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Jurnal Club May 16 2019 - YAN MARDIAN.pptx
- 1. Dynamic expression of ZNF382 and its tumor-suppressor role in hepatitis B virus-related hepatocellular carcinogenesis Dang S, Zhou J, Chen Y, Chen P, Ji M, Shi B, et al. Dynamic expression of ZNF382 and its tumor-suppressor role in hepatitis B virus-related hepatocellular carcinogenesis. Oncogene. 2019; https://doi.org/10.1038/s41388-019-0759-9 Journal Club Virology Lab May 16th, 2019 Prepared by: Yan Mardian
- 2. HCC Overview
- 3. Introduction Transcription factor Zinc-Finger protein 382 (ZNF382) frequently silenced by promoter methylation functions as a tumor suppressor •Gene located in chromosome 19q13.13, encoding 64kDa protein •Usually expressed at very early embryonic stage, relatively low in adult liver tissue •Frequently methylate in multiple human cancers (breast, colon, esophageal, gastric, and nasopharyngeal cancers) potential tumor suppressor Hepatitis B virus X protein (HBx) weak oncogene, can initiate and progress HCC with other cofactors •154 aa, act as a nuclear coactivator that activates signal transduction by several pathways •alters the expression oh host gene (activating protein 1/2, NFKB, Wnt/β-catenin, etc) and dysregulating several miRNAs (miR3188, miR122, etc.) Promoting cell growth and survival Liver tumorigenesis is a complex process •Involves in multiple numerous genetic and epigenetic abnormalities •Promoter methylation is a major epigenetic event silencing of tumor suppressor gene Background of the study: •ZNF382 role in HCC remain largely unknown •No previous study tested association of HBx levels with ZNF382 expression
- 4. HBx Protein
- 5. Material and Methods Clinical samples • 13 normal liver tissues, 20 HBV-infected liver cirrhosis tissues, and 39 HBV-infected HCC tissues obtained from the First Affiliated Hospital of Xi’an Jiaotong University. • All patients did not receive chemotherapy and radiotherapy, and signed an informed consent before surgery. Cell lines (in vitro studies) • HepG2, HepG2.2.15, H7402, Hep3B, SMMC7721 In vitro functional studies • Four- to 5-week-old female athymic nude mice were obtained from Shanghai SLAC Laboratory Animal Co. Ltd., and were randomly divided into two groups (five mice per group) without blind design. • Next, 3 × 106 HepG2 cells stably expressing ZNF382 and control cells were subcutaneously inoculated into the right armpit region of mice to establish xenograft tumor model. • From 3 days after injection, tumor size was measured every 2 days. The formula (length × width2 × 0.5) was used to calculated tumor volumes. • After day 13 post-injection, tumors were isolated from the sacrificed mice and weighted.
- 6. Results - 1 a. mRNA expression of ZNF382 in HBV-negative normal liver tissues (N, n = 13), HBV- positive liver cirrhosis tissues (LC, n = 20), and HBV-positive hepatocellular carcinoma (HCC) tissues (HCC, n = 39) was investigated by quantitative real-time RT-PCR (qRT-PCR) assay with 18S rRNA as an internal control. b Protein expression of ZNF382 in the above tissues was determined by western blot analysis with GAPDH as a loading control. Shown in c is quantitative analysis of protein expression of ZNF382 by densitometry.
- 7. Results – 1 – Cont.’ Transcription activator-like effector nucleases (TALENs) targeting the conserved regions of HBV genome were constructed and transfected into HepG2.2.15 cells, and five cell clones (C1–C5) were then selected to assess the levels of HBV DNA by qPCR assay ZNF382 expression in the above cell clones was detected by qRT-PCR assay (h) with 18S rRNA as an internal control, and western blot analysis (i) with GAPDH as a loading control, respectively.
- 8. Results – 1 – Conclusion ZNF382 expression is associated with HBV infection • These data indicate upregulation of ZNF382 by HBV at posttranscriptional levels.
- 9. Results – 2 ZNF382 methylation in a number of HCCs (T) and non-cancerous liver tissues (N) from The Cancer Genome Atlas (TCGA) dataset.
- 10. Results – 2 – Cont.’ b. 5-Aza-2′-dC (5 μM) or SAHA (7.5 μM) were used to treat the indicated HCC cells for 5 or 3 days, respectively, and mRNA expression of ZNF382 was then assessed by quantitative real-time RT-PCR (qRT-PCR) assay with 18S rRNA as a reference gene. c. 5-Aza-2′-dC was used to treat the indicated cells, and the pyrosequencing was then performed to confirm demethylation of ZNF382 promoter. 5-Aza 5-Aza-2′-dC.
- 11. Results – 2 – Conclusion ZNF382 expression is associated with HBV infection ZNF382 is frequently downregulated by promoter methylation in HCCs •These findings indicate that epigenetic abnormalities such as promoter methylation is a principal mechanism for transcriptional inactivation of ZNF382 in HCCs.
- 12. Results - 3 Upregulation of zinc- finger protein 382 (ZNF382) by hepatitis B virus X protein (HBx) at posttranscriptional levels. The associations between HBx protein levels with mRNA (a) and protein (b) expression of ZNF382 were tested by linear regression analysis.
- 13. Results – 3 – Cont.’ Dox-inducible HBx expression and the effect of HBx on ZNF382 protein levels in HepG2 and H7402 cells were determined by western blot analysis (left panels). Quantitative analysis for protein expression of ZNF382 is shown in middle panels. The impact of HBx on ZNF382 mRNA levels in HepG2 and H7402 cells was assessed by qRT-PCR assay (right panels) with 18S rRNA as a reference gene.
- 14. Results – 3 – Cont.’ HBx knockdown by two different siRNAs (si-HBx#1 and #2) and its effect on ZNF382 protein levels in HepG2.2.15 and Hep3B cells were tested by western blot analysis (left panels). Protein expression of ZNF382 is quantified by densitometry in middle panels. The impact of HBx knockdown on ZNF382 mRNA levels in HepG2.2.15 and Hep3B cells were assessed by quantitative real-time RT-PCR (qRT-PCR) assay (right panels) with 18S rRNA as a reference gene.
- 15. Results – 3 – Conclusion ZNF382 expression is associated with HBV infection ZNF382 is frequently downregulated by promoter methylation in HCCs HBx upregulates ZNF382 expression at posttranscriptional levels
- 16. Results - 4 a. HBx-mediated ZNF382 upregulation was assessed by western blot analysis with GAPDH as a loading control. b Promoter activity of ZNF382 upon ectopic expression of HBx was evaluated by the dual- luciferase reporter system in HepG2 and H7402 cells with empty vector as the control. The ratio of the Luc/Renilla activity was indicated as mean ± SD of three independent assays.
- 17. Results – 4 – Cont.’ c. miR-6867 expression in HepG2 and H7402 cells expressing HBx induced by Dox were assessed by quantitative real-time RT-PCR (qRT-PCR), and miR-6867 expression was normalized to U6 levels. d miR-6867 expression was validated by qRT- PCR assay in HepG2 and H7402 cells transfected with miR-6867 mimics or negative control (NC). e The impact of miR-6867 mimics on ZNF382 protein expression in the above cells was assessed by western blot assay.
- 18. Results – 4 – Cont.’ g. Western blot analysis was performed to determine HBx-mediated upregulation of ZNF382 proteins via miR-6867 in HepG2 and H7402 cells. GAPDH was used as loading control. h HBx-mediated upregulation of ZNF382 proteins via Erk activation was demonstrated in HepG2 and H7402 cells by western blot analysis with GAPDH as a loading control. i. qRT-PCR was performed to determine HBx- mediated inhibition of mature miR-6867 via Erk activation in HepG2 and H7402 cells. U6 levels were used to normalize miR-6867 expression. The data were presented as mean ± SD. **P < 0.01; ***P < 0.001
- 19. Results – 4 – Conclusion ZNF382 expression is associated with HBV infection ZNF382 is frequently downregulated by promoter methylation in HCCs HBx upregulates ZNF382 expression at posttranscriptional levels Upregulation of ZNF382 by HBx is caused by Erk- mediated miR-6867 inhibition
- 20. Results – 5 a. HepG2 and H7402 cells were transfected with the indicated constructs, and western blot analysis was used to detect protein expression of ZNF382 with GAPDH as a loading control. b. Inhibitory effect of ZNF382 on the proliferation of the indicated HCC cells. c. Inhibitory effect of ZNF382 on colony formation of the indicated HCC cells. Shown are the representative images of colony formation (left panel) and quantitative analysis of colony numbers (right panel).
- 21. Results – 5 – Cont.’ g. Tumor growth curves were compared between HepG2 cells stably expressing ZNF382 and control cells in nude mice. Tumor cells were injected at day 0, and data are shown as mean ± SD (n = 5/ group). h. Photographs (left panel) and scatter diagram of tumor weight (right panel) of dissected tumors from ZNF382 overexpression and control mice. ZNF382 expression in dissected tumors was then validated by quantitative real-time RT-PCR (qRT-PCR) (i) and western blot (j) assays.
- 22. Results – 5 – Cont.’ The effect of ZNF382 on the expression of its potential downstream targets in HepG2 and H7402 cells was assessed by quantitative real-time RT- PCR (qRT-PCR) assay (a) with 18S rRNA as a reference gene, and western blot analysis (b) with GAPDH and histone H3 as loading controls, respectively. AP-1 family members (FOS and JUN) and downstream targets of AP-1 (CCND1 and MMP1) FZD1 and DVL2 modulate the activity of Wnt/β-catenin cascade through downregulating these two genes IN VITRO ASSAY
- 23. Results – 5 – Cont.’ The effect of ZNF382 on its downstream targets expression in the xenograft tumors was investi gated by qRT-PCR (f), western blot (g), and IHC (h) assays. IN VIVO ASSAY
- 24. Results – 5 – Conclusion ZNF382 expression is associated with HBV infection ZNF382 is frequently downregulated by promoter methylation in HCCs HBx upregulates ZNF382 expression at posttranscriptional levels Upregulation of ZNF382 by HBx is caused by Erk- mediated miR-6867 inhibition ZNF382 is a potent tumor suppressor in HCC • ZNF382 inhibits HCC tumorigenesis through impairing the activities of AP-1 and Wnt/β-catenin pathways and activating p53 signaling
- 26. ZNF382 is dynamically expressed in HBV-related liver carcinogenesis. • Initially, ZNF382 expression is relatively low in normal liver tissues, whereas it is dramatically elevated by HBx through Erk/miR-6867 axis in occurrence and progress of HBV-related liver cirrhosis. High expression of ZNF382 inhibits cell growth and invasiveness • through blocking the activities of AP-1 and Wnt/β-catenin pathways and activating p53 signaling, thereby preventing liver cirrhosis from progressing further to HCC. When ZNF382 is inactivated by promoter methylation in some liver cirrhosis tissues. • it will promote the progression of liver cirrhosis to HCC. Final Conclusion
- 27. Terima kasih ありがとうございます [thank you very much]
Editor's Notes
- Slide 3: Responsible globally for approximately 800,000 deaths each year, primary liver cancer is the second leading cause of cancer-related death and fifth-most-common cancer worldwide. Primary liver cancer comprises a heterogeneous group of malignant tumors that do not include metastases to the liver from other sites. Liver carcinogenesis is a multifactorial process, and predisposing factors for the various liver cancer subtypes differ. Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer, accounting for 80–90% of cases, with major geographical differences in prevalence2. Intrahepatic cholangiocarcinoma and extrahepatic bile-duct carcinoma (perihilar or distal cholangiocarcinoma) account for 6–15% of liver cancers. A distinction between intrahepatic cholangiocarcinoma and extrahepatic bile-duct carcinoma is important, as inflammatory risk factors for intrahepatic cholangiocarcinoma have been found to be similar to those known for HCC. Thus, in the development of intrahepatic cholangiocarcinoma as well as that of HCC, geographical differences in incidence probably reflect differences in genetic, environmental and infectious risk factors. Other liver cancers include rare non-epithelial tumors and pediatric hepatoblastoma. Known causes of chronic liver disease (CLD) and HCC include alcohol abuse and rare disorders such as α-1 antitrypsin deficiency and hemochromatosis. HCC is less common in liver cirrhosis caused by autoimmune hepatitis, Wilson disease or cholestatic liver disorder (Table 1). However, chronic infection with hepatitis B virus (HBV) remains the leading cause of HCC worldwide, and chronic infection with hepatitis C virus (HCV) is currently the leading cause of end-stage liver disease and HCC in the Western world. The incidence of liver cancer, including HCC, has risen in areas with historically low rates, including Western Europe and North America. This might be due in part to the rising prevalence of metabolic syndrome and non-alcoholic steatohepatitis (NASH). Non-alcoholic fatty liver disease (NAFLD), characterized by increased intrahepatic lipid storage and a non-symptomatic diminished ability of the liver to metabolize several substrates, is on the rise. NAFLD can progress to NASH, in which metabolic stress within hepatocytes initiates the death of liver cells, the production of damage-associated molecules (DAMPs) and the influx of activated immune cells (i.e., ‘sterile’ chronic inflammation). Thus, not only in chronic viral hepatitis but also in alcoholic or metabolic liver disease, chronic inflammation, and an altered immune response are associated with the development of HCC.
- Q arm = long arm of the chromosome
- HepG2 = hepatoblastoma-derived cell line, wild type P53 H7402 = Human hepatoma cell line Hep3B = human hepatoma cell line, contain integrated HBV, point mutation p53 SMMC7721 = heptocellular carcinoma cell line One of the most widely used models is the human tumor xenograft. In this model, human tumor cells are transplanted, either under the skin or into the organ type in which the tumor originated, into immunocompromised mice that do not reject human cells.
- Fig. 1a, mRNA levels of ZNF382 in HBV- positive liver cirrhosis tissues were not significantly different from HBV-negative normal tissues, whereas it was dramatically elevated compared with that in HBV-positive HCC tissues (P = 0.0013). Fig. 1b, c, protein levels of ZNF382 were significantly increased in HBV-positive liver cirrhosis tissues relative to HBV-negative normal liver tissues (P < 0.001) and HBV-positive HCC tissues (P < 0.001),
- It was showed that decline in ZNF382 expression was closely related to poor survival in early-stage HCC patients (HR = 1.816, P = 0.045 by log-rank test) (Fig. 1d). HBV DNA levels were not significantly correlated with mRNA levels of ZNF382 (P = 0.29, r = 0.16; Pearson’s correlation coefficient) (Fig. 1e), whereas were strongly correlated with protein levels of ZNF382 (P < 0.01, r = 0.39; Pearson’s correlation coefficient) (Fig. 1f). Note: r = 0.1- 0.3 weak r = 0.3 - 0.5 moderate r = 0.5 - 1.0 strong
- TALENs significantly downregulated the HBV DNA levels in five cell clones relative to control cells (Fig. 1g). As expected, relative to control cells, mRNA levels of ZNF382 were not significantly changed (Fig. 1h), whereas protein levels of ZNF382 were markedly decreased in these cell clones (Fig. 1i). Note: Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations.
- Downregulation of zinc-finger protein 382 (ZNF382) by promoter methylation in hepatocellular carcinomas (HCCs). The methylation-specific PCR (MSP) assay was used to detect ZNF382 methylation in HCCs and non-cancerous live tissues. In vitro methylated DNA, bisulfite-treated normal leukocyte DNA and H2O were used as positive control (PC), negative control (NC) and blank control, respectively. Mk DNA marker, M methylated gene, U unmethylated gene. Shown in a is the MSP results of two representative HCC samples (HCC-1 and -2) As shown in Fig. 2d, methylation levels of ZNF382 were markedly increased in HCCs relative to control subjects. Moreover, we found that methylation levels of ZNF382 promoter were significantly negatively correlated with its mRNA expression (P < 0.0001, r = 0.59; Pearson’s correlation coefficient) (Fig. 2e). Note: CpG sites, region in DNA where C is followed by G, the C can be methylaed to form 5-methylcytosines methylation of CpG sites in the promoter CpG island to cause the stable silencing of the gene
- As shown in Fig. 2b, ZNF382 expression was dramatically resorted upon 5-Aza-2′-dC or SAHA treatment in all cell lines. In addition, we also found that 5-Aza-2′-dC treatment decreased methylation levels of all seven CpG sites within ZNF382 promoter in the indicated cell lines compared with the controls by pyrosequencing analysis (Fig. 2c).
- CpG sites, region in DNA where C is followed by G, the C can be methylaed to form 5-methylcytosines methylation of CpG sites in the promoter CpG island to cause the stable silencing of the gene
- We first tested the association of HBx levels with ZNF382 expression in HBV-positive liver tissues. Our results showed that HBx levels were not significantly correlated with mRNA expression of ZNF382 (P = 0.77, r = 0.04; Pearson’s correlation coefficient) (Fig. 3a), whereas were strongly associated with protein expression of ZNF382 (P < 0.001, r = 0.58; Pearson’s correlation coefficient)
- These observations suggest that ZNF382 expression may be regulated by HBx at posttranscriptional levels. To do this, we established doxycycline (Dox)-controlled inducible system (Tet-on) to ectopically express HBx in HepG2 and H7402 cells. As shown in Fig. 3c, Dox treatment remarkably induced HBx expression (left panels), and upregulated ZNF382 expression at protein levels (left and middle panels), but not at mRNA levels (right panels) in these two cell lines. Note: The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from basic biological research to biotechnology and gene therapy applications. These systems are based on regulatory elements that control the activity of the tetracycline-resistance operon in bacteria. The Tet-Off system allows silencing of gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system allows activation of gene expression by dox.
- Conversely, we knocked down HBx (Supplementary Fig. 1 and Fig. 3d, left panels), and determine the effect of HBx depletion on ZNF382 expression in HepG2.2.15 and Hep3B cells, which have been demonstrated to express HBx. As shown in Fig. 3d, HBx knockdown significantly reduced ZNF382 protein levels, but not its mRNA levels (middle and right panels).
- To further explore the mechanism of HBx regulating ZNF382 expression, we first established HepG2 and H7402 cells stably expressing HBx using lentivirus expression system (Fig. 4a). Next, promoter activity of ZNF382 upon ectopic expression of HBx was evaluated by the dual-luciferase reporter system in these cells. Our data showed that ectopic expression of HBx almost did not influence promoter activity of ZNF382 in these two cells. These data further support that HBx regulates ZNF382 expression at posttranscriptional levels. Note: In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest.
- It is clear that microRNAs (miRNAs) can post-transcriptionally modulate the expression of protein-coding genes through targeting their mRNAs. In addition, there is evidence demonstrating that a number of miRNAs can be modulated by HBx. Thus, we speculate that HBx may upregulate ZNF382 by suppressing certain miRNAs, which potentially target ZNF382 mRNA. Using target prediction softwares such as miRDB, miR- anda, and TargetScan, we identified a panel of candidate miRNAs that potentially target ZNF382 gene, including miR-30a, miR-30b, miR-30d, miR-30e, miR-190a, miR- 190b, miR-233, miR-374a, miR-410, miR-483, miR-539, and miR-6867. We next assessed the effect of HBx on the expression of these miRNAs in HepG2 and H7402 cells using a Tet-on system. The results showed that, of these miRNAs, only miR-6867 was dramatically downregulated by HBx in both cell lines (Fig. 4c). To determine regulatory effect of miR-6867 on ZNF382 expression, miR-6867 mimics were synthesized and transfected into HepG2 and H7402 cells (Fig. 4d). The results showed that, compared with negative control (NC), miR-6867 mimics markedly decreased protein levels of ZNF382 (Fig. 4e). Note: A microRNA (abbreviated miRNA) is a small non-coding RNA molecule (containing about 22 nucleotides) found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression. Pre-miRNAs, pri-miRNAs and genes that lead to 100% identical mature miRNAs but that are located at different places in the genome are indicated with an additional dash-number suffix. For example, the pre-miRNAs hsa-mir-194-1 and hsa-mir-194-2 lead to an identical mature miRNA (hsa-miR-194) but are from genes located in different genome regions.
- To determine whether the regulation of ZNF382 by HBx is mediated by miR-6867, miR-6867 mimics, or NC were transfected into HepG2 and H7402 cells inducibly expressing HBx by Dox. As expected, HBx-mediated upregulation of ZNF382 proteins was markedly reversed by miR- 6867 mimics in these two cells (Fig. 4g), whereas it did not affect mRNA expression of ZNF382 relative to NC (Supplementary Fig. 8). Collectively, our data indicate that upregulation of ZNF382 by HBx is mediated by miR-6867.
- Evidently, activated Erk suppresses the nucleocytoplasmic export of the precursor miRNAs (pre-miRNAs) through phosphorylating exportin-5, thereby resulting in the reduction of mature miRNAs [31]. In addition, it is the fact that HBx is able to activate the Erk cascade in HCC cells. Thus, we speculate that HBx upregulates ZNF382 expression at posttranscriptional levels through repressing miR-6867 maturation via Erk activation in HCC cells. To validate this, we first used MEK inhibitor GSK1120212 to treat HepG2 and H7402 cells expressing HBx induced by Dox. The results showed that inducible expression of HBx dramatically upregulated protein expression of ZNF382 and Erk phosphorylation in these two cell lines, whereas GSK1120212 treatment reversed promoting effects of HBx. on Erk phosphorylation and ZNF382 protein levels (Fig. 4h), but did not affect mRNA expression of ZNF382 (Supplementary Fig. 9). These data indicate that upregulation of ZNF382 by HBx is caused by Erk activation. Next, we attempted to validate the impact of activated Erk on miR-6867 maturation. The results showed that Dox-induced HBx expression significantly decreased the expression of mature miR-6867 in HepG2 and H7402 cells, and GSK1120212 treatment remarkably reversed the regulatory effect of HBx on mature miR-6867 (Fig. 4i), but not pri/pre- miR-6867 in these two cell lines (Fig. 4j). Altogether, our data indicate that HBx upregulates ZNF382 expression at posttranscriptional levels through Erk-mediated miR-6867 inhibition. Note: A MEK inhibitor is a chemical or drug that inhibits the mitogen-activated protein kinase kinase enzymes MEK1 and/or MEK2. They can be used to affect the MAPK/ERK pathway which is often overactive in some cancers. MEK inhibitors suppress ERK activity in normal cells.
- Given frequent downregulation of ZNF382 by promoter methylation in HBV-positive HCC tissues relative to control subjects, we speculate that ZNF382 may be a putative tumor suppressor in HCC tumorigenesis. Thus, a series of in vitro and in vivo studies were performed to determine the biological function of ZNF382 in HCC. First, we used qRT-PCR (Supplementary Fig. 10a) and western blot (Fig. 5a) assays to validate ectopic expression of ZNF382 in HepG2 and H7402 cells. The results showed that ZNF382 over- expression significantly suppressed cell proliferation (Fig. 5b) and colony formation (Fig. 5c) relative to empty vector.
- On the other hand, ZNF382 knockdown in Hep3B and SMMC7721 cells by two different specific siRNAs (si- ZNF382 #1 and si-ZNF382 #2) significantly promoted cell proliferation and colony formation compared with control siRNA (si-NC) (Fig. 5d–f).
- Next, xenograft mouse model was established to test in vivo tumor-suppressor role of ZNF382. As shown in Fig. 5g, tumor induced by HepG2 cells stably expressing ZNF382 displayed a significantly lower growth rate compared with tumors induced by control cells. Next, we isolated and weighted the xenograft tumors 13 days after injection. As shown in Fig. 5h, mean weight of xenograft tumors from ZNF382 overexpression mice was significantly less compared with control mice. Moreover, ZNF382 expression in the above xenograft tumors was further confirmed by qRT-PCR (Fig. 5i) and western blot (Fig. 5j) assays. Overall, these findings demonstrate that ZNF382 is a potent suppressor in HCC tumorigenesis and progression.
- It has been reported that ZNF382 represses transcriptional activities of AP-1 in colon cancer cell line HCT116; so, we ectopically expressed ZNF382 in HepG2 and H7402 cells and determined the effect of ZNF382 overexpression on the expression of two AP-1 family members (FOS and JUN). The results showed that, relative to the vector, ZNF382 transfection significantly inhibited the expression of FOS and JUN (Fig. 6a, b). Moreover, there is evidence showing that JUN can control anti-proliferation activity of p53 by directly inhibiting its transcription. As expected, ectopic expression of ZNF382 markedly decreased the expression of CCND1 and MMP1, and increased p53 and its downstream target p21 expression in these two cell lines (Fig. 6a, b). Notably, a recent study has identified FZD1 and DVL2 genes as potential targets of ZNF382 in esophageal squamous cell carcinoma (ESCC) cells and demonstrated that ZNF382 modulates the activity of Wnt/β-catenin cascade through downregulating these two genes. Thus, we speculate that ZNF382 also blocks Wnt/β-catenin signaling in HCC via transcriptional repression of its potential targets such as FZD1 and DVL2. Expectedly, the expression of FZD1 and DVL2 and nuclear accumulation of β-catenin were markedly repressed upon ectopic expression of ZNF382. Collectively, our data demonstrated suppressing effect of ZNF382 on Wnt/β-catenin cascade in HCC cells.
- As shown in Fig. 6f–h and Supplementary Fig. 14, the expression of FOS, JUN, DVL2, and FZD1 was dramatically downregulated in ZNF382 overexpression tumors relative to control tumors. Accordingly, we found that the expression of CCND1 and MMP1 was decreased, whereas the expression of p53 and its downstream target p21 was increased in the former compared with the latter. In addition, using immunofluorescence assay, we also observed nucleus translocation of β-catenin in ZNF382 overexpression tumors and control tumors (Supplementary Fig. 15), further demonstrating inhibitory effect of ZNF382 on Wnt/β-catenin signaling.
- Fig. 7 A schematic model illustrating the mechanisms underlying dynamic expression of zinc-finger protein 382 (ZNF382) and its tumor-suppressor role in hepatitis B virus (HBV)-related hepatocellular carcinogenesis. In general, mature miR-6867 represses the translation of ZNF382 through binding to its 3′-UTR in hepatocytes from the adult liver tissue, thereby leading to its relatively low expression (left panel). In the process of HBV-related liver cirrhosis, ZNF382 is dramatically elevated by HBV at posttranscriptional levels through hepatitis B virus X protein (HBx)/Erk/miR-6867 axis, and increased expression of ZNF382 exerts as a potent tumor suppressor through inhibiting the activities of AP-1 and Wnt/β-catenin signaling pathways and activating p53 signaling via transcriptional repression of its downstream targets such as FOS, JUN, FZD1, and DVL2. This will prevent liver cirrhosis from progressing further to hepatocellular car- cinoma (HCC) (middle penal). However, ZNF382 is inactivated by promoter methylation in some liver cirrhosis tissues, which is thought to be a potential hit to drive the progression of liver cirrhosis to HCC (right panel)