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RACK1 is a candidate gene associated with the prognosis of patients with early stage non-small cell lung cancer.

Choi YY, Lee SY, Lee WK, Jeon HS, Lee EB, Lee HC, Choi JE, Kang HG, Lee EJ, Bae EY, Yoo SS, Lee J, Cha SI, Kim CH, Kim IS, Lee MH, Kim YT, Jheon S, Park JY - Oncotarget (2015)

Bottom Line: Fifty six SNPs which were associated with both overall survival (OS) and disease-free survival (DFS) with log-rank P values < 0.05 in discovery set were selected for validation.Among those, five SNPs (RACK1 rs1279736C>A and rs3756585T>G, C3 rs2287845T>C, PCAF rs17006625A>G, and PCM1 rs17691523C>G) were found to be significantly associated with survival in the same direction as the discovery set.In combined analysis, the rs1279736C>A and rs3756585T>G were most significantly associated with OS and DFS in multivariate analysis (P for OS = 4 × 10⁻⁵ and 7 × 10⁻⁵, respectively; and P for DFS = 0.003, both; under codominant model).

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Cell Biology, Kyungpook National University, Daegu, Republic of Korea.

ABSTRACT

Background: This study was conducted to identify genetic polymorphisms associated with the prognosis of patients with early stage NSCLC.

Materials and methods: We genotyped 1,969 potentially functional single nucleotide polymorphisms (SNPs) of 1,151 genes involved in carcinogenesis in 166 NSCLC patients who underwent curative surgery, using the Affymetrix custom-made GeneChip. A replication study was performed in an independent cohort of 626 patients.

Results: Fifty six SNPs which were associated with both overall survival (OS) and disease-free survival (DFS) with log-rank P values < 0.05 in discovery set were selected for validation. Among those, five SNPs (RACK1 rs1279736C>A and rs3756585T>G, C3 rs2287845T>C, PCAF rs17006625A>G, and PCM1 rs17691523C>G) were found to be significantly associated with survival in the same direction as the discovery set. In combined analysis, the rs1279736C>A and rs3756585T>G were most significantly associated with OS and DFS in multivariate analysis (P for OS = 4 × 10⁻⁵ and 7 × 10⁻⁵, respectively; and P for DFS = 0.003, both; under codominant model). In vitro promoter assay and electrophoretic mobility shift assay revealed that the rs3756585 T-to-G change increased promoter activity and transcription factor binding of RACK1.

Conclusions: We identified five SNPs, especially RACK1 rs3756585T>G, as markers for prognosis of patients with surgically resected NSCLC.

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Related in: MedlinePlus

Effect of rs1279736C>A and rs3756585T>G (−283 and −123 from transcription start site, respectively) polymorphisms on RACK1 promoter(A) Transcription activity analysis of the haplotypes of rs1279736C>A and rs3756585T>G polymorphisms. The transcription activity of −283C/−123T haplotype and −283A/−123G haplotype was measured using Dual-Luciferase Reporter Assay System in H1299 cell line. The −283A/−123G haplotype had significantly increased promoter activity compared with the −283C/−123T haplotype. The results were confirmed in three independent experiments in triplicate. (B) Electrophoretic mobility shift assay with H1299 cell nuclear extracts using rs3756585T and rs3756585G oligonucleotides. Competition assays were performed using unlabeled rs3756585T or rs3756585G oligonucleotides. Each binding reaction contained 10 μg of nuclear extracts except lane 1 and 6, and labeled rs3756585T (lanes 1–5) or rs3756585G (lanes 6–10) oligonucleotides. Excess unlabeled rs3756585T (5- and 50-fold) and rs3756585G (5-fold) oligonucleotides were included in the binding reactions as competitors for labeled rs3756585T oligonucleotide (lanes 3–4 and 5, respectively). In addition, excess unlabeled rs3756585G (5- and 50-fold) and rs3756585T (5-fold) oligonucleotides were used to compete with rs3756585G oligonucleotide (lanes 8–9 and 10, respectively). *NE, nuclear extracts.
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Figure 2: Effect of rs1279736C>A and rs3756585T>G (−283 and −123 from transcription start site, respectively) polymorphisms on RACK1 promoter(A) Transcription activity analysis of the haplotypes of rs1279736C>A and rs3756585T>G polymorphisms. The transcription activity of −283C/−123T haplotype and −283A/−123G haplotype was measured using Dual-Luciferase Reporter Assay System in H1299 cell line. The −283A/−123G haplotype had significantly increased promoter activity compared with the −283C/−123T haplotype. The results were confirmed in three independent experiments in triplicate. (B) Electrophoretic mobility shift assay with H1299 cell nuclear extracts using rs3756585T and rs3756585G oligonucleotides. Competition assays were performed using unlabeled rs3756585T or rs3756585G oligonucleotides. Each binding reaction contained 10 μg of nuclear extracts except lane 1 and 6, and labeled rs3756585T (lanes 1–5) or rs3756585G (lanes 6–10) oligonucleotides. Excess unlabeled rs3756585T (5- and 50-fold) and rs3756585G (5-fold) oligonucleotides were included in the binding reactions as competitors for labeled rs3756585T oligonucleotide (lanes 3–4 and 5, respectively). In addition, excess unlabeled rs3756585G (5- and 50-fold) and rs3756585T (5-fold) oligonucleotides were used to compete with rs3756585G oligonucleotide (lanes 8–9 and 10, respectively). *NE, nuclear extracts.

Mentions: The effect of the haplotype of rs1279736C>A and rs3756585T>G polymorphisms on the promoter activity of the RACK1 gene was investigated using a luciferase assay. In H1299 cells, the rs1279736A-rs3756585G haplotype significantly increased promoter activity compared to the rs1279736C-rs3756585T haplotype (P = 0.001, Figure 2A).


RACK1 is a candidate gene associated with the prognosis of patients with early stage non-small cell lung cancer.

Choi YY, Lee SY, Lee WK, Jeon HS, Lee EB, Lee HC, Choi JE, Kang HG, Lee EJ, Bae EY, Yoo SS, Lee J, Cha SI, Kim CH, Kim IS, Lee MH, Kim YT, Jheon S, Park JY - Oncotarget (2015)

Effect of rs1279736C>A and rs3756585T>G (−283 and −123 from transcription start site, respectively) polymorphisms on RACK1 promoter(A) Transcription activity analysis of the haplotypes of rs1279736C>A and rs3756585T>G polymorphisms. The transcription activity of −283C/−123T haplotype and −283A/−123G haplotype was measured using Dual-Luciferase Reporter Assay System in H1299 cell line. The −283A/−123G haplotype had significantly increased promoter activity compared with the −283C/−123T haplotype. The results were confirmed in three independent experiments in triplicate. (B) Electrophoretic mobility shift assay with H1299 cell nuclear extracts using rs3756585T and rs3756585G oligonucleotides. Competition assays were performed using unlabeled rs3756585T or rs3756585G oligonucleotides. Each binding reaction contained 10 μg of nuclear extracts except lane 1 and 6, and labeled rs3756585T (lanes 1–5) or rs3756585G (lanes 6–10) oligonucleotides. Excess unlabeled rs3756585T (5- and 50-fold) and rs3756585G (5-fold) oligonucleotides were included in the binding reactions as competitors for labeled rs3756585T oligonucleotide (lanes 3–4 and 5, respectively). In addition, excess unlabeled rs3756585G (5- and 50-fold) and rs3756585T (5-fold) oligonucleotides were used to compete with rs3756585G oligonucleotide (lanes 8–9 and 10, respectively). *NE, nuclear extracts.
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Related In: Results  -  Collection

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Figure 2: Effect of rs1279736C>A and rs3756585T>G (−283 and −123 from transcription start site, respectively) polymorphisms on RACK1 promoter(A) Transcription activity analysis of the haplotypes of rs1279736C>A and rs3756585T>G polymorphisms. The transcription activity of −283C/−123T haplotype and −283A/−123G haplotype was measured using Dual-Luciferase Reporter Assay System in H1299 cell line. The −283A/−123G haplotype had significantly increased promoter activity compared with the −283C/−123T haplotype. The results were confirmed in three independent experiments in triplicate. (B) Electrophoretic mobility shift assay with H1299 cell nuclear extracts using rs3756585T and rs3756585G oligonucleotides. Competition assays were performed using unlabeled rs3756585T or rs3756585G oligonucleotides. Each binding reaction contained 10 μg of nuclear extracts except lane 1 and 6, and labeled rs3756585T (lanes 1–5) or rs3756585G (lanes 6–10) oligonucleotides. Excess unlabeled rs3756585T (5- and 50-fold) and rs3756585G (5-fold) oligonucleotides were included in the binding reactions as competitors for labeled rs3756585T oligonucleotide (lanes 3–4 and 5, respectively). In addition, excess unlabeled rs3756585G (5- and 50-fold) and rs3756585T (5-fold) oligonucleotides were used to compete with rs3756585G oligonucleotide (lanes 8–9 and 10, respectively). *NE, nuclear extracts.
Mentions: The effect of the haplotype of rs1279736C>A and rs3756585T>G polymorphisms on the promoter activity of the RACK1 gene was investigated using a luciferase assay. In H1299 cells, the rs1279736A-rs3756585G haplotype significantly increased promoter activity compared to the rs1279736C-rs3756585T haplotype (P = 0.001, Figure 2A).

Bottom Line: Fifty six SNPs which were associated with both overall survival (OS) and disease-free survival (DFS) with log-rank P values < 0.05 in discovery set were selected for validation.Among those, five SNPs (RACK1 rs1279736C>A and rs3756585T>G, C3 rs2287845T>C, PCAF rs17006625A>G, and PCM1 rs17691523C>G) were found to be significantly associated with survival in the same direction as the discovery set.In combined analysis, the rs1279736C>A and rs3756585T>G were most significantly associated with OS and DFS in multivariate analysis (P for OS = 4 × 10⁻⁵ and 7 × 10⁻⁵, respectively; and P for DFS = 0.003, both; under codominant model).

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Cell Biology, Kyungpook National University, Daegu, Republic of Korea.

ABSTRACT

Background: This study was conducted to identify genetic polymorphisms associated with the prognosis of patients with early stage NSCLC.

Materials and methods: We genotyped 1,969 potentially functional single nucleotide polymorphisms (SNPs) of 1,151 genes involved in carcinogenesis in 166 NSCLC patients who underwent curative surgery, using the Affymetrix custom-made GeneChip. A replication study was performed in an independent cohort of 626 patients.

Results: Fifty six SNPs which were associated with both overall survival (OS) and disease-free survival (DFS) with log-rank P values < 0.05 in discovery set were selected for validation. Among those, five SNPs (RACK1 rs1279736C>A and rs3756585T>G, C3 rs2287845T>C, PCAF rs17006625A>G, and PCM1 rs17691523C>G) were found to be significantly associated with survival in the same direction as the discovery set. In combined analysis, the rs1279736C>A and rs3756585T>G were most significantly associated with OS and DFS in multivariate analysis (P for OS = 4 × 10⁻⁵ and 7 × 10⁻⁵, respectively; and P for DFS = 0.003, both; under codominant model). In vitro promoter assay and electrophoretic mobility shift assay revealed that the rs3756585 T-to-G change increased promoter activity and transcription factor binding of RACK1.

Conclusions: We identified five SNPs, especially RACK1 rs3756585T>G, as markers for prognosis of patients with surgically resected NSCLC.

Show MeSH
Related in: MedlinePlus