Abstract
Hepatic apoptosis and the initiated liver inflammation play the initial roles in inflammation-induced hepatocarcinogenesis. Molecular mechanisms underlying the regulation of hepatocyte apoptosis and their roles in hepatocarcinogenesis have attracted much attention. A set of microRNAs (miRNAs) have been determined to be dysregulated in hepatocellular carcinoma (HCC) and participated in cancer progression, however, the roles of these dysregulated miRNAs in carcinogenesis are still poorly understood. We previously analyzed the dysregulated miRNAs in HCC using high-throughput sequencing, and found that miR-199a/b-3p was abundantly expressed in human normal liver while markedly decreased in HCC, which promotes HCC progression. Whether miR-199a/b-3p participates in HCC carcinogenesis is still unknown up to now. Hence, we focused on the role and mechanism of miR-199a/b-3p in hepatocarcinogenesis in this study. Hepatic miR-199a/b-3p was determined to be expressed by miR-199a-2 gene in mice, and we constructed miR-199a-2 knockout and hepatocyte-specific miR-199a-2 knockout mice. Diethylnitrosamine (DEN)-induced hepatocarcinogenesis were markedly increased by hepatocyte-specific miR-199a-3p knockout, which is mediated by the enhanced hepatocyte apoptosis and hepatic injury by DEN administration. In acetaminophen (APAP)-induced acute hepatic injury model, hepatocyte-specific miR-199a-3p knockout also aggravated hepatic apoptosis. By proteomic screening and reporter gene validation, we identified and verified that hepatic programed cell death 4 (PDCD4), which promotes apoptosis, was directly targeted by miR-199a-3p. Furthermore, we confirmed that miR-199a-3p-suppressed hepatocyte apoptosis and hepatic injury by targeting and suppressing PDCD4. Thus, hepatic miR-199a-3p inhibits hepatocyte apoptosis and hepatocarcinogenesis, and decreased miR-199a-3p in hepatocytes may aggravate hepatic injury and HCC development.
Similar content being viewed by others
Introduction
Hepatocellular carcinoma (HCC) is a kind of lethal malignant primary liver cancer and ranks fourth on the cancer-related death list and sixth in the terms of incident cases worldwide, which has a low 5-year survival rate1,2. Liver diseases, such as chronic hepatitis caused by hepatitis B or C virus (HBV or HCV), alcoholic liver disease and non-alcoholic fatty liver disease, increase the risk of hepatocellular carcinoma development3. Generally, HCC carcinogenesis is caused by the repeated cycle of hepatic injury, induced liver inflammation, and compensatory hepatocyte proliferation4. The liver injury plays a critical role in this vicious cycle, and death of hepatocytes is able to release damage-associated molecular patterns (DAMPs), which activates immune cells in the liver, recruits circulatory inflammatory cells, and initiates liver inflammation. The induced liver inflammation stimulates compensatory hepatocyte proliferation, while hepatic inflammatory responses also worsen hepatic damage and aggravate liver inflammation. Sustained hepatic injury induces the chronic liver inflammation and repeated compensatory hepatocyte proliferation, which eventually lead to hepatocarcinogenesis5,6. The underlying regulatory mechanisms of hepatocarcinogenesis have attracted much attention but remain largely unknown, which needs further investigation.
Cell death plays pivotal roles in the initiation of hepatocarcinogenesis. Apoptosis, necroptosis, pyroptosis, and ferroptosis are the intensively explored types of cell death during tissue damage, and different types of hepatocyte death eventually lead to different types of liver cancer7. For instance, the necroptosis of hepatocytes incubates an environment, which determines intrahepatic cholangiocarcinoma (ICC) outgrowth. However, if hepatocytes are in the environment created by apoptotic hepatocytes, they are inclined to become HCC8. Hepatocyte-specific deletion of the IκB kinase (IKK) subunit NEMO/IKKγ sensitizes hepatocyte apoptosis by NF-κB inhibition, which spontaneously forms mouse HCC in 12 months9. Similarly, mice lacking the anti-apoptotic myeloid cell leukemia-1 (Mcl-1) in hepatocytes have severe liver damage caused by spontaneous apoptosis, and tumor formation is observed in over 50% of mice in 8 months10. Besides, mice with combined knockout of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Rel-like domain-containing protein A (RelA) in hepatocytes show increased hepatocyte apoptosis and develop spontaneous HCC11. Furthermore, other proteins regulating hepatocyte apoptosis, such as TGF-β-activated kinase 1(TAK1), TNF receptor-associated factor 2 (TRAF2), and IκB kinase subunit beta (IKKβ), also participate in hepatocarcinogenesis12,13. Therefore, hepatocyte apoptosis is critical in the initiation of hepatocarcinogenesis, and the molecular regulatory mechanisms of hepatocyte apoptosis and their roles in hepatocarcinogenesis have raised widespread concerns.
MicroRNAs (miRNAs) are a class of single-strand RNA with approximately 22 nucleotides in length. The functions of miRNAs have been widely investigated, and they are determined to play critical roles in cancer progression, especially in HCC. A set of miRNAs, such as miR-330-5p, miR-520a, and miR-483-3p, have been suggested to participate in cancer progression, by modulating proliferation, apoptosis, migration, and invasion41. All animal experiments were undertaken in accordance with National Institutes of Health’s Guide for the Care and Use of Laboratory Animals with the approval of the institutional research ethics committee of Second Military Medical University, Shanghai, China. HL-7702 and BNL CL.2 cell lines were obtained from cell bank of Chinese Academy of Sciences (Shanghai, China). All cell lines have been authenticated using STR profiling and tested for mycoplasma contamination by Genechem (Shanghai, China). The knockout cell lines were constructed using CRISPR/Cas9 method (Supplementary Table S2)42. HL-7702 was cultured in RPMI 1640 with 10% Fetal Bovine Serum (FBS), and BNL CL.2 was cultured in dulbecco’s modified eagle medium (DMEM) with 10% FBS and 1% non-essential animo acid (NAA).
Reagents
Antibodies specific to caspase-3 (9662), cleaved caspase-3 (9661), caspase-8 (4790), human cleaved caspase-8 (9496), mouse cleaved caspase-8 (9429), BAK (12105), BAX (2772), BCL-2 (3498), BCL-XL (2764), PDCD4 (9535), and horseradish peroxidase-coupled secondary antibodies (7074 and 7076) were from Cell Signaling Technology (Danvers, MA). Antibody specific to β-actin (A5441), DEN (N0258), DHE (D7008), CHX (239764), LPS (L3024) and MEM NAA (M7145) were from Sigma-Aldrich (St. Louis, MO). Antibody specific to cytochrome C (sc-13156) was from Santa Cruz Biotechnology (CA, USA). Recombinant human TNF-α (300-01A) and murine TNF-α (315-01 A) were from Pepro Tech (Rocky Hill, NJ). TUNEL assay kit (11684817910) was from Roche (Shanghai, China). CCl4 (CAS: 56-23-5, C805329) was from MAKLIN (Shanghai, China). Olive oil (CAS: 8001-25-0, A502795-0100), APAP (CAS: 103-90-2, A506808), and D-hanks buffer (B548148-0500) were from Sangon Biotech (Shanghai, China). Cell mitochondria isolation kit (C3601) and tissue mitochondria isolation kit (C3606) were from Beyotime (Shanghai, China). Percoll solution (17-0891-01) was from GE Healthcare Life Science (Little Chalfont, UK). Type IV collagenase (LS004140) was from Worthington Biochemical Corporation (Lakewood, NJ). Protease inhibitor cocktail (539134-1SML) and apoptosis assay kit (PF032) were from Calbiochem (Darmstadt, Germany).
Establishment of hepatocarcinogenesis model
To construct DEN-induced hepatocarcinogenesis model, the 2-week-old male mice were injected with 25 mg/kg body weight DEN intraperitoneally43. Mice were sacrificed 8 months later and tumors were analyzed. As for DEN plus CCl4 model, the 2-week-old male mice were injected with 25 mg/kg body weight DEN intraperitoneally, and 4 weeks later, mice were injected with CCl4 (0.5 ml/kg body weight, dissolved in olive oil at a ratio of 1:3) weekly lasting for 15 weeks19. Mice were sacrificed 8 weeks after last CCl4 injection.
Establishment of acute hepatic injury model
To constructed DEN-induced acute hepatic injury model, 8-week-old male mice were injected with DEN (100 mg/kg) intraperitoneally and mice were sacrificed according to the time points. As for APAP-induced acute hepatic injury model, eight-week-old male mice were fasted overnight (16 h) and then injected with APAP (400 mg/kg) intraperitoneally. Mice were fed immediately after injection and sacrificed as indicated. Serum ALT and AST were measured by automatic biochemical analyzer FDC-7000i (Shanghai, China).
In vivo AAV8 administration
The rAAV (serotype 8) vector expressing PDCD4 under promoter CAG was constructed as previously described18. For AAV8 administration, 1 × 1012 vg AAV8 per mouse in 200 μl saline buffer was injected into the mice through tail vein. Four weeks later, the protein expression of PDCD4 was measured by Western blot.
Isolation of primary hepatocytes
Primary hepatocytes were isolated from mouse liver using two-step collagenase perfusion procedure44. Mice were anesthetized and first perfused with 45 ml D-hanks buffer and then with type IV collagenase digestion (1 mg/ml) for 5 min at a rate of 2 ml/min through the portal vein. After digestion, the liver was excised, minced, filtered through 70-micron membrane, spin for 5 min at 50 × g, and purified using 50% Percoll solution. The obtained hepatocytes were resuspended in DMEM, and trypan blue exclusion assays indicated that cell viability was >95%. Primary hepatocytes were cultured in DMEM with 10% FBS and 1% Penicillin-Streptomycin.
RNA isolation and quantitative PCR analysis
Total RNA was extracted from mouse liver tissues or cell lines using RNAiso Plus reagent (Takara, Dalian, China) according the recommended protocol. Real-time quantitative RT-PCR (qRT-PCR) analysis was performed using LightCycler 2.0 (Roche, Switzerland) and SYBR RT-PCR kit (Takara) as previously described18. The relative expression level of the individual genes was normalized to that of internal control by using 2-ΔΔCt cycle threshold method in each sample (Supplementary Table S3)45.
Western blot
Cells or tissues were lysed on ice with cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Calbiochem) at a ratio of 1:200. Protein concentrations of the extracts were measured with bicinchoninic acid (BCA) assay (Pierce). Equal amount of the extracts was loaded to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane for immunoblot analysis as described previously20.
Histology
For HE, IHC and TUNEL, liver tissues were fixed in 4% paraformaldehyde for 48 h, embedded by paraffin and sliced up into 5 μm thick sections, which were processed to HE, IHC, and TUNEL as kit protocols described. For reactive oxygen species (ROS) measurement, fresh liver tissues were embedded by Tissue-Tek® O.C.T compound and immediately sliced up into liver sections. The slices were incubated in DHE (1 μM) for 30 min. After washing with PBS for three times, the red fluorescence was measured by fluorescence microscope.
Dual-luciferase reporter assay
The PDCD4 luciferase reporter was made by amplifying the mouse pdcd4 mRNA by PCR and cloned into the 3ʹUTR region of pMIR-promoter-Firefly plasmids. The luciferase reporter plasmids, RL-TK-Renilla plasmids, and indicated miRNAs (final concentration 20 nM) were co-transfected into HEK 293T cells. After 24 h, the activities of Firefly and Renilla luciferases were measured using the Dual-Luciferase Reporter Assay System (Promega) as previously described46. Data was normalized for transfection efficiency by dividing Firefly luciferase activity with that of Renilla luciferase.
Flow cytometry
The control and knockout cells were treated with APAP (10 mM) for 24 h. Cells were collected and labeled by apoptosis assay kit and subjected to flow cytometry analysis on LSRII. Data were analyzed using FACSDiva software (Becton Dickinson).
Statistical analysis
As indicated in the figure legends, sample size of the experiments depended on the assay type. There were no blind experiments for the investigators both in cells and mice experiments. The mice were randomly distributed to the experimental group. For all groups that are statistically compared, the variance within each group was similar. Data are shown as mean ± SD from one representative of three independent experiments. Statistical comparisons between experimental groups were analyzed by unpaired Student’s t-test or chi-square test in GraphPad Prism 8.0, and a two-tailed P < 0.05 was taken to indicate statistical significance.
References
Villanueva, A. Hepatocellular carcinoma. N. Engl. J. Med. 380, 1450–1462 (2019).
Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2020. CA. Cancer J. Clin. 70, 7–30 (2020).
Forner, A., Reig, M. & Bruix, J. Hepatocellular carcinoma. Lancet 391, 1301–1314 (2018).
Grivennikov, S. I., Greten, F. R. & Karin, M. Immunity, inflammation, and cancer. Cell 140, 883–899 (2010).
Ringelhan, M., Pfister, D., O’Connor, T., Pikarsky, E. & Heikenwalder, M. The immunology of hepatocellular carcinoma. Nat. Immunol. 19, 222–232 (2018).
Kubes, P. & Jenne, C. Immune responses in the liver. Annu. Rev. Immunol. 36, 247–277 (2018).
Green, D. R. The coming decade of cell death research: five riddles. Cell 177, 1094–1107 (2019).
Seehawer, M. et al. Necroptosis microenvironment directs lineage commitment in liver cancer. Nature 562, 69–75 (2018).
Luedde, T. et al. Deletion of NEMO/IKKγ in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell 11, 119–132 (2007).
Weber, A. et al. Hepatocyte-specific deletion of the antiapoptotic protein myeloid cell leukemia-1 triggers proliferation and hepatocarcinogenesis in mice. Hepatology 51, 1226–1236 (2010).
Van, T. M. et al. Kinase-independent functions of RIPK1 regulate hepatocyte survival and liver carcinogenesis. J. Clin. Invest. 127, 2662–2677 (2017).
Inokuchi, S. et al. Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis. Proc. Natl Acad. Sci. USA 107, 844–849 (2010).
Schneider, A. T. et al. RIPK1 suppresses a TRAF2-dependent pathway to liver cancer. Cancer Cell 31, 94–109 (2017).
**ao, S. et al. miR-330-5p targets SPRY2 to promote hepatocellular carcinoma progression via MAPK/ERK signaling. Oncogenesis 7, 90 (2018).
Huang, J. et al. An ANCCA/PRO2000-miR-520a-E2F2 regulatory loop as a driving force for the development of hepatocellular carcinoma. Oncogenesis 30, e229 (2016).
Pepe, F. et al. Regulation of miR-483-3p by the O-linked N-acetylglucosamine transferase links chemosensitivity to glucose metabolism in liver cancer cells. Oncogenesis 8, e328 (2017).
Tsai, W. C. et al. MicroRNA-122 plays a critical role in liver homeostasis and hepatocarcinogenesis. J. Clin. Invest. 122, 2884–2897 (2012).
Hou, J. et al. Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. Cancer Cell 19, 232–243 (2011).
Hernandez, C. et al. HMGB1 links chronic liver injury to progenitor responses and hepatocarcinogenesis. J. Clin. Invest. 128, 2436–2451 (2018).
Hou, J. et al. Hepatic RIG-I predicts survival and interferon-α therapeutic response in hepatocellular carcinoma. Cancer Cell 25, 49–63 (2014).
Bunchorntavakul, C. & Reddy, K. R. Acetaminophen (APAP or N-Acetyl-p-Aminophenol) and acute liver failure. Clin. Liver Dis. 22, 325–346 (2018).
Priem, D. et al. A20 protects cells from TNF-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms. Cell Death Dis. 10, 692 (2019).
**g, Z. T. et al. AKT activator SC79 protects hepatocytes from TNF-α-mediated apoptosis and alleviates d-Gal/LPS-induced liver injury. Am. J. Physiol. Gastrointest. Liver Physiol. 316, G387–G396 (2019).
Deng, Y. et al. Oridonin ameliorates lipopolysaccharide/D-galactosamine-induced acute liver injury in mice via inhibition of apoptosis. Am. J. Transl. Res. 9, 4271–4279 (2017).
Liwak, U. et al. Tumor suppressor PDCD4 represses internal ribosome entry site-mediated translation of antiapoptotic proteins and is regulated by S6 kinase 2. Mol. Cell Biol. 32, 1818–1829 (2012).
White, K. et al. Endothelial apoptosis in pulmonary hypertension is controlled by a microRNA/programmed cell death 4/caspase-3 axis. Hypertension 64, 185–194 (2014).
Luedde, T. & Schwabe, R. F. NF-κB in the liver-linking injury, fibrosis and hepatocellular carcinoma. Nat. Rev. Gastroenterol. Hepatol. 8, 108–118 (2011).
Kondylis, V. et al. NEMO prevents steatohepatitis and hepatocellular carcinoma by inhibiting RIPK1 kinase activity-mediated hepatocyte apoptosis. Cancer Cell 28, 582–598 (2015).
Cheng, L. et al. microRNA-148a deficiency promotes hepatic lipid metabolism and hepatocarcinogenesis in mice. Cell Death Dis. 8, e2916 (2017).
Yang, L. et al. Transforming growth factor-β signaling in hepatocytes promotes hepatic fibrosis and carcinogenesis in mice with hepatocyte-specific deletion of TAK1. Gastroenterology 144, 1042–1054.e4 (2013).
Fuchs, C. D. et al. Colesevelam attenuates cholestatic liver and bile duct injury in Mdr2-/- mice by modulating composition, signalling and excretion of faecal bile acids. Gut 67, 1683–1691 (2018).
Nakagawa, H. et al. ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development. Cancer Cell 26, 331–343 (2014).
van der Windt, D. J. et al. Neutrophil extracellular traps promote inflammation and development of hepatocellular carcinoma in nonalcoholic steatohepatitis. Hepatology 68, 1347–1360 (2018).
Dow, M. et al. Integrative genomic analysis of mouse and human hepatocellular carcinoma. Proc. Natl Acad. Sci. USA 115, E9879–E9888 (2018).
Liu, J. et al. miR-199a-3p acts as a tumor suppressor in clear cell renal cell carcinoma. Pathol. Res. Pract. 214, 806–813 (2018).
**, H. et al. Restoration of mutant K-Ras repressed miR-199b inhibits K-Ras mutant non-small cell lung cancer progression. J. Exp. Clin. Cancer Res. 38, 165 (2019).
Wan, D. et al. Aberrant expression of miR-199a-3p and its clinical significance in colorectal cancers. Med. Oncol. 30, 378 (2013).
Werneck-de-Castro, J. P., Blandino-Rosano, M., Hilfiker-Kleiner, D. & Bernal-Mizrachi, E. Glucose stimulates microRNA-199 expression in murine pancreatic β-cells. J. Biol. Chem. 295, 1261–1270 (2020).
Hilliard, A. et al. Translational regulation of autoimmune inflammation and lymphoma genesis by programmed cell death 4. J. Immunol. 177, 8095–8102 (2006).
Wang, L. et al. PDCD4 deficiency aggravated colitis and colitis-associated colorectal cancer via promoting IL-6/STAT3 pathway in mice. Inflamm. Bowel Dis. 22, 1107–1118 (2016).
Zheng, Q., Hou, J., Zhou, Y., Li, Z. & Cao, X. The RNA helicase DDX46 inhibits innate immunity by entrap** mA-demethylated antiviral transcripts in the nucleus. Nat. Immunol. 18, 1094–1103 (2017).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Han, Y. et al. Tumor-induced generation of splenic erythroblast-like Ter-cells promotes tumor progression. Cell 173, 634–648.e12 (2018).
Sun, P. et al. Caspase recruitment domain protein 6 protects against hepatic steatosis and insulin resistance by suppressing apoptosis signal-regulating kinase 1. Hepatology 68, 2212–2229 (2018).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408 (2001).
Hou, J. et al. MicroRNA-146a feedback inhibits RIG-I-dependent Type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2. J. Immunol. 183, 2150–2158 (2009).
Acknowledgements
We thank Yuchao Yin, Tingting Fang, and Yan Li for technical support, and Prof. Taoyong Chen, **ngguang Liu, and Chaofeng Han for helpful discussion. This work was supported by Grants from the National 135 Major Project of China (2018ZX10302205 and 2017ZX10102032), the National Natural Science Foundation of China (91842104, 81871229, 81671564, 81701566, 81672798, 81872232), Program of Shanghai Academic Research Leader (19XD1424900), Shanghai Sailing Project (17YF1424600), and Shanghai Chen Guang Project (18CG39).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Li, Z., Zhou, Y., Zhang, L. et al. microRNA-199a-3p inhibits hepatic apoptosis and hepatocarcinogenesis by targeting PDCD4. Oncogenesis 9, 95 (2020). https://doi.org/10.1038/s41389-020-00282-y
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41389-020-00282-y
- Springer Nature Limited
This article is cited by
-
miR-199a/b-3p inhibits HCC cell proliferation and invasion through a novel compensatory signaling pathway DJ-1\Ras\PI3K/AKT
Scientific Reports (2024)
-
Interferon-α stimulates DExH-box helicase 58 to prevent hepatocyte ferroptosis
Military Medical Research (2024)
-
Mitochondrial IRG1 traps MCL-1 to induce hepatocyte apoptosis and promote carcinogenesis
Cell Death & Disease (2023)
-
Circ-ZEB1 promotes PIK3CA expression by silencing miR-199a-3p and affects the proliferation and apoptosis of hepatocellular carcinoma
Molecular Cancer (2022)
-
JMJD4-demethylated RIG-I prevents hepatic steatosis and carcinogenesis
Journal of Hematology & Oncology (2022)