Construction of miR-199a-3p knockout mice
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 progression18. In order to elucidate the roles of miR-199a/b-3p in hepatocarcinogenesis, we intended to knock out its expression in mouse liver. As miR-199a/b-3p is expressed mainly by miR–199a–2 gene (Chr 1) in human liver, we first examined the expression of miR-199a gene in mouse liver. miR-199a-3p was found to be abundantly expressed in mouse liver while the miR-199a-5p strand was nearly not expressed (Fig. 1a), which is similar to those in human liver. There are two gene loci of miR-199a, miR–199a–1 and miR–199a–2, which may both express miR-199a-3p. We then constructed the miR–199a–1 or miR–199a–2 conventional knockout mice, respectively, to determine which one is mature miR-199a-3p primarily from (Supplementary Fig. S1A, B). These generated knockout mice are fertile and seem normal. Among them, mature miR-199a-3p expression was not influenced by knockout of miR–199a–1 gene (Fig. 1b), while knockout of miR–199a–2 gene deleted the expression of miR-199a-3p in the liver (Fig. 1c). Therefore, we determined that the mature miR-199a-3p was mainly expressed from miR–199a–2 gene in mouse liver.
In order to investigate the role of miR-199a-3p exclusively in hepatocytes and hepatocarcinogenesis, we then constructed the miR–199a–2 floxed mice and crossed them with Alb-cre mice to generate hepatocyte-specific miR–199a–2 knockout mice (Supplementary Fig. S1, C). There is no obvious difference in body weight and size between miR-199a-2f/f and miR-199a-2hep-/- mice, and they are both fertile. In 24-week-old miR-199a-2f/f and miR-199a-2hep-/- mice, serum ALT, AST, and GGT levels are similar, suggesting no spontaneous liver injury by hepatic miR-199a-2 knockout (Supplementary Fig. S1D). The livers of miR-199a-2f/f and miR-199a-2hep-/- mice were examined by HE staining, and no abnormal hepatic structure was found in miR-199a-2hep-/- mice (Supplementary Fig. S1E). Altogether, no obvious spontaneous hepatic disease is induced in miR-199a-2hep-/- mice. In miR-199a-2hep-/- mice, miR-199a-3p expression was markedly decreased in the liver tissue, while abolished in the isolated hepatocytes (Fig. 1d). Thus, we constructed the genetic mouse model of hepatic miR-199a-3p knockout, so as to investigate the role of miR-199a-3p in hepatocarcinogenesis.
Hepatocyte-specific miR–199a–2 knockout promotes hepatocarcinogenesis
To study the role of miR-199a-3p in hepatocarcinogenesis, the DEN-induced hepatocarcinogenesis model and DEN plus CCl4 model were applied (Supplementary Fig. S1F)19,20. In the DEN model, mice were intraperitoneally injected with DEN 14 days post birth, and were sacrificed after 8 months. In contrast to miR–199a–2f/f mice, the induced hepatocarcinogenesis was markedly enhanced in miR–199a–2hep-/- mice (Fig. 1e), including increased tumor incidence, number, and maximal diameter (Fig. 1f). Similarly, in the DEN plus CCl4 model, hepatocyte-specific miR-199a-3p knockout also markedly promoted hepatocarcinogenesis, including the increased tumor incidence, number, and maximal diameter (Fig. 1g, h). Hence, we concluded that hepatocyte-specific miR-199a-3p knockout remarkably promoted hepatocarcinogenesis in vivo.
Hepatic miR–199a–2 knockout promotes hepatocyte apoptosis and hepatic injury
To illustrate the corresponding mechanism responsible for hepatic miR-199a-3p knockout-promoted hepatocarcinogenesis, miR–199a–2f/f and miR–199a–2hep-/- mice were injected with high-dose DEN to induce acute hepatic injury and liver inflammation. After DEN challenge, ballooning degeneration and coagulative necrosis were observed in the peri-central hepatocytes, which were more severe in miR–199a–2hep-/- mice than those in miR–199a–2f/f mice (Fig. 2a). Besides, the level of serum ALT and AST post DEN injection in miR–199a–2hep-/- mice were also significantly higher than those in miR–199a–2f/f mice (Fig. 2b). Correspondingly, TUNEL staining and cleaved caspase-3 IHC staining determined that hepatocyte apoptosis in miR–199a–2hep-/- mice was significantly more severe than that in miR–199a–2f/f mice, which was confirmed by the increased cleaved caspase-3 in the liver of miR–199a–2hep-/- mice (Fig. 2c, d). These data suggest that hepatocyte-specific miR-199a-3p knockout promoted the hepatocyte apoptosis and hepatic injury following DEN injection.
The infiltration of inflammatory cells and compensatory proliferation of hepatocytes following acute DEN injection were also examined in the liver of miR–199a–2hep-/- mice, and they were only slightly increased as compared to those in miR–199a–2f/f mice (Fig. 2e and Supplementary Fig. S2A). Furthermore, the damage of DEN, determined by the hepatocyte DNA damage, showed no significant difference in the livers between miR–199a–2f/f and miR–199a–2hep-/- mice (Fig. 2f). Thus, the increased hepatic injury and hepatocyte apoptosis in the liver of miR–199a–2hep-/- mice may be mediated by the direct enhancement of apoptosis under miR-199a-3p knockout.
To further determine the role of miR-199a-3p in hepatocyte apoptosis and hepatic injury, we applied the hepatic injury mouse model using acetaminophen (APAP), an anti-pyretic drug causing liver injury or even hepatic failure by misuse21. In the APAP-challenged miR–199a–2f/f and miR–199a–2hep-/- mice, serum ALT and AST indicating liver injury were significantly increased by hepatocyte-specific miR-199a-3p knockout (Fig. 3a). Hepatic injury around the central vein were also much heavier in the liver of in miR–199a–2hep-/- mice than that in miR–199a–2f/f mice (Fig. 3b). Besides, TUNEL and cleaved caspase-3 staining in the liver sections showed significantly more apoptotic peri-central hepatocytes in miR–199a–2hep-/- mice, which was confirmed by western blot (Fig. 3c, d). The infiltrated inflammatory cells were increased in the damaged hepatic region of miR–199a–2hep-/- mice, and the compensatory hepatocyte proliferation was also increased (Fig. 3e and Supplementary Fig. S2B). Additionally, the direct damage of APAP, which is the induced reactive oxygen species (ROS) in hepatocytes, was similar in the liver between miR–199a–2f/f and miR–199a–2hep-/- mice (Fig. 3f). Together, all these data suggest that hepatocyte apoptosis were enhanced by miR-199a-3p knockout, which may be responsible for the increased hepatocarcinogenesis.
Hepatic miR-199a-3p knockout promotes apoptosis in vitro
In order to confirm the pro-apoptotic function of miR-199a-3p knockout in hepatocytes, we used the APAP-induced apoptosis model in vitro, and the miR–199a–2 knockout hepatocyte cell lines were constructed and verified (Fig. 4a). As compared to those in control cell lines, miR–199a–2 knockout human hepatocyte HL-7702 cells and mouse hepatocyte BNL CL.2 cells exhibited markedly increased apoptosis, respectively, following APAP administration (Fig. 4b, c). The APAP-induced cleaved caspase-3 was also significantly enhanced by miR-199a-3p knockout in these hepatocyte cell lines (Fig. 4d). Moreover, the primary hepatocytes were isolated from miR–199a–2f/f and miR–199a–2hep-/- mice, respectively, and the APAP-induced cleaved caspase-3 was confirmed to be significantly promoted by miR–199a–2 knockout (Fig. 4e). Thus, miR-199a-3p knockout promotes apoptosis in both primary hepatocytes and hepatocyte cell lines in vitro.
Hepatic miR-199a-3p knockout promotes apoptosis through the mitochondrial pathway
We next intended to figure out the hepatic miR-199a-3p knockout-promoted apoptosis was through death receptor pathway or mitochondrial pathway. The cleavage and activation of caspase-8, characteristic of death receptor apoptosis pathway, were examined in both DEN and APAP-induced hepatocyte apoptosis. Using death receptor pathway activator TNF-α plus CHX in vitro and LPS plus D-gal in vivo as the positive control22,23,24, we did not find the caspase-8 cleavage and death receptor pathway activation in hepatocytes upon DEN or APAP administration (Supplementary Fig. S3A–D). Thus, death receptor apoptosis pathway is not activated in DEN or APAP-induced hepatocyte apoptosis, and miR-199a-3p is less likely to inhibit hepatocyte apoptosis through suppressing death receptor pathway.
In mitochondrial apoptosis pathway, the classical pro-apoptotic BAK and BAX and anti-apoptotic BCL-2 and BCL-XL were examined in DEN or APAP-induced hepatocyte apoptosis. In APAP-induced hepatocyte apoptosis, the levels of these four proteins were not changed both in vitro and in vivo (Supplementary Fig. S3E, F, G). In DEN-induced hepatocyte apoptosis, the levels of both BAX and BCL-XL were increased upon DEN injection (Supplementary Fig. S3H). In miR-199a-2hep-/- mice, the level of hepatic BCL-XL was significantly decreased as compared to that in miR-199a-2 f/f mice, and similar results were obtained in hepatocyte cell lines (Supplementary Fig. S3E–H). These data suggest that miR-199a-2 knockout may inhibit BCL-XL expression, so as to enhance the activation of hepatic mitochondrial apoptosis pathway.
Furthermore, we also examined the release of cytochrome C into the cytosol, which is the characteristic of mitochondrial apoptosis pathway activation. In both DEN and APAP-induced release of cytochrome C, cytosol cytochrome C was significantly increased by hepatic miR-199a-2 knockout (Supplementary Fig. S3I–L), suggesting that miR-199a-3p knockout promotes the activation of mitochondrial apoptosis pathway in hepatocytes upon DEN or APAP administration.
Hepatic PDCD4 expression is directly targeted by miR-199a-3p
The corresponding mechanism responsible for miR-199a-3p knockout-promoted apoptosis was then investigated. As miRNAs function mainly through the inhibition of their target genes expression, we screened the differential proteins between the liver tissues from miR–199a–2f/f and miR–199a–2hep-/- mice using proteomic tandem mass tags (TMT) mass spectrometry (MS) analysis. Among the identified differentially expressed proteins in the liver of miR–199a–2f/f and miR–199a–2hep-/- mice, eight of them with good reproducibility were chosen for further analysis (Fig. 4f and Supplementary Fig. S4A). By sequence alignment and functional annotation, we focused on the increased PDCD4 in miR–199a–2hep-/- liver, which bears miR-199a-3p conserved target sequence and is known for its ability to promote apoptosis (Supplementary Fig. S4B)25,26. The direct targeting of PDCD4 mRNA by miR-199a-3p was validated using dual-luciferase reporter assay (Fig. 4g). The mRNA level of PDCD4 was not influenced by miR-199a-3p knockout (Supplementary Fig. S4,C, D), while the protein level of PDCD4 was significantly increased by miR-199a-3p knockout in liver tissues, primary hepatocytes, and hepatocyte cell lines (Fig. 4h, i). Additionally, the pro-apoptotic function of PDCD4 was validated in hepatocyte cell lines, as PDCD4 knockout inhibited while PDCD4 overexpression promoted APAP-induced apoptosis (Fig. 4j, k and Supplementary Fig. S4,E). As PDCD4 is known to repress internal ribosome entry site-mediated translation of BCL-XL and suppress its expression25, the decreased BCL-XL expression was also determined above in the miR-199a-3p knockout hepatocyte cell lines and liver tissues (Supplementary Fig. S3E–H). Altogether, these data suggest that miR-199a-2 knockout may increase PDCD4 to inhibit BCL-XL expression, so as to enhance the activation of hepatic mitochondrial apoptosis pathway.
miR-199a-3p suppresses hepatocyte apoptosis by targeting PDCD4
We next examined whether miR-199a-3p-suppressed hepatocyte apoptosis was dependent on PDCD4. miR-199a-3p mimics was transfected into the control and PDCD4 knockout hepatocyte cell lines, and miR-199a-3p-mediated inhibition of APAP-induced cleaved caspase-3 was abolished in PDCD4 knockout cell lines (Supplementary Fig. S5A). Moreover, PDCD4 was also overexpressed in control and miR–199a–2 knockout hepatocyte cell lines, and miR-199a-3p knockout-mediated increase of cleaved caspase-3 was abolished in PDCD4 overexpressed cell lines (Supplementary Fig. S5B). Thus, the miR-199a-3p-mediated inhibition of hepatocyte apoptosis was dependent on PDCD4 in vitro.
To analyze whether miR-199a-3p-suppressed hepatocyte apoptosis was dependent on PDCD4 in vivo, PDCD4 was overexpressed in the liver of miR–199a–2f/f and miR–199a–2hep-/- mice through AAV8-mediated gene delivery. miR-199a-3p knockout-mediated increase of DEN-induced hepatocyte apoptosis and hepatic injury, suggested by the elevated serum ALT and AST, TUNEL and cleaved caspase-3 staining, and cleaved caspase-3 blot, were abolished in the PDCD4 overexpressed livers (Fig. 5a–c). Similarly, in the APAP-induced hepatic injury model, miR-199a-3p knockout-promoted hepatocyte apoptosis and hepatic injury were also abolished in the PDCD4 overexpressed livers (Fig. 6a–c). Altogether, these data determine that miR-199a-3p suppresses hepatocyte apoptosis and hepatic injury by targeting PDCD4.