Hepatology. 2014 Feb;59(2):505-17.

Autophagy suppresses tumorigenesis of hepatitis B virus-associated hepatocellular carcinoma through degradation of microRNA-224.

Lan SH, Wu SY, Zuchini R, Lin XZ, Su IJ, Tsai TF, Lin YJ, Wu CT, Liu HS.

Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan.



In hepatocellular carcinoma (HCC), dysregulated expression of microRNA-224 (miR-224) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. In this study we determined that autophagy is down-regulated and inversely correlated with miR-224 expression in hepatitis B virus (HBV)-associated HCC patient specimens. These results were confirmed in liver tumors of HBV X gene transgenic mice. Furthermore, miR-224 was preferentially recruited and degraded during autophagic progression demonstrated by real-time polymerase chain reaction and miRNA in situ hybridization electron microscopy after extraction of autophagosomes. Our in vitro study demonstrated that miR-224 played an oncogenic role in hepatoma cell migration and tumor formation through silencing its target gene Smad4. In HCC patients, the expression of low-Atg5, high-miR-224, and low-Smad4 showed significant correlation with HBV infection and a poor overall survival rate. Autophagy-mediated miR-224 degradation and liver tumor suppression were further confirmed by the autophagy inducer amiodarone and miR-224 antagonist using an orthotopic SD rat model. CONCLUSION: A noncanonical pathway links autophagy, miR-224, Smad4, and HBV-associated HCC. These findings open a new avenue for the treatment of HCC.

PMID: 23913306


Additional information:

Currently, many papers reported  that diverse microRNAs regulate autophagic related genes to affect tumorigenesis [1]. Differently, we revealed that autophagy regulates an oncogenic miR-224 through the selective autophagic degradation pathway to affect the tumorigenesis of HBV-associated hepatocellular carcinoma (HCC). We found that induced-autophagy showed no effect on miR-224 transcription (precursor miR-224) as well as its secretion to the extracellular medium. Instead, only the mature form miR-224 was preferably recruited and degraded by the autophagic degradation pathway. To further confirm that autophagosomes selectively recruit miR-224, a ubiquitously expressed microRNA let-7a was used as a general control. We purified the autophagosomes based on Senlen’s protocol with modification [2]  followed by detection of miR-224 and let-7a using immune-gold labeling of microRNAs under the transmission electron microscopy (Fig. 1). Current knowledge of selective autophagy degradation is through ubiquitin conjugation mediated degradation. Further study is needed to clarify the underlying mechanism of selective degradation of microRNAs and whether it is a general event of microRNA regulation by autophagy. Balgi et al., identified amiodarone (an antiarrhythmic agent for cardiac dysrhythmias) as a potential autophagy inducer among 3584 small molecules [3]. We are the first to use amiodarone both in vitro and in vivo to induce autophagic activity and further demonstrated its tumor suppressive effect through increased autophagic activity. Autophagy plays pivotal roles in innate immunity including clearance pathogens like bacteria and virus through its degradation pathway. In contrast, many pathogens counteract autophagy to evade host immune surveillance. Despite autophagy is induced during HBV infection by HBx protein and HBV replication is promoted, our data from HBx transgenic mice and clinical HBV-associated HCC specimens consistently showed that autophagic activity is decreased during liver tumor tumorigenesis. These results indicate that HBV-induced autophagy promotes its replication and HBV-related suppression of autophagy contributes to HCC tumorigenesis. Surprisingly, this phenomenon was not detected in HCV-related HCC. In summary, autophagy inducer amiodarone has the potential to become the anticancer drug against HBV-associated HCC.



  1. Frankel, L.B. and A.H. Lund, MicroRNA regulation of autophagy. Carcinogenesis, 2012. 33(11): p. 2018-25.
  2. Seglen, P.O. and M.F. Brinchmann, Purification of autophagosomes from rat hepatocytes. Autophagy, 2010. 6(4): p. 542-7.
  3. Balgi, A.D., et al., Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC1 signaling. PLoS One, 2009. 4(9): p. e7124.


Additional Figure: fig 1

Figure 1. Preferential accumulation of miR-224 was demonstrated in the autophagosome. Hep 3B cells were treated with amiodarone to induce autophagy and chloroquine was used to block fusion of autophagosome and lysosome. (A) Accumulation of immune-gold labeled miR-224 (18 nm gold beads) in the purified double membrane autophagosomes was detected by miRNA in situ hybridization under TEM. (B) Accumulation of immune-gold labeled let-7a (18 nm gold beads) in the autophagosomes. Scale bar = 100 nm.



This study was supported by grants from the Headquarters of University Advancement, National Cheng Kung University (D100~101-35001); the National Science Council (NSC-99-2745-B-006-002, NSC100-2320-B-006-022 and NSC101-2320-B006-025-MY3), and from the National Science and Technology Development Fund (No. 97-EC-17-A-31-F1-0695)



Hsiao-Sheng Liu, Ph.D. Corresponding author, Department of Microbiology and Immunology College of Medicine,  National Cheng Kung University, Tainan, Taiwan, R. O. C.

FAX -886-6-2082705; e-mail: a713@mail.ncku.edu.tw


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