Abstract
Purpose
Genome-wide association studies have identified SMAD7 as a colorectal cancer (CRC) susceptibility gene. However, its underlying mechanism has not yet been characterized. This study screened functional SNPs (fSNPs) related to colorectal cancer through Reel-seq and obtained regulatory proteins on functional SNPs.
Methods
The candidate fSNPs on the SMAD7 locus were screened by Reel-seq method. Eight SNPs such as rs8085824 were identified as functional SNPs by luciferase reporter assay and EMSA, SDCP-MS and AIDP-WB revealed that HNRNPK can specifically bind to the rs8085824-C allele. The knockdown of HNRNPK by RNAi proved that HNRNPK could affect cell function by regulating SMAD7.
Results
Eight functional SNPs was found on the SMAD7 locus in linkage disequilibrium (LD) with R2 > 0.8, i.e., rs12953717, rs7227023, rs34007497, rs58920878, rs8085824, rs4991143, rs4939826, and rs7227023. We also identified allele-imbalanced binding of HNRNPK to rs8085824, H1-3 to rs12953717, THOC6 to rs7227023, and DDX21 to rs58920878. Further functional analysis revealed that these proteins are the regulatory proteins that modulate the expression of SMAD7 in the human colorectal cancer cell line DLD1. In particular, we discovered that siRNA knockdown of HNRNPK inhibits cell proliferation and cell clonal formation by downregulating SMAD7, as the decreased cell proliferation and cell clonal formation in the siRNA HNRNPK knockdown cells was restored by SMAD7 overexpression.
Conclusion
Our findings reveal a mechanism which underlies the contribution of the fSNP rs8085824 on the SMD7 locus to CRC susceptibility.
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Data availability
The data underlying this article are available in the article and in its online supplementary material.
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Acknowledgements
We thank Dr. Gang Li (University of Pittsburgh, USA) for scientific discussions concerning this work.
Funding
This work was supported by Science and Technology Commission of Shanghai Municipality (23141901800, 23141903300) and the National Nature Science Foundation of China (82173099, 32270960).
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MQ and HR supervised the project. ZL, HS, HM, YW and CT performed the experiments and analyzed the data. MQ, YZ and TF supervised the Reel-seq experiments. BD and JQ provided advice and technical expertise. HR and ZL wrote the manuscript. All authors revised the manuscript and approved its final version.
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432_2023_5402_MOESM2_ESM.tif
Supplementary file2 Supplementary Figure 2. The luciferase reporter gene demonstrated that HNRNPK could regulate luciferase activity through rs8085824-C, through knockdown or overexpression of HNRNPK in DLD1 cells(n>3). RLA relative luciferase activity, P value was calculated using Student’s t test with two tails. Error bars, SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (TIF 4403 KB)
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Liu, Z., Zhao, Y., Song, H. et al. Identification and characterization of colorectal-cancer-associated SNPs on the SMAD7 locus. J Cancer Res Clin Oncol 149, 16659–16668 (2023). https://doi.org/10.1007/s00432-023-05402-w
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DOI: https://doi.org/10.1007/s00432-023-05402-w