Background

Autophagy plays multifaceted roles in regulating hepatocellular carcinoma (HCC) and the mechanisms involved are under-explored. Regulatory microRNAs (miRNAs) have been reported to target autophagy proteins but their roles in HCC is not well studied. Using HCC patient tissues, this study aims to investigate the association of autophagy with several clinicopathological parameters as well as identifying the autophagy-related miRNAs and the possible pathways.

Methods and results

Autophagy level in the HCC patient-derived cancer and non-cancer tissues was determined by immunohistochemistry (IHC) targeting SQSTM1, LC3A and LC3B proteins. Significance tests of clinicopathological variables were tested using the Fisher's exact or Chi-square tests. Gene and miRNA expression assays were carried out and analyzed using Nanostring platform and software followed by validation of other online bioinformatics tools, namely String and miRabel. Autophagy expression was significantly higher in cancerous tissues compared to adjacent non-cancer tissues. High LC3B expression was associated with advanced tumor histology grade and tumor location. Nanostring gene expression analysis revealed that SQSTM1, PARP1 and ATG9A genes were upregulated in HCC tissues compared to non-cancer tissues while SIRT1 gene was downregulated. These genes are closely related to an autophagy pathway in HCC. Further, using miRabel tool, three downregulated miRNAs (hsa-miR-16b-5p, hsa-miR-34a-5p, and hsa-miR-660-5p) and one upregulated miRNA (hsa-miR-539-5p) were found to closely interact with the abovementioned autophagy-related genes. We then mapped out the possible pathway involving the genes and miRNAs in HCC tissues.

Conclusions

We conclude that autophagy events are more active in HCC tissues compared to the adjacent non-cancer tissues. We also reported the possible role of several miRNAs in regulating autophagy-related genes in the autophagy pathway in HCC. This may contribute to the development of potential therapeutic targets for improving HCC therapy. Future investigations are warranted to validate the target genes reported in this study using a larger sample size and more targeted molecular technique.

1. Abdel-Moety, A., Baddour, N., Salem, P., El-Khouly, M., & El-Kalla, I. (2022). SQSTM1 expression in hepatocellular carcinoma and relation to tumor recurrence after radiofrequency ablation. Journal of Clinical and Experimental Hepatology, 12(3), 774–784. https://doi.org/10.1016/j.jceh.2021.12.001
2. Akkoc, Y., & Gozuacik, D. (2018). Autophagy and liver cancer. The Turkish Journal of Gastroenterology, 29(3), 270–282. https://doi.org/10.5152/tjg.2018.150318
3. Awi, N. J., Armon, S., Peh, K. B., Peh, S. C., & Teow, S. Y. (2020). High expression of LC3A, LC3B, and p62/SQSTM1 autophagic proteins in human colonic ganglion cells. The Malaysian Journal of Pathology, 42(1), 85–90.
4. Awi, N. J., Yap, H. Y., Armon, S., Peh, K. B., Peh, S. C., & Teow, S. Y. (2021). Association between autophagy and KRAS mutation with clinicopathological variables in colorectal cancer patients. The Malaysian Journal of Pathology, 43(2), 269–279.
5. Chan, H. Y., Ramasamy, T. S., Chung, F. F., & Teow, S. Y. (2024). Role of sirtuin 1 (SIRT1) in regulation of autophagy and nuclear factor-kappa Beta (NF-ĸβ) pathways in sorafenib-resistant hepatocellular carcinoma (HCC). Cell Biochemistry and Biophysics. Advance online publication. https://doi.org/10.1007/s12013-024-01247-3
6. Chava, S., Lee, C., Aydin, Y., Dash, S., & Wu, T. (2017). Chaperone-mediated autophagy compensates for impaired macroautophagy in the cirrhotic liver to promote hepatocellular carcinoma. Oncotarget, 8(25), 40019–40036. https://doi.org/10.18632/oncotarget.16685
7. Cicchini, M., Karantza, V., & Xia, B. (2015). Molecular pathways: Autophagy in cancer—a matter of timing and context. Clinical Cancer Research, 21(3), 498–504. https://doi.org/10.1158/1078-0432.CCR-13-2438
8. Dash, S., Chava, S., Chandra, P. K., Aydin, Y., Balart, L. A., & Wu, T. (2016). Autophagy in hepatocellular carcinomas: From pathophysiology to therapeutic response. Hepatoma Research, 8, 9–20. https://doi.org/10.2147/HMER.S63700
9. Gozuacik, D., Akkoc, Y., Ozturk, D. G., & Kocak, M. (2017). Autophagy-regulating microRNAs and cancer. Frontiers in Oncology, 7, Article 65. https://doi.org/10.3389/fonc.2017.00065
10. Gu, H., Gu, S., Zhang, X., Zheng, Z., Jiang, B., Lin, C., & Chen, G. (2019). miR-106b-5p promotes aggressive progression of hepatocellular carcinoma via targeting RUNX3. Cancer Medicine, 8(15), 6756–6767. https://doi.org/10.1002/cam4.2511
11. Guardia, C. M., Tan, X. F., & Lian, T. (2020). Structure of human ATG9A, the only transmembrane protein of the core autophagy machinery. Cell Reports, 31(13), Article 107837. https://doi.org/10.1016/j.celrep.2020.107837
12. Hao, C., Zhu, P. X., Yang, X., Zhang, P. X., Kang, F. X., Liu, B., ... & Wang, T. G. (2014). Overexpression of SIRT1 promotes metastasis through epithelial-mesenchymal transition in hepatocellular carcinoma. BMC Cancer, 14, Article 978. https://doi.org/10.1186/1471-2407-14-978
13. Hennig, P., Fenini, G., Di Filippo, M., & Beer, H. D. (2021). The pathways underlying the multiple roles of p62 in inflammation and cancer. Biomedicines, 9(7), Article 707. https://doi.org/10.3390/biomedicines9070707
14. Kessler, S. M., Pokorny, J., Zimmer, V., Laggai, S., Lammert, F., Bohle, R. M., ... & Kiemer, A. K. (2013). IGF2 mRNA binding protein p62/IMP2-2 in hepatocellular carcinoma: Anti-apoptotic action is independent of IGF2/PI3K signaling. American Journal of Physiology-Gastrointestinal and Liver Physiology, 304(4), G328–G336. https://doi.org/10.1152/ajpgi.00005.2012
15. Kimkong, I., & Kunanopparat, A. (2020). Autophagy related protein 9A increase in hepatitis B virus-associated hepatocellular carcinoma and the role in apoptosis. World Journal of Hepatology, 12(12), 1367–1371. https://doi.org/10.4254/wjh.v12.i12.1367
16. Kobayashi, M., Yasukawa, H., Arikawa, T., Yoshimaru, T., Komori, M., & Hashimoto, S. (2021). Trehalose induces SQSTM1/p62 expression and enhances lysosomal activity and antioxidative capacity in adipocytes. FEBS Open Bio, 11(1), 185–194. https://doi.org/10.1002/2211-5463.13055
17. Li, Y., Xu, S., Li, J., Zheng, L., Feng, M., Wang, X., ... & Zhang, Z. (2016). SIRT1 facilitates hepatocellular carcinoma metastasis by promoting PGC-1α-mediated mitochondrial biogenesis. Oncotarget, 7(20), 29255–29274. https://doi.org/10.18632/oncotarget.8711
18. Li, X., He, S., & Ma, B. (2020). Autophagy and autophagy-related proteins in cancer. Molecular Cancer, 19(1), Article 12. https://doi.org/10.1186/s12943-020-1138-4
19. Liu, L., Liao, J., He, X., & Li, P. (2017). The role of autophagy in hepatocellular carcinoma: Friend or foe. Oncotarget, 8(34), 57707–57722. https://doi.org/10.18632/oncotarget.17202
20. Liu, Y. M., Cao, Y., Zhao, P. S., Wang, Y., & Zhang, J. H. (2022). Erratum: CircCCNB1 silencing acting as a miR-106b-5p sponge inhibited GPM6A expression to promote HCC progression by enhancing DYNC1I1 expression and activating the AKT/ERK signaling pathway. International Journal of Biological Sciences, 18(6), 2652–2654. https://doi.org/10.7150/ijbs.72651
21. Liu, S., Zhang, H., Yan, J., Zhao, X., & Wang, W. (2023). FOXP3 and SQSTM1/P62 correlate with prognosis and immune infiltration in hepatocellular carcinoma. Pathology - Research and Practice, 242, Article 154292. https://doi.org/10.1016/j.prp.2022.154292
22. Llovet, J. M., Kelley, R. K., Villanueva, A., Singal, A. G., Pikarsky, E., Roayaie, S., ... & Finn, R. S. (2021). Hepatocellular carcinoma. Nature Reviews Disease Primers, 7, Article 6. https://doi.org/10.1038/s41572-020-00240-3
23. Luna, A., Aladjem, M. M., & Kohn, K. W. (2013). SIRT1/PARP1 crosstalk: Connecting DNA damage and metabolism. Genome Integrity, 4(1), Article 6. https://doi.org/10.1186/2041-9414-4-6
24. Manley, S., Williams, J. A., & Ding, W. X. (2013). Role of p62/SQSTM1 in liver physiology and pathogenesis. Experimental Biology and Medicine, 238(5), 525–538. https://doi.org/10.1177/1535370213489446
25. Meng, Y. C., Lou, X. L., Yang, L. Y., Chen, Y., & Oyang, Q. (2020). Role of the autophagy-related marker LC3 expression in hepatocellular carcinoma: A meta-analysis. Journal of Cancer Research and Clinical Oncology, 146(5), 1103–1113. https://doi.org/10.1007/s00432-020-03174-1
26. Morishita, A., Oura, K., Tadokoro, T., Fujita, K., Tani, J., & Masaki, T. (2021). MicroRNAs in the pathogenesis of hepatocellular carcinoma: A review. Cancers, 13(3), Article 514. https://doi.org/10.3390/cancers13030514
27. Niu, X., Wei, N., Pen, L., & Zhang, J. (2022). miR-34a-5p plays an inhibitory role in hepatocellular carcinoma by regulating target gene VEGFA. The Malaysian Journal of Pathology, 44(1), 39–52.
28. Nomura, F., Yaguchi, M., Togawa, A., Miyamoto, M., Harada, M., Noda, M., ... & Ohto, M. (2000). Enhancement of poly-adenosine diphosphate-ribosylation in human hepatocellular carcinoma. Journal of Gastroenterology and Hepatology, 15(5), 529–535. https://doi.org/10.1046/j.1440-1746.2000.02193.x
29. Pang, S. W., Armon, S., Chook, J. B., Peh, K. B., Peh, S. C., & Teow, S. Y. (2024). Association of Fusobacterium nucleatum infection with the clinicopathological characteristics in colorectal cancer patients. Molecular Biology Reports, 51(1), Article 124. https://doi.org/10.1007/s11033-023-09150-5
30. Parzych, K. R., & Klionsky, D. J. (2014). An overview of autophagy: Morphology, mechanism, and regulation. Antioxidants & Redox Signaling, 20(3), 460–473. https://doi.org/10.1089/ars.2013.5371
31. Qiu, D., Wang, G., Chen, L., Li, Y., Zhang, F., & Zhou, D. (2014). The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance. BMC Cancer, 14, Article 327. https://doi.org/10.1186/1471-2407-14-327
32. Qiu, G., Li, X., Che, X., Wei, C., He, S., Lu, J., ... & Wang, J. (2015). SIRT1 is a regulator of autophagy: Implications in gastric cancer progression and treatment. FEBS Letters, 589(16), 2034–2042. https://doi.org/10.1016/j.febslet.2015.05.042
33. Rodríguez-Vargas, J. M., Rodríguez, M. I., Majuelos-Melguizo, J., García-Diaz, A., del Río-Moreno, M., & Oliver, F. J. (2016). Autophagy requires poly(ADP-ribosyl)ation-dependent AMPK nuclear export. Cell Death & Differentiation, 23(12), 2007–2018. https://doi.org/10.1038/cdd.2016.80
34. Santana-Codina, N., Mancias, J. D., & Kimmelman, A. C. (2017). The role of autophagy in cancer. Annual Review of Cancer Biology, 1, 19–39. https://doi.org/10.1146/annurev-cancerbio-041816-122338
35. Schläfli, A. M., Berezowska, S., Adams, O., Langer, R., & Tschan, M. P. (2015). Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry. European Journal of Histochemistry, 59(2), Article 2481. https://doi.org/10.4081/ejh.2015.2481
36. Schreiber, V., Dantzer, F., Ame, J. C., & de Murcia, G. (2006). Poly(ADP-ribose): Novel functions for an old molecule. Nature Reviews Molecular Cell Biology, 7(7), 517–528. https://doi.org/10.1038/nrm1963
37. Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 71(3), 209–249. https://doi.org/10.3322/caac.21660
38. Takamura, A., Komatsu, M., Hara, T., Sakamoto, A., Ichimura, Y., Mizushima, J. W., ... & Mizushima, N. (2011). Autophagy-deficient mice develop multiple liver tumors. Genes & Development, 25(8), 795–800. https://doi.org/10.1101/gad.2016211
39. Tobita, T., Guzman-Lepe, J., Takeishi, K., Han, B., Dorko, K., & Soto-Gutierrez, A. (2016). SIRT1 disruption in human fetal hepatocytes leads to increased accumulation of glucose and lipids. PLOS ONE, 11(2), Article e0149344. https://doi.org/10.1371/journal.pone.0149344
40. Umemura, A., He, F., Taniguchi, K., Nakagawa, H., Yamachika, S., Font-Burgada, J., ... & Karin, M. (2016). p62, upregulated during preneoplasia, induces hepatocellular carcinogenesis by maintaining survival of stressed HCC-initiating cells. Cancer Cell, 29(6), 935–948. https://doi.org/10.1016/j.ccell.2016.04.006
41. Wang, R. H., Sengupta, K., Li, C., Kim, H. S., Cao, L., Xiao, C., ... & Deng, C. X. (2008). Impaired DNA damage response, genome instability, and tumorigenesis in SIRT1 mutant mice. Cancer Cell, 14(4), 312–323. https://doi.org/10.1016/j.ccr.2008.09.001
42. Wang, Y., Cui, R., Zhang, X., Lu, Z., Sheng, H., & Zhou, W. (2016). SIRT1 increases YAP- and MKK3-dependent p38 phosphorylation in mouse liver and human hepatocellular carcinoma. Oncotarget, 7(10), 11284–11298. https://doi.org/10.18632/oncotarget.7022
43. White, E. (2015). The role of autophagy in cancer. The Journal of Clinical Investigation, 125(1), 42–46. https://doi.org/10.1172/JCI73941
44. Wong, M. M. T., Chan, H. Y., Aziz, N. A., Teow, S. Y., & Ramasamy, T. S. (2021). Interplay of autophagy and cancer stem cells in hepatocellular carcinoma. Molecular Biology Reports, 48(8), 6135–6157. https://doi.org/10.1007/s11033-021-06334-9
45. Wu, D. H., Jia, C. C., Chen, J., Cheng, M. T., Lin, Z. M., Jiang, A. Z., ... & Lin, T. X. (2014). Autophagic LC3B overexpression correlates with malignant progression and predicts a poor prognosis in hepatocellular carcinoma. Tumour Biology, 35(12), 12225–12233. https://doi.org/10.1007/s13277-014-2531-7
46. Xi, S. Y., Lu, J. B., Chen, J. W., Cao, Y., & Mukaida, N. (2013). The stone-like pattern of LC3A expression and its clinicologic significance in hepatocellular carcinoma. Biochemical and Biophysical Research Communications, 431(4), 760–766. https://doi.org/10.1016/j.bbrc.2012.12.151
47. Yang, Z., & Klionsky, D. J. (2009). An overview of the molecular mechanism of autophagy. Current Topics in Microbiology and Immunology, 335, 1–32. https://doi.org/10.1007/978-3-642-00302-8_1
48. Yang, X. D., Kong, F. E., Qi, L., Zhou, C. Y., Wang, M. H., & Zhang, J. H. (2021). PARP inhibitor Olaparib overcomes Sorafenib resistance through reshaping the pluripotent transcriptome in hepatocellular carcinoma. Molecular Cancer, 20(1), Article 20. https://doi.org/10.1186/s12943-021-01315-9
49. Yu, L. X., Zhang, B. L., Yang, M. Y., & Cui, M. M. (2019). MicroRNA-106b-5p promotes hepatocellular carcinoma development via modulating FOG2. OncoTargets and Therapy, 12, 5639–5647. https://doi.org/10.2147/OTT.S203382