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Pancreatic cancer risk variant in LINC00673 creates a miR-1231 binding site and interferes with PTPN11 degradation

Nature Genetics volume 48, pages 747–757 (2016)Cite this article

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Abstract

Genome-wide association studies have identified several loci associated with pancreatic cancer risk; however, the mechanisms by which genetic factors influence the development of sporadic pancreatic cancer remain largely unknown. Here, by using genome-wide association analysis and functional characterization, we identify a long intergenic noncoding RNA (lincRNA), LINC00673, as a potential tumor suppressor whose germline variation is associated with pancreatic cancer risk. LINC00673 is able to reinforce the interaction of PTPN11 with PRPF19, an E3 ubiquitin ligase, and promote PTPN11 degradation through ubiquitination, which causes diminished SRC–ERK oncogenic signaling and enhanced activation of the STAT1-dependent antitumor response. A G>A change at rs11655237 in exon 4 of LINC00673 creates a target site for miR-1231 binding, which diminishes the effect of LINC00673 in an allele-specific manner and thus confers susceptibility to tumorigenesis. These findings shed new light on the important role of LINC00673 in maintaining cell homeostasis and how its germline variation might confer susceptibility to pancreatic cancer.

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Figure 1: Functional relevance of the rs11655237 SNP.
Figure 2: LINC00673 inhibits PDAC cell proliferation and cell cycle progression.
Figure 3: LINC00673 interacts with and promotes PTPN11 degradation via ubiquitination.
Figure 4: LINC00673 promotes PTPN11 ubiquitination by strengthening PTPN11–PRPF19 interaction.
Figure 5: LINC00673 suppresses cell proliferation via PTPN11 degradation in an allele-specific manner.
Figure 6: LINC00673 regulates the PTPN11 downstream signaling pathway.
Figure 7: Proposed functional action of LINC00673 in modulating PDAC risk.

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Acknowledgements

This research was supported by the Recruitment Program of Global Youth Experts (C. Wu), grants (91229126 to D.L. and 81490753 to W.T.) from the National Natural Science Foundation of China, a grant (2014AA020609) from the National High-Tech Research and Development Program of China (C. Wu) and the Chinese Academy of Medical Sciences Intramural Funds (C. Wu, X.C. and J.Z.).

Author information

Author notes

  1. Jian Zheng and Xudong Huang: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Jian Zheng, Xudong Huang, Zhongli Du, Jiang Chang, Lixuan Wei, Yaling Han & Chen Wu

  2. Department of Etiology and Carcinogenesis, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Wen Tan, Dianke Yu & Dongxin Lin

  3. Department of Abdominal Surgery, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

    Chengfeng Wang & Xu Che

  4. Department of Genetics, Medical College of Soochow University, Suzhou, China

    Yifeng Zhou

  5. School of Public Health, Tongji Medical College, Huazhong University of Sciences and Technology, Wuhan, China

    Xiaoping Miao

  6. Department of Radiation Oncology, Cancer Hospital, Fudan University, Shanghai, China

    Guoliang Jiang

  7. Department of Pancreas and Hepatobiliary Surgery, Cancer Hospital, Fudan University, Shanghai, China

    Xianjun Yu

  8. Department of Pathology, Shengjing Hospital, China Medical University, Shenyang, China

    Xianghong Yang

  9. Department of Epidemiology, Second Military Medical University, Shanghai, China

    Guangwen Cao

  10. Department of Gastroduodenal and Pancreatic Surgery, Hunan Province Tumor Hospital, Changsha, China

    Chaohui Zuo

  11. Department of Gastroenterology, First Affiliated Hospital, Second Military Medical University, Shanghai, China

    Zhaoshen Li

  12. Union Hospital, Tongji Medical College, Huazhong University of Sciences and Technology, Wuhan, China

    Chunyou Wang

  13. Department of Surgery, University of Hong Kong, Hong Kong, China

    Siu Tim Cheung

  14. Department of Pathology, Affiliated Hospital, Inner Mongolia School of Medicine, Huhhot, China

    Yongfeng Jia

  15. Department of Pathology, Fujian Provincial Cancer Hospital, Fuzhou, China

    Xiongwei Zheng

  16. Department of Epidemiology and Biostatistics, Cancer Center, Nanjing Medical University, Nanjing, China

    Hongbing Shen

  17. Collaborative Innovation Center for Cancer Medicine, Guangzhou, China

    Dongxin Lin

Authors
  1. Jian Zheng
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  2. Xudong Huang
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  3. Wen Tan
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  4. Dianke Yu
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Contributions

D.L. conceptualized and supervised this study. C. Wu, J.Z. and X.H. contributed to the study design. C. Wu and W.T. supervised the genome-wide association analysis. J.Z. and X.H. performed and analyzed the data from most functional assays. D.Y., J.C., Z.D., L.W. and Y.H. performed sample preparation, genotyping assays and association analysis. Chengfeng Wang, X.C., Y.Z., X.M., G.J., X. Yu, X. Yang, G.C., C.Z., Z.L., Chunyou Wang, S.T.C., Y.J., X.Z. and H.S. contributed human data and samples. D.L., J.Z. and C. Wu were involved in manuscript preparation.

Corresponding authors

Correspondence to Chen Wu or Dongxin Lin.

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Integrated supplementary information

Supplementary Figure 1 Regional plots of association results and recombination rates within the significant susceptibility locus and null association between rs11655237 genotypes and survival in patients with PDAC.

(a) The negative log10-transformed P value (y axis) for each SNP is presented according to the chromosomal position of the SNP (x axis). The estimated recombination rates using 1000 Genomes Project March 2012 release samples as the reference (NCBI Build 37) are shown as light blue lines. The genomic locations of genes within the region of interest were annotated to NCBI Build 37 of the human genome assembly from the UCSC Genome Browser, and genes are shown as arrows. Both genotyped and imputed SNPs are shown, and the top SNP is labeled by rs ID. Imputation quality was estimated by masking 2% of the genotypes at random and then imputing them and comparing the imputed values with the original genotypes to estimate genotypic error rates; this yielded an error rate of 1.49% per allele. Linkage (r2) is shown for each SNP with the tagging SNP. The purple diamond represents the association of the tagging SNP identified in the GWAS stage, and the purple circle represents association of the tagging SNP in the combined genome-wide discovery and replication sample. (b) Kaplan–Meier estimates of survival time in individuals with PDAC stratified by rs11655237 (G>A) genotype. Genome-wide genotype data and survival information were available for 600 cases. After exclusion of 237 cases without disease stage information, 363 cases were available for the final analysis. The median survival time was 7.1 months. rs11655237 genotype was not significantly associated with survival. The mean survival time for individuals with the GG, GA and AA genotypes at rs11655237 were 7, 6 and 6 months, respectively, with a hazard ratio (HR) of 1.05 (95% CI = 0.89−1.25).

Supplementary Figure 2 Downregulation of LINC00673 in human cancer tissues and null association between LINC00673 expression levels and survival in patients with PDAC.

(a) LINC00673 expression is significantly lower in PDAC cells than in immortalized human pancreatic duct epithelial (HPDE6-C7) cells. RNA levels were determined by qRT–PCR; results are shown as means ± s.e.m. normalized to GAPDH. P values are from two-sided Student’s t tests. (b) LINC00673 expression, as determined by qRT–PCR, is significantly lower in PDAC tissues than in their adjacent normal tissues (n = 74). Results are shown as means ± s.e.m. normalized to GAPDH; P values are based on two-sided Student's t tests. (c) Expression levels of LINC00673 in 39 pairs of PDAC tissues and adjacent normal tissues from the GEO database (78921667). Centered values represent medians and bars represent minimum and maximum values for each group. P values are from two-sided t tests. (d,e) Downregulation of LINC00673 in human esophageal squamous cell carcinoma (ESCC) (d) and hepatocellular carcinoma (HCC) (e) in comparison with their adjacent non-tumor tissues. Results are shown as means ± s.e.m. normalized to GAPDH as determined by qRT–PCR. All P values are from two-sided t tests. (f) LINC00673 expression levels vary across different cancer types in the TCGA database. PAAD, pancreatic adenocarcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; BLCA, bladder urothelial carcinoma; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma. (g) Null association of LINC00673 expression, as determined by qRT–PCR, with survival in patients with PDAC. The hazard ratio (HR) was calculated using Cox regression under a log additive genetic model with age, sex and disease stage as covariates. (h) Null association of LINC00673 expression with survival in patients with PAAD in the TCGA database.

Supplementary Figure 3 Prediction of the effects of the rs11655237 G>A change on LINC00673 folding structure and interaction with miR-1231.

(a,b) Predicted folding structures and mountain plots for LINC00673 with rs11655237[G] or rs11655237[A]. Each mountain plot is an x–y graph representing the secondary structures of LINC00673, including the minimum free energy (MFE) structure, the partition function (PF) of the thermodynamic ensemble of RNA structures and the centroid structure (Centroid) in height versus position. (c) In silico prediction of interaction between miR-1231 and LINC00673 shows differences in binding within the seed region. We used three publicly available software programs, including FINDTAR3, RegRNA and RegRNA 2.0 (see URLs). (d) Relative expression levels of miR-1231 as determined by qRT–PCR in BXPC-3 and CFPAC-1 cells. Results are shown as means ± s.e.m. normalized to U6 expression. (e−g) Correlations between miR-1231 expression levels and LINC00673 expression levels as determined by qRT–PCR in normal pancreatic tissues adjacent to tumors with different rs11655237 genotypes. The r values and P values are from Pearson’s correlation analysis. (h) Copy numbers per cell of LINC00673 and miR-1231 in BXPC-3 and CFPAC-1 cells as determined by qRT–PCR. Data are shown as means ± s.e.m.

Supplementary Figure 4 Effect of LINC00673 on apoptosis, migration and invasion in pancreatic cancer cells.

(a) Relative expression levels of LINC00673 as determined by qRT–PCR in BXPC-3 and CFPAC-1 cells transfected with LINC00673[G] or LINC00673-targeting shRNAs. Results are shown as means ± s.e.m. relative to lentiviral vector control. (b) Effect of LINC00673 on apoptosis in BXPC-3 and CFPAC-1 cells as detected by flow cytometry. Results are shown as means ± s.e.m. from three experiments, each with six replications. No significant difference was found between cells transfected with LINC00673[G] and those with lentiviral vector. (c) Representative visual fields of BXPC-3 and CFPAC-1 cells treated with LINC00673[G], LINC00673[A] or shRNA to LINC00673 as compared with control cells with empty vector. Scale bars, 100 μm. (d,e) Cell counts under the microscope at 100× magnification. Data are shown as means ± s.e.m. from three random fields. The experiments were performed in triplicate. P values are from two-sided t tests.

Supplementary Figure 5 Xenograft tumor growth of BXPC-3 and CFPAC-1 cells with stable overexpression or knockdown of LINC00673[G] in nude mice.

Supplementary Figure 6 Effects of LINC00673 on the phenotypes of immortalized human pancreatic duct epithelial (HPDE6-C7) cells.

(a) Relative expression levels of LINC00673 in HPDE6-C7 cells transfected with LINC00673[A], LINC00673[G] or LINC00673-targeting shRNAs as determined by qRT–PCR. Results are shown as means ± s.e.m. relative to lentiviral vector control. (b) Overexpression of LINC00673 substantially reduced HPDE6-C7 cell proliferation. (c) Knockdown of LINC00673 significantly enhanced HPDE6-C7 cell proliferation. Cells were seeded in 96-well plates after transfection with LINC00673[A], LINC00673[G], LINC00673-targeting shRNA or control lentivirus vector, and cell number was determined every 24 h for 96 h using CCK-8 assays. Results are shown as means ± s.e.m. from three experiments, each with six replicates. *P < 0.05, compared with control on the basis of two-sided Student's t tests. (d) Effect of LINC00673 overexpression or knockdown on the cell cycle progression of HPDE6-C7 cells. Results are shown as means ± s.e.m. from three experiments. (e) Effect of LINC00673 overexpression or knockdown on colony formation of HPDE6-C7 cells (left); values are colony formation ability relative to control (set to 100%) from three experiments (right). *P < 0.05, compared with control on the basis of two-sided Student's t tests. (f) Effect of LINC00673 knockdown on colony formation of HPDE6-C7 cells in soft agar. Cells (4 × 103) were cultured in soft agar medium for 6 weeks, but no colony formation was observed. (g,h) Effect of LINC00673 overexpression or knockdown on the migration and invasion of HPDE6-C7 cells. Scale bars, 100 μm. Cell counts were determined under the microscope at 100× magnification. Data are shown as means ± s.e.m. from three random fields. The experiments were performed in triplicate. P values are from two-sided Student's t tests.

Supplementary Figure 7 Effects of altered LINC00673 levels on PTPN11 ubiquitination but not PRPF19 expression.

(a,b) BXPC-3 and CFPAC-1 cells with stable overexpression or knockdown of LINC00673[G] were treated with MG132 (5 μM) for 24 h. Cell lysates were immunoprecipitated with either control IgG or antibody to ubiquitin, followed by immunoblot analysis with antibody to PTPN11. Bottom, input of cell lysates. (c) Levels of PRPF19 mRNA as determined by qRT–PCR in BXPC-3 and CFPAC-1 cells with stable overexpression or knockdown of LINC00673[G]. Results are shown means ± s.e.m. relative to GAPDH; P values were obtained from two-sided t tests. (d) Immunoblot analysis of PRPF19 protein in control BXPC-3 and CFPAC-1 cells and in cells stably overexpressing LINC00673[G].

Supplementary Figure 8 Effects of overexpression or knockdown of LINC00673[G] on RNA levels of some genes downstream of PTPN11 in BXPC-3 and CFPAC-1 cells.

RNA levels as determined by qRT–PCR are presented as means ± s.e.m. relative to GAPDH; P values were obtained from two-sided t tests.

Supplementary Figure 9 Effects of LINC00673 on cell proliferation and cell cycle progression via PTPN11.

(a,b) Proliferation profiles of control BXPC-3 and CFPAC-1 cells and cells overexpressing LINC00673[G] transiently transfected with or without pCDNA3.1-PTPN11. Values are means ± s.e.m. from three experiments, each with six replicates. *P < 0.05, compared with cells overexpressing both LINC00673[G] and PTPN11 on the basis of two-sided t tests. (c,d) Proliferation profiles of control BXPC-3 and CFPAC-1 cells and cells expressing LINC00673 shRNA transiently transfected with or without PTPN11 siRNA. Values are means ± s.e.m. from three experiments, each with six replicates. *P < 0.05, compared with cells expressing LINC00673 shRNA and transfected with PTPN11 siRNA on the basis of a two-sided t test. (e,f) Cell cycle profiles of control BXPC-3 and CFPAC-1 cells and cells overexpressing LINC00673[G] transiently transfected with or without pCDNA3.1-PTPN11 or of control cells and cells expressing LINC00673 shRNA transiently transfected with or without PTPN11.

Supplementary Figure 10 Correlation between LINC00673 levels and the levels of some genes downstream of PTPN11 in clinical pancreatic tissues.

mRNA levels were measured by qRT–PCR in PDAC tissues (n = 74) and normalized to GAPDH. The r and P values were obtained from Pearson’s correlation analysis.

Supplementary Figure 11 Characterization of full-length human LINC00673 and cellular localization of LINC00673 in PDAC cell lines.

(a,b) Representative images of the PCR products from 5′-RACE (a) and 3′-RACE (b) (left). Sequencing of PCR products identified the boundary between the universal anchor primer and LINC00673 sequences (right). (c) The nucleotide sequence of full-length human LINC00673. (d) RNA blotting of LINC00673 extracted from BXPC-3 and CFPAC-1 cells, showing its expected molecular size. (e) Relative nuclear and cytoplasmic levels of LINC00673 in PDAC cells as determined by qRT–PCR with U6 or GAPDH as a nuclear or cytoplasmic marker, respectively. Results are presented as the percent ± s.e.m. of the total level of each molecule.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11 and Supplementary Tables 1–9. (PDF 2070 kb)

Supplementary Data 1

Genotyping data for rs11655237 in replication III samples. (XLSX 27 kb)

Supplementary Data 2

Quantification overview (111 proteins). (ZIP 6257 kb)

Supplementary Data 3

Quantification overview (34 proteins). (ZIP 10143 kb)

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Zheng, J., Huang, X., Tan, W. et al. Pancreatic cancer risk variant in LINC00673 creates a miR-1231 binding site and interferes with PTPN11 degradation. Nat Genet 48, 747–757 (2016). https://doi.org/10.1038/ng.3568

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