Home Liver DiseasesLiver Cancer microRNA-199a-3p inhibits hepatic apoptosis and hepatocarcinogenesis by targeting PDCD4

microRNA-199a-3p inhibits hepatic apoptosis and hepatocarcinogenesis by targeting PDCD4

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microRNA-199a-3p inhibits hepatic apoptosis and hepatocarcinogenesis by targeting PDCD4

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 miR199a2 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, miR199a1 and miR199a2, which may both express miR-199a-3p. We then constructed the miR199a1 or miR199a2 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 miR199a1 gene (Fig. 1b), while knockout of miR199a2 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 miR199a2 gene in mouse liver.

Fig. 1: Hepatocyte-specific miR199a2 knockout promotes hepatocarcinogenesis.

a The expression of miR-199a-5p and miR-199a-3p was detected by qRT-PCR in mouse liver tissues (n = 8). b The expression of miR-199a-3p was detected by qRT-PCR from liver tissues of miR199a1+/+, miR199a1+/- and miR199a1-/- mice (n = 4). c The expression of miR-199a-3p was detected by qRT-PCR from liver tissues of miR199a2+/+, miR199a2+/- and miR199a2-/- mice (n = 4). d The expression of miR-199a-3p was detected by qRT-PCR from liver tissues and primary hepatocytes of miR199a2f/f and miR199a2hep-/- mice (n = 4). e Two-week-old miR199a2f/f and miR199a2hep-/- male mice were intraperitoneally injected with a single dose of DEN (25 mg/kg) and sacrificed 8 months later. The livers were dissected and photographed. f Tumor incidence (chi-square test), tumor number per mouse (unpaired t-test), and maximal tumor diameter per mouse (unpaired t-test) of the DEN-induced HCC in miR199a2f/f and miR199a2hep-/- mice were shown, respectively, as indicated (n = 15). g Two-week-old miR199a2f/f and miR199a2hep-/- male mice were intraperitoneally injected with DEN and then CCl4. The livers with DEN plus CCl4-induced HCC were dissected and photographed. h Tumor incidence (chi-square test), tumor number per mouse (unpaired t-test), and maximal tumor diameter per mouse (unpaired t-test) of the DEN plus CCl4-induced HCC in miR199a2f/f and miR199a2hep-/- mice were shown, respectively, as indicated (n = 15). Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. P > 0.05; *P < 0.05; **P < 0.01.

In order to investigate the role of miR-199a-3p exclusively in hepatocytes and hepatocarcinogenesis, we then constructed the miR199a2 floxed mice and crossed them with Alb-cre mice to generate hepatocyte-specific miR199a2 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 miR199a2 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 miR199a2f/f mice, the induced hepatocarcinogenesis was markedly enhanced in miR199a2hep-/- 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 miR199a2 knockout promotes hepatocyte apoptosis and hepatic injury

To illustrate the corresponding mechanism responsible for hepatic miR-199a-3p knockout-promoted hepatocarcinogenesis, miR199a2f/f and miR199a2hep-/- 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 miR199a2hep-/- mice than those in miR199a2f/f mice (Fig. 2a). Besides, the level of serum ALT and AST post DEN injection in miR199a2hep-/- mice were also significantly higher than those in miR199a2f/f mice (Fig. 2b). Correspondingly, TUNEL staining and cleaved caspase-3 IHC staining determined that hepatocyte apoptosis in miR199a2hep-/- mice was significantly more severe than that in miR199a2f/f mice, which was confirmed by the increased cleaved caspase-3 in the liver of miR199a2hep-/- mice (Fig. 2c, d). These data suggest that hepatocyte-specific miR-199a-3p knockout promoted the hepatocyte apoptosis and hepatic injury following DEN injection.

Fig. 2: Hepatocyte-specific miR-199a-3p knockout promotes DEN-induced hepatocyte apoptosis and hepatic injury.
figure2

Eight-week-old miR199a2f/f and miR199a2hep-/- male mice were intraperitoneally injected with DEN (100 mg/kg). Livers and serum were collected at the indicated time points. HE staining of liver sections (a), Serum ALT and ALT (n = 4) (b), TUNEL and cleaved caspase-3 IHC staining (c), cleaved caspase-3 blot (d), Ly6G staining for inflammatory cell infiltration (e), and pH2AX staining for DNA damage (f) were measured in the indicated time points post DEN challenge. Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. **P < 0.01. Scale bars, 100 μm.

The infiltration of inflammatory cells and compensatory proliferation of hepatocytes following acute DEN injection were also examined in the liver of miR199a2hep-/- mice, and they were only slightly increased as compared to those in miR199a2f/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 miR199a2f/f and miR199a2hep-/- mice (Fig. 2f). Thus, the increased hepatic injury and hepatocyte apoptosis in the liver of miR199a2hep-/- 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 miR199a2f/f and miR199a2hep-/- 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 miR199a2hep-/- mice than that in miR199a2f/f mice (Fig. 3b). Besides, TUNEL and cleaved caspase-3 staining in the liver sections showed significantly more apoptotic peri-central hepatocytes in miR199a2hep-/- mice, which was confirmed by western blot (Fig. 3c, d). The infiltrated inflammatory cells were increased in the damaged hepatic region of miR199a2hep-/- 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 miR199a2f/f and miR199a2hep-/- 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.

Fig. 3: Hepatocyte-specific miR-199a-3p knockout promotes APAP-induced hepatocyte apoptosis and hepatic injury.
figure3

Eight-week-old miR199a2f/f and miR199a2hep-/- male mice were fasted for 16 h and then intraperitoneally injected with APAP (400 mg/kg). Livers and serum were obtained at the indicated time points. Serum ALT and ALT (n = 4) (a), HE staining (b), TUNEL and cleaved caspase-3 IHC staining (c), cleaved caspase-3 blot (d), Ly6G staining for inflammatory cell infiltration (e), and DHE staining for ROS (f) were measured in the indicated time points post APAP injection. Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. *P < 0.05. Scale bars, 100 μm.

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 miR199a2 knockout hepatocyte cell lines were constructed and verified (Fig. 4a). As compared to those in control cell lines, miR199a2 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 miR199a2f/f and miR199a2hep-/- mice, respectively, and the APAP-induced cleaved caspase-3 was confirmed to be significantly promoted by miR199a2 knockout (Fig. 4e). Thus, miR-199a-3p knockout promotes apoptosis in both primary hepatocytes and hepatocyte cell lines in vitro.

Fig. 4: Loss of miR-199a-3p promotes hepatocyte apoptosis by directly targeting pro-apoptotic PDCD4.
figure4

a The expression of miR-199a-3p was detected by qRT-PCR in control and miR-199a-2-/- cell lines. be Control or miR199a2 knockout hepatocyte cell lines were treated by APAP (10 mM) for 24 h as indicated. Cell apoptosis was analyzed by flow cytometry (b, c), and cleaved caspase-3 was examined by Western blot (d, e). f The comparative differential proteins in liver tissues from miR199a2f/f and miR-199a2hep-/- mice were shown as indicated, which were from four biological repeats. g The indicated Firefly luciferase reporter plasmids and miR-199a-3p or control mimics were transfected into the HEK 293 T cells. After 24 h, Firefly luciferase activity was measured and normalized by Renilla luciferase activity (n = 10). h Protein level of PDCD4 in liver tissues or primary hepatocytes from miR199a2f/f and miR-199a2hep-/- mice was measured by Western blot. i Protein level of PDCD4 in control or miR199a2 knockout hepatocyte cell lines was measured by western blot. j, k Control and pdcd4 knockout hepatocyte cell lines were treated with APAP for the indicated time points. PDCD4, caspase-3, and cleaved caspase-3 were measured by Western blot (j), and cell apoptosis was measured by flow cytometry (k). Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. P > 0.05; **P < 0.01.

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-2f/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 miR199a2f/f and miR199a2hep-/- mice using proteomic tandem mass tags (TMT) mass spectrometry (MS) analysis. Among the identified differentially expressed proteins in the liver of miR199a2f/f and miR199a2hep-/- 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 miR199a2hep-/- 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 miR199a2 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 miR199a2f/f and miR199a2hep-/- 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.

Fig. 5: miR-199a-3p inhibits DEN-induced hepatocyte apoptosis and hepatic injury by targeting PDCD4 in vivo.
figure5

Control AAV8 or AAV8-PDCD4 were injected into miR199a2f/f or miR-199a2hep-/- mice through tail vein at a dose of 1 × 1012 vg per mouse, and DEN (100 mg/kg) was injected 4 weeks later. Serum ALT and AST (n = 4) (a), TUNEL and cleaved caspase-3 IHC staining (b), and cleaved caspase-3 blot (c) were measured in the indicated time points post DEN administration. Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. P > 0.05; **P < 0.01.

Fig. 6: miR-199a-3p inhibits APAP-induced hepatocyte apoptosis and hepatic injury by targeting PDCD4 in vivo.
figure6

Control AAV8 or AAV8-PDCD4 were injected into miR199a2f/f or miR-199a2hep-/- mice through tail vein at a dose of 1 × 1012 vg per mouse, and APAP (400 mg/kg) was injected 4 weeks later. Serum ALT and AST (n = 4) (a), TUNEL and cleaved caspase-3 IHC staining (b), and cleaved caspase-3 blot (c) were measured in the indicated time points post APAP administration. Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. P > 0.05; *P < 0.05; **P < 0.01.

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