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Biotechnology Letters

, Volume 40, Issue 5, pp 809–818 | Cite as

Gene expression profiling of human hepatocytes grown on differing substrate stiffness

  • Tingting Xia
  • Runze Zhao
  • Fan Feng
  • Yijiang Song
  • Yu Zhang
  • Lili Dong
  • Yonggang Lv
  • Li Yang
Original Research Paper

Abstract

Objective

To study the effects of different substrate stiffness on human hepatocytes using RNA sequencing (RNA-Seq) technology. The stiffness was corresponding to physiology and pathology stiffness of liver tissues.

Results

With the aid of RNA-Seq technology, our study characterizes the transcriptome of hepatocytes cultured on soft, moderate, stiff and plastic substrates. Compared to soft substrate, our RNA-Seq results revealed 1131 genes that were up-regulated and 2534 that were down-regulated on moderate substrate, 1370 genes that were up-regulated and 2677 down-regulated genes on stiff substrate. Functional enrichment analysis indicated that differentially expressed genes were associated with the regulation of actin cytoskeleton, focal adhesion, tight junction, adherens junction as well as antigen processing and presentation. RNA-Seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction.

Conclusion

Our study provides a comprehensive picture of the gene expression landscape in hepatocytes grown on different substrate stiffness, offering insights into the role of substrate stiffness in hepatic pathology.

Keywords

Differentially expressed gene Hepatocyte RNA-sequencing Substrate stiffness 

Notes

Acknowledgements

This work was supported by National Natural Science Foundation of China (31270990, 11532004) and Innovation and Attracting Talents Program for College and University (“111” Project) (B06023).

Supporting information

Supplementary Table 1—Primer sequence of selected genes designed for qRT-PCR.

Supplementary Table 2—Quality examination of sequence data.

Supplementary Fig. 1—GO functional classification on DEGs in soft, moderate and stiff substrates.

Supplementary Fig. 2—Statistical representation of DEG pathway enrichment in soft vs moderate, soft vs stiff and soft vs plastic groups.

Supplementary Fig. 3—Statistics of pathway enrichment of DEGs expressed in soft, moderate and stiff groups.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

10529_2018_2536_MOESM1_ESM.docx (874 kb)
Supplementary material 1 (DOCX 874 kb)

References

  1. Begnaud S, Chen TC, Delacour D, Mege RM, Ladoux B (2016) Mechanics of epithelial tissues during gap closure. Curr Opin Cell Biol 42:52–62.  https://doi.org/10.1016/j.ceb.2016.04.006 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Canver AC, Ngo O, Urbano RL, Clyne AM (2016) Endothelial directed collective migration depends on substrate stiffness via localized myosin contractility and cell–matrix interactions. J Biomech 49:1369–1380.  https://doi.org/10.1016/j.jbiomech.2015.12.037 CrossRefPubMedGoogle Scholar
  3. Chiang MYM, Yangben YZ, Lin NJ, Zhong JLL, Yang L (2013) Relationships among cell morphology, intrinsic cell stiffness and cell–substrate interactions. Biomaterials 34:9754–9762.  https://doi.org/10.1016/j.biomaterials.2013.09.014 CrossRefPubMedGoogle Scholar
  4. Das N et al (2017) Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice. J Pineal Res.  https://doi.org/10.1111/jpi.12404 PubMedGoogle Scholar
  5. Deegan DB, Zimmerman C, Skardal A, Atala A, Shupe TD (2016) Stiffness of hyaluronic acid gels containing liver extracellular matrix supports human hepatocyte function and alters cell morphology. J Mech Behav Biomed Mater 55:87–103.  https://doi.org/10.1016/j.jmbbm.2015.10.016 CrossRefGoogle Scholar
  6. Giorgi C, Yeo GW, Stone ME, Katz DB, Burge C, Turrigiano G, Moore MJ (2007) The EJC factor eIF4AIII modulates synaptic strength and neuronal protein expression. Cell 130:179–191.  https://doi.org/10.1016/j.cell.2007.05.028 CrossRefPubMedGoogle Scholar
  7. Humphrey JD, Dufresne ER, Schwartz MA (2014) Mechanotransduction and extracellular matrix homeostasis. Nat Rev Mol Cell Biol 15:802–812.  https://doi.org/10.1038/nrm3896 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Jerrell RJ, Parekh A (2014) Cellular traction stresses mediate extracellular matrix degradation by invadopodia. Acta Biomater 10:1886–1896.  https://doi.org/10.1016/j.actbio.2013.12.058 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Kamimura M, Sugawara M, Yamamoto S, Yamaguchi K, Nakanishi J (2016) Dynamic control of cell adhesion on a stiffness-tunable substrate for analyzing the mechanobiology of collective cell migration. Biomater Sci UK 4:933–937.  https://doi.org/10.1039/c6bm00100a CrossRefGoogle Scholar
  10. Mao AS, Shin JW, Mooney DJ (2016) Effects of substrate stiffness and cell–cell contact on mesenchymal stem cell differentiation. Biomaterials 98:184–191.  https://doi.org/10.1016/j.biomaterials.2016.05.004 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Mui KL, Chen CS, Assoian RK (2016) The mechanical regulation of integrin–cadherin crosstalk organizes cells, signaling and forces. J Cell Sci 129:1093–1100.  https://doi.org/10.1242/jcs.183699 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Nagayama K, Matsumoto T (2010) Estimation of single stress fiber stiffness in cultured aortic smooth muscle cells under relaxed and contracted states: its relation to dynamic rearrangement of stress fibers. J Biomech 43:1443–1449.  https://doi.org/10.1016/j.jbiomech.2010.02.007 CrossRefPubMedGoogle Scholar
  13. Patro R, Mount SM, Kingsford C (2014) Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms. Nat Biotechnol 32:462–464.  https://doi.org/10.1038/nbt.2862 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Sahin H et al (2012) Chemokine Cxcl9 attenuates liver fibrosis-associated angiogenesis in mice. Hepatology (Baltimore Md) 55:1610–1619.  https://doi.org/10.1002/hep.25545 CrossRefGoogle Scholar
  15. Saw TB, Jain S, Ladoux B, Lim CT (2015) Mechanobiology of collective cell migration. Cell Mol Bioeng 8:3–13.  https://doi.org/10.1007/s12195-014-0366-3 CrossRefGoogle Scholar
  16. Shukla VC, Higuita-Castro N, Nana-Sinkam P, Ghadiali SN (2016) Substrate stiffness modulates lung cancer cell migration but not epithelial to mesenchymal transition. J Biomed Mater Res A 104:1182–1193.  https://doi.org/10.1002/jbm.a.35655 CrossRefPubMedGoogle Scholar
  17. Takeda T et al (2006) Usefulness of noninvasive transient elastography for assessment of liver fibrosis stage in chronic hepatitis C. World J Gastroenterol 12:7768–7773CrossRefPubMedPubMedCentralGoogle Scholar
  18. Tu T et al (2015) Hepatocytes in liver injury: victim, bystander, or accomplice in progressive fibrosis? J Gastroenterol Hepatol 30:1696–1704.  https://doi.org/10.1111/jgh.13065 CrossRefPubMedGoogle Scholar
  19. Weinberg SH, Mair DB, Lemmon CA (2017) Mechanotransduction dynamics at the cell–matrix interface. Biophys J 112:1962–1974.  https://doi.org/10.1016/j.bpj.2017.02.027 CrossRefPubMedGoogle Scholar
  20. Xia T et al (2018) Effect of substrate stiffness on hepatocyte migration and cellular Young’s modulus. J Cell Physiol.  https://doi.org/10.1002/jcp.26491 Google Scholar
  21. Yang L, Inokuchi S, Roh YS, Song J, Loomba R, Park EJ, Seki E (2013) Transforming growth factor-beta signaling in hepatocytes promotes hepatic fibrosis and carcinogenesis in mice with hepatocyte-specific deletion of TAK1. Gastroenterology 144(1042–1054):e1044.  https://doi.org/10.1053/j.gastro.2013.01.056 Google Scholar
  22. Yangben YZ et al (2013) Relative rigidity of cell–substrate effects on hepatic and hepatocellular carcinoma cell migration. J Biomater Sci Polym E 24:148–157.  https://doi.org/10.1163/156856212x627856 Google Scholar
  23. You J et al (2013) Characterizing the effects of heparin gel stiffness on function of primary hepatocytes. Tissue Eng A 19:2655–2663.  https://doi.org/10.1089/ten.tea.2012.0681 CrossRefGoogle Scholar
  24. Zhang XW et al (2017) Antagonism of Interleukin-17A ameliorates experimental hepatic fibrosis by restoring the IL-10/STAT3-suppressed autophagy in hepatocytes. Oncotarget 8:9922–9934.  https://doi.org/10.18632/oncotarget.14266 PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering CollegeChongqing UniversityChongqingChina

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