the nuclear extraction with empty vector (pcDNA3), p42 C/EBPα, p30 C/EBPα or C/EBPβ expression constructs. As shown in Fig. 3, p42 C/EBPα and C/EBPβ bind strongly to the site, whereas the binding of p30 C/EBPα is weaker. The specificity of C/EBPs binding was confirmed either by lack of the shift in the presence of the nuclear extract from control (pcDNA3) and by the super shift (or an abrogation of the bands) when specific antibodies against the C/EBP proteins were used. To investigate the effect of C/EBPα and C/EBPβ on the promoter activity, COS-7 cells were transfected with an expression vector harboring cDNA of C/EBPα (p30 and p42 isoforms) or C/EBPβ together with the reporter construct containing the promoter region of the PTCSC3 with the rs944289[T] or rs944289[C] allele (Fig. 3). We observed an increase in the relative luciferase activity by both p42 C/EBPα and C/EBPβ (C allele: P = 0.0009 and P = 0.0014, respectively; T allele: P = 0.0019 and P = 0.0048, respectively) but not for p30 C/EBPα (P = 0.107 and P = 0.327 for C and T allele, respectively). The risk allele (T) decreased the PTCSC3 promoter activity compared with nonrisk allele (C) for both p42 C/EBPα and C/EBPβ (P = 0.026 and P = 0.048, respectively).

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Fig. 3.

Transcriptional activity of the PTCSC3 promoter. (A) Electrophoretic mobility shift assay analysis of binding of C/EBP transcription factors to the rs944289 site. C/EBPα (p30 and p42 isoforms) and C/EBPβ bind to the rs944289[C] allele site as demonstrated by a shift (S) in the presence of nuclear extract from 293T cells transfected with C/EBPα (p30 or p42) and C/EBPβ constructs but not when cells were transfected with empty vector (pcDNA3). Specificity of the binding complex was confirmed by super shifts (SS) with anti-C/EBPα and anti-C/EBPβ antibodies. (B) Dual luciferase assay in COS-7 cells cotransfected with reporters containing PTCSC3 promoter having either the C or T allele of the SNP sequence and C/EBPα or C/EBPβ expression vectors or empty vector. The p42 C/EBPα and C/EBPβ transcription factors stimulate transcription (C allele: P = 0.0009 and P = 0.0014, respectively; T allele: P = 0.0019 and P = 0.0048, respectively); the C allele being more active than the risk allele (T) (P = 0.026 and P = 0.048, respectively). Results are mean ± SEM of six independent experiments. *P < 0.05; **P = 0.01–0.001; ***P < 0.001; ns, not significant.

No Germline or Somatic Mutations Were Observed in the PTCSC3 Gene.

We searched for previously undefined variants in the gene by cDNA Sanger sequencing of PTCSC3 in 33 PTC normal/tumor tissue pairs. No mutations or new polymorphisms were found.

Thyroid tissue samples.

All samples included in this study were collected at The Ohio State University as part of ongoing studies approved by the Institutional Review Board at OSU.

Cell lines.

TPC-1 and BCPAP cells were a kind gift from Matthew D. Ringel and Motoyasu Saji (The Ohio State University, Columbus, OH). COS-7 and 293T cells were purchased from ATCC.

In Silico Analysis.

To search for the genes and ESTs in the vicinity of rs944289, we used genome browser (http://genome.ucsc.edu/cgi-bin/hgGateway, assembly February 2009, GRCh37/hg19). The potential transcription factor binding sites around the SNP were searched for by using the MATCH program (43).

Real-Time PCR Measurement of PTCSC3 Expression.

SYBR Green real-time PCR system (Applied Biosystems) was used to assess PTCSC3 expression in PTC patients. The Master mix for each reaction of 10 μL total volume was prepared as follows: 2× SYBR Green Master mix 5 μL, forward primer 200 nM (5′-TCAAACTCCAGGGCTTGAAC-3′), reverse primer 200 nM (5′-ATTACGGCTGGGTCTACCT-3′), cDNA 100 ng. For each plate, a dissociation curve was obtained to monitor any additional double stranded DNA. GAPDH was used as an internal control and the formula 2^−ΔCt, where ΔCt = Ct_(GENE) − Ct_(GAPDH) was used to calculate the relative mRNA level.

RNA Ligase Mediated Rapid Amplification of cDNA Ends.

RNA Ligase Mediated Rapid Amplification of cDNA Ends kit (RLM-RACE, Ambion) was used to establish the full structure of CB987890 according to the protocol of the manufacturer. The CB987890 specific primers used in 3′-RLM-RACE were as follows: outer 5′-TGGGACCTGTTGTTTTTCTTG-3′; inner 5′-TCAAACTCCAGGGCTTGAAC-3′. For 5′-RLM-RACE, the CB987890 specific primers were as follows: outer 5′-CATTTTGCCACCATGGTCTA-3′; inner 5′-TCTTCTGCTTGGCCTTTGAC-3′. The Expand Long Template PCR system (Roche) was used for nested PCR reactions for both 5′- and 3′-RLM-RACE, and the TOPO TA kit (Invitrogen) was used to clone nested amplicons into pCR4-TOPO vector. The clones were sequenced using M13 primers.

Northern Blot Analysis.

Total RNA was isolated from 293T cells, 293T cells transfected with PTCSC3 expression vector, and thyroid tissue by using TRIzol solution (Invitrogen). Total RNA (20 μg) was separated on a 1% formaldehyde gel and transferred onto Hybond N membrane (GE Healthcare Life Sciences) and crosslinked (Stratalinker 1800 UV, Stratagene). The membrane was hybridized at 42 °C in UltraHyb Ultrasensitive buffer (Ambion). A 183-bp DNA probe corresponding to 125–307 nucleotides of PTCSC3 cDNA served as probe. Following overnight hybridization, the membrane was washed in 2xSSC/0.2% SDS at 42 °C.

Constructs.

The cDNA of PTCSC3 was amplified by PCR (forward primer: 5′-gtacggtaccCTCCTTCAGACTTCTCAGTACTC-3′; reverse primer: 5′-tcgactcgagATTGCTACTGTGAGCATAACCTAC-3′), and the amplicons were ligated between Kpn-I and Xho-I sites of the pcDNA3 vector (Invitrogen) to create the expression construct for PTCSC3. The construct was confirmed by Sanger sequencing. The promoter region of PTCSC3 containing rs944289 was amplified by using forward (5′-atcaggtaccGGCAATTGAAGTTCCCAAAA-3′) and reverse (5′-atcactcgagGCCTCCAGACTTGGACTGAG-3′) primers from DNA of homozygous C and homozygous T patients. The amplicons were ligated between Kpn-I and Xho-I sites of the pGL4.24 vector (Promega) and the obtained constructs (C allele: rs944289-C and T allele: rs944289-T) were confirmed by sequencing. C/EBPα (p30 and p42) and C/EBPβ were cloned into pcDNA3-FLAG vector kindly provided by Daniel G. Tenen (Harvard Medical School, Boston, MA).

Luciferase Assay.

COS-7 cells were seeded in quadruplicate into 24-well plates, and after 24 h were transfected as shown in Fig. 3 using Lipofectamine 2000 (Invitrogen). The cells were harvested 24 h after transfection using Passive Lysis Buffer (Promega). The promoter activity was determined as Firefly/Renilla ratio relative to the ratio obtained in cells transfected with the empty vector.

Electrophoretic Mobility Shift Assay.

293T cells were transfected with an empty vector (pcDNA3-FLAG), p30 C/EBPα, p42 C/EBPα or C/EBPβ expression vectors. After 24 h, cells were harvested and nuclear extract was prepared. For the electrophoretic mobility shift assay (EMSA), the double-stranded oligonucleotide probe for the C allele (5′-GGAAAGATAGTCATTGCAGATTTGTAATA-3′) was labeled by filling in the ends with [α-P³²]-dCTP by Klenow. The nuclear extracts were incubated with radiolabeled oligonucleotide on ice for 20 min. For the supershift, 1 μL of polyclonal anti-C/EBP (α or β) antibody (Santa Cruz) was added to the binding reaction mixture. Binding reaction products were resolved on a 4% (wt/vol) nondenaturing polyacrylamide gel and electrophoresed at 150 V at 4 °C for 2 h.

PTCSC3 cDNA resequencing.

RNA from unaffected thyroid and tumor tissue (extracted by TRIzol solution) was DNase-1 treated (Ambion), and High Capacity Reverse transcriptase kit (Applied Biosystems) was used to obtain cDNA. After amplification (forward primer: 5′-AAAAACTCCTTCAGACTTCTCAGT-3′; reverse primer: 5′-GTCCCAGTCCCAAAACTGAA-3′), the PCR amplicons were Sanger sequenced.

Microarray Hybridization Following Transfection with PTCSC3.

TPC-1 cells (with no endogenous expression of PTCSC3) were plated in a 12-well dish and transfected in quadruplicate with 50 ng PTCSC3 expression construct, and with 50 ng empty vector (pcDNA3) as control using 2 μL Lipofectamine 2000 reagent (Invitrogen). At 24 h after transfection total RNA was extracted (TRIzol reagent, Invitrogen) and DNase-1 treated (Ambion). SurePrint G3 Human Gene Expression 8 × 60,000 Arrays (AMADID 028004; Agilent Technologies) were used to assess gene expression according to manufacturer’s protocol (Agilent Technologies).

The authors thank Stephan Tanner and Agnieszka Bronisz for helpful technical advice, Matthew D. Ringel and Motoyasu Saji for providing the TPC-1 and BCPAP cell lines, Daniel G. Tenen for providing the C/EBP constructs, and Krystian Jazdzewski for critical reading of the manuscript. This work was supported by National Cancer Institute Grants P30CA16058 and P01CA124570.