Hepatitis B virus regulates apoptosis and tumorigenesis through the microRNA-15a-Smad7-transforming growth factor beta pathway - PubMed (original) (raw)
Hepatitis B virus regulates apoptosis and tumorigenesis through the microRNA-15a-Smad7-transforming growth factor beta pathway
Ningning Liu et al. J Virol. 2015 Mar.
Abstract
Hepatitis B virus (HBV) infection causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). Previously, we found that HBV mRNAs can absorb microRNA-15a (miR-15a) to affect apoptosis through the Bcl-2 pathway. We asked whether HBV could inhibit apoptosis and promote tumorigenesis through different pathways. In this study, we found that the transforming growth factor β (TGF-β) pathway-inhibitory factor Smad7 is a novel target of miR-15a. We demonstrated that HBV can upregulate the level of Smad7 by downregulating miR-15a. Furthermore, we examined the level of Smad7 in liver samples from HBV-infected HCC patients and found that HBV mRNAs are positively correlated with the level of Smad7. By taking the approach of using immunoblotting and luciferase reporter assays, we revealed that HBV can abrogate TGF-β signaling via upregulating Smad7. By using annexin V staining and caspase 3/7 activity assays, we found that HBV can inhibit TGF-β-induced apoptosis of HepG2 cells. We also showed that HBV can promote tumor growth in BALB/c nude mice through upregulating the expression of Smad7. In conclusion, we demonstrated that HBV can upregulate Smad7 expression and inhibit TGF-β signaling, which makes the cells resistant to TGF-β-induced apoptosis and promotes tumorigenesis.
Importance: Hepatitis B virus (HBV) infection causes chronic hepatitis, which can eventually lead to hepatocellular carcinoma (HCC). TGF-β signaling is closely linked to liver fibrosis, cirrhosis, and subsequent HCC progression and plays a unique role in the pathogenesis of HCC. At the early stage of tumor formation, TGF-β functions as a tumor suppressor that inhibits cell proliferation and induces apoptosis. Previously, we found that HBV mRNAs can sponge off miR-15a to affect apoptosis through the Bcl-2 pathway. In this study, we identified that the TGF-β-inhibitory factor Smad7 is a novel target of miR-15a. We reveal that HBV can abrogate TGF-β signaling via upregulating Smad7, inhibit TGF-β-induced apoptosis, as well as promote tumor development. Our study provides evidence to support the idea that viral RNAs can exert their functions as competing endogenous RNAs (ceRNAs) toward microRNA and participate in important cellular processes.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Figures
FIG 1
MiR-15a downregulates Smad7 expression by targeting its 3′ UTR. (A) HepG2 cells were transfected with miR-15a or a randomized oligonucleotide as a control for 48 h. Total RNA was extracted and quantified by real-time PCR for the expression of predicted target genes. (B) HepG2 cells were transfected with miR-15a or a randomized oligonucleotide as a control for 48 h. Cell lysates were harvested for immunoblotting with rabbit anti-human Smad7 antibody. (C) The predicted miR-15a-binding sequences are located in the 3′ UTR of Smad7 mRNA. The matched nucleotides are indicated with a line. (D) Mutations on the 3′ UTR of Smad7 were made in the predicted region of miR-15a-binding sites (named Smad7-3′ UTR-mut). The mutated nucleotides are shown in boldface italic type. (E) HepG2 cells were cotransfected with the miR-15a mimic and pGLO-Smad7-3′UTR or pGLO-Smad7-3′UTR-mut. The cell lysates were harvested for dual-luciferase assays. The relative luciferase activity was quantified. **, P < 0.01.
FIG 2
HBV upregulates Smad7 expression by downregulating miR-15a in hepatocytes. (A and B) Total mRNAs of HepG2, HepG2-C5, and HepG2-4D14 cells were extracted and subjected to real-time PCR for miR-15a (A) and Smad6 and Smad7 mRNAs (B, left), and the cell lysates from the above-described cell lines were harvested for immunoblotting with Smad6 and Smad7 antibodies (B, right). (C and D) HepG2 and L-02 cells were transfected with pHBV1.3, pHBV1.3-mut, or pcDNA3.1 as the control for 48 h. The amount of Smad7 mRNA was quantified by real-time PCR (C), and the protein level of Smad7 was detected by immunoblotting (D). **, P < 0.01.
FIG 3
HBV upregulates Smad7 expression by downregulating miR-15a in vivo. (A) Total RNAs extracted from liver tissues of HBV transgenic BALB/c mice (n = 3) or control BALB/c mice (n = 3) were subjected to real-time PCR to detect the amount of Smad7 mRNA. (B) Total RNAs from liver specimens of HCC patients were extracted and subjected to real-time PCR for miR-15a and Smad7 mRNA. The correlation between miR-15a and Smad7 mRNA in samples from HCC patients was analyzed by a Spearman rank test. Correlation coefficient (R) values were calculated. (C) Total RNAs from liver specimens of HCC patients were extracted and subjected to real-time PCR for Smad7 mRNA and HBV transcripts. The correlation between HBV transcripts and Smad7 mRNA in HCC patient samples was analyzed by a Spearman rank test. R values are also shown. (D) Protein levels of Smad7 were detected by immunoblotting (top), and the relative amount of HBV mRNA was detected by real-time PCR (bottom) in tumor tissues (T) and adjacent nontumorous liver tissues (ANLTs). **, P < 0.01.
FIG 4
MiR-15a enhances TGF-β signaling by downregulating Smad7. (A) HepG2 cells were transfected with an miR-15a mimic, an miR-15a inhibitor, or a randomized oligonucleotide as a control, together with the TGF-β luciferase reporter p3TP-luc and renilla luciferase plasmid pRL-TK. The cell lysates were harvested for firefly luciferase and renilla luciferase activity assays. The relative luciferase activity was quantified. (B) HepG2 cells were transfected with pcDNA3.1 or pCMV-FLAG-Smad7 for 24 h and then treated with TGF-β1 for 24 h. The total cell lysate and the nuclear fraction were subjected to immunoblotting with the indicated antibodies. (C) HepG2 cells were transfected with the miR-15a mimic or a randomized oligonucleotide as a control for 24 h and then treated with TGF-β1 for 24 h. Immunoblotting was performed as described above. (D) HepG2 cells were transfected with the miR-15a mimic with or without FLAG-Smad7 for 24 h and then treated with TGF-β1 for 24 h. Immunoblotting was performed as indicated. The relative amount of Smad2 in the nucleus was quantified. *, P < 0.05.
FIG 5
HBV inhibits TGF-β signaling by downregulating miR-15a. (A) HepG2 cells were transfected with pHBV1.3, pHBV1.3-mut, or pcDNA3.1 as the control, together with the TGF-β luciferase reporter p3TP-luc and renilla luciferase plasmid pRL-TK. The cell lysates were harvested for firefly luciferase and renilla luciferase activity assays. The relative luciferase activity was quantified. (B) HepG2 cells were transfected with pHBV1.3 or pcDNA3.1 as the control, with or without the miR-15a mimic, for 24 h and then treated with TGF-β1 for 24 h. The total cell lysate and nuclear fraction were subjected to immunoblotting with the indicated antibodies. (C) HepG2 cells were transfected with pHBV1.3 or pHBV1.3-mut for 24 h and then treated with TGF-β1 for 24 h. Immunoblotting was performed as indicated. (D) HepG2 cells were transfected with pHBV1.3 with or without the miR-15a mimic for 24 h and then treated with TGF-β1 for 24 h. The cell lysates were subjected to immunoblotting. (E) HepG2 cells were transfected with pHBV1.3 and Myc-Smad2 for 24 h and then treated with TGF-β1 for 24 h. Immunoblotting was performed as described above. The relative amount of Smad2 in the nucleus was quantified. **, P < 0.01.
FIG 6
HBV makes HepG2 cells resistant to TGF-β-induced apoptosis. (A) HepG2 cells were treated with or without TGF-β1 for 48 h. The cell lysates were harvested for immunoblotting with PARP-1 antibody or β-actin as an internal control. (B) HepG2, HepG2-C5, and HepG2-4D14 cells were treated with or without TGF-β1 for 48 h. The cell lysates were harvested for immunoblotting with the indicated antibodies. (C) HepG2 cells were transfected with pHBV1.3 or pHBV1.3-mut for 24 h and then treated with TGF-β1 for 24 h. The cell lysates were harvested for immunoblotting with the indicated antibodies. (D) HepG2 cells were transfected with pHBV1.3 or pHBV1.3-mut for 24 h and then treated with TGF-β1 for 24 h. The cell lysates were harvested for immunoblotting with the indicated antibodies. (E) HepG2 (left) and L-02 (right) cells were transfected with pHBV1.3 or pcDNA3.1 for 24 h and then treated with TGF-β1 for 24 h. The cell lysates were harvested and subjected to a caspase 3/7 activity assay. (F) HepG2 cells were transfected with pHBV1.3, pHBV1.3-mut, or pcDNA3.1 as a control for 24 h and then treated with TGF-β1 for 48 h. (Left) The cells were subjected to annexin V and PI staining for flow cytometry analysis. (Right) The percentage of apoptotic cell was calculated. *, P < 0.05; **, P < 0.01.
FIG 7
HBV promotes tumorigenesis through inhibiting TGF-β signaling by upregulating Smad7. (A) HepG2 and HepG2-4D14 cells were treated with TGF-β1 for 12 h, 24 h, and 48 h. Cell viability was detected by a CCK-8 assay. (B and C) HepG2, HepG2-C5, and HepG2-4D14 cells were injected subcutaneously into BALB/c nude mice (5 mice/group). (B, left) The size of tumors was measured at the indicated times after injection. (Right) At day 30, the tumors were dissected and photographed. (C, left) The RNAs of tumors generated from HepG2 and HepG2-4D14 cells were subjected to real-time PCR for miR-15a and Smad7 mRNA. (Right) Immunofluorescence analysis of tumor tissues was performed with Smad7 antibody or rabbit IgG as a negative control. (D) Smad7 knockdown HepG2-4D14 cells (HepG2-4D14 shSmad7#1 [shSmad7#1 in short] and HepG2-4D14 shSmad7#2 [shSmad7#2 in short]) or control (HepG2-4D14 sh-NC [sh-NC in short]) cells were generated as described in Materials and Methods. The cells were treated with TGF-β1 for the indicated times. (Top) Cell viability was detected by a CCK-8 assay. (Bottom) The protein level of Smad7 in cells was checked by immunoblotting. (E) HepG2-4D14 Sh-NC, HepG2-4D14 shSmad7#1, or HepG2-4D14 shSmad7#2 cells were injected subcutaneously into BALB/c nude mice. (Top) The size of tumors was measured at the indicated times after injection. The results are presented as means ± standard deviations from 5 mice for each group. (Bottom) The tumors were dissected at day 30 and photographed.
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