Changliang Shan

Upregulated MicroRNA-29a by Hepatitis B Virus X Protein Enhances Hepatoma Cell Migration by Targeting PTEN in Cell Culture Model

Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3′UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.

Hepatitis B virus X protein modulates oncogene yes-associated protein by CREB to promote growth of hepatoma cells†

Hepatitis B virus X protein (HBx) plays critical roles in the development of hepatocellular carcinogenesis (HCC). Yes‐associated protein (YAP), a downstream effector of the Hippo‐signaling pathway, is an important human oncogene. In the present article, we report that YAP is involved in the hepatocarcinogenesis mediated by HBx. We demonstrated that the expression of YAP was dramatically elevated in clinical HCC samples, hepatitis B virus (HBV)‐infected hepatoma HepG2.2.15 cell line, and liver cancer tissues of HBx‐transgenic mice. Meanwhile, we found that overexpression of HBx resulted in the up‐regulation of YAP in stably HBx‐transfected HepG2/H7402 hepatoma cell lines, whereas HBx RNA interference reduced YAP expression in a dose‐dependent manner in the above‐mentioned cell lines, suggesting that HBx up‐regulates YAP. Then, we investigated the mechanism underlying the up‐regulation of YAP by HBx. Luciferase reporter gene assays revealed that the promoter region of YAP regulated by HBx was located at nt −232/+115 containing cyclic adenosine monophosphate response element‐binding protein (CREB) element. Chromatin immunoprecipitation (ChIP) demonstrated that HBx was able to bind to the promoter of YAP, whereas it failed to work when CREB was silenced. Moreover, we confirmed that HBx activated the YAP promoter through CREB by electrophoretic mobility shift assay and luciferase reporter gene assays. Surprisingly, we found that YAP short interfering RNA was able to remarkably block the HBx‐enhanced growth of hepatoma cells in vivo and in vitro. Conclusion: YAP is a key driver gene in HBx‐induced hepatocarcinogenesis in a CREB‐dependent manner. YAP may serve as a novel target in HBV‐associated HCC therapy. (HEPATOLOGY 2012;56:2051–2059)

MicroRNA-520e suppresses growth of hepatoma cells by targeting the NF-κB-inducing kinase (NIK)

MicroRNAs (miRNAs) are small, non-coding RNAs that can act as oncogenes or tumor suppressor genes in human cancer. Emerging evidence indicates that deregulation of miRNAs contributes to the hepatocarcinogenesis. In the present study, we demonstrated that the levels of miR-520e were dramatically decreased in examined hepatoma cell lines and clinical hepatocellular carcinoma (HCC) tissues. Moreover, we found that DNA hypermethylation in the upstream region of miR-520e resulted in the downregulation of miR-520e. Next, we demonstrated that introduction of miR-520e dramatically suppressed the growth of hepatoma cells in vitro and in vivo, whereas silencing the expression of miR-520e by anti-miR-520e resulted in a promoted cell proliferation, suggesting that miR-520e may be a novel tumor suppressor. Further studies revealed that NF-κB-inducing kinase (NIK) was one of the direct target genes of miR-520e, as miR-520e directly bound to the 3′untranslated region of NIK, which reduced the expression of NIK at the levels of mRNA and protein. Moreover, silencing of NIK was able to inhibit the growth of hepatoma cells, similar to the effect of miR-520e overexpression on growth of hepatoma cells. Meanwhile, the knockdown of NIK expression reversed the enhanced proliferation mediated by anti-miR-520e. In addition, miR-520e significantly decreased the phosphorylation of ERK1/2 (p-ERK1/2) and depressed the transcriptional activity and nuclear translocation of nuclear factor κB (NF-κB) (p65). These results suggest that miR-520e suppresses the growth of hepatoma cells by targeting NIK involving the NIK/p-ERK1/2/NF-κB signaling pathway. Finally, we showed that the intratumoral injection with miR-520e was able to directly repress the growth of hepatoma cells in the nude mice. Thus, our finding provides new insight into the mechanism of hepatocarcinogenesis, indicating a therapeutic potential of miR-520e in the treatment of HCC.

Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-ERK1/2.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma. Here, we sought to identify the mechanisms by which HBx mediates liver cell proliferation. We found that HBx upregulated the levels of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and phosphorylated extracellular signal-regulated protein kinases 1/2 (p-ERK1/2) in liver cells. HBx-induced p-ERK1/2 was abolished by inhibition of Gi/o proteins, COX or LOX. In addition, HBx increased the amounts of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) released from cell lines derived from hepatocytes. Moreover, these released arachidonic acid metabolites were able to activate ERK1/2. Interestingly, activated ERK1/2 could upregulate the expression of COX-2 and 5-LOX in a positive feedback manner. In conclusion, HBx enhances and maintains liver cell proliferation via a positive feedback loop involving COX-2, 5-LOX, released arachidonic acid metabolites, Gi/o proteins and p-ERK1/2.

Identification of a natural mutant of HBV X protein truncated 27 amino acids at the COOH terminal and its effect on liver cell proliferation

AbstractAim:To identify mutants of the hepatitis B virus (HBV) X (HBx) gene and investigate the effect of the natural mutant on liver cell proliferation.Methods:We identified natural mutants of the HBx gene from 188 sera and 48 tissues of Chinese patients infected with HBV by PCR, respectively. Based on the identification of the mutants of HBx gene, we cloned the fragments of the mutants into the pcDNA3 vector. The biological activities of the mutants were investigated.Results:We identified a natural mutant of the HBx gene with deletion from 382 to 401 base pairs from 3 sera out of 188 patients, which resulted in the expression deletion of the HBx protein from the 128th amino acid at the COOH terminal. The similar mutant with deletion from 382 base pair at the COOH terminal was identified from 5 cases of genomes out of 48 hepatocellular carcinoma tissues. Regarding the biological activities of the mutant, we found that the mutant of the HBx protein failed to induce apoptosis by transient transfection, but promoted proliferation of human liver immortalized L-O2 cells by stable transfection, compared with the wild-type HBx protein. The data showed that the proliferation of the mutant stably-transfected L-O2-X-Sera cells and fragment stably-transfected L-O2-XΔ127 cells was enhanced by the BrdU incorporation assay and flow cytometry analysis. Lu-ciferase reporter gene assay showed that the transcriptional activities of NF-κB, survivin, and human telomerase reverse transcriptase were upregulated, and Western blot analysis revealed that the expression levels of c-Myc and proliferating cell nuclear antigen (PCNA) were upregulated in the cells.Conclusion:Our findings suggest that the natural HBx mutant truncated 27 amino acids at the COOH terminal promotes cell proliferation.

NF-κB downregulation may be involved the depression of tumor cell proliferation mediated by human mesenchymal stem cells

AbstractAim:It has been reported that stem cells are able to home to tumorigenesis and inhibit the proliferation of tumor cells. The purpose of our study was to demonstrate the molecular mechanism of the inhibitory proliferation of hepatoma cells and breast cancer cells mediated by human mesenchymal stem cells (hMSCs).Methods:The proliferation of H7402 human hepatoma cells and MCF-7 human breast cancer cells was measured by the 5-bromodeoxyuridine (BrdU) incorporation assay and flow cytometry assay after the treatment with conditioned media from hMSCs culture, such as Z3 cells or BMMS-03 cells. The role of NF-κB or the phosphorylation of inhibitor κBα (p-IκBα) in the depression of hepatoma or breast cancer cells treated with conditioned media from Z3 cells or BMMS-03 cells was examined by reporter gene assay, quantitative real-time PCR, and Western blot analysis, respectively.Results:The proliferation of H7402 cells and MCF-7 cells was decreased significantly by the BrdU incorporation assay and flow cytometry assay after treatment. The transcriptional activity and mRNA level of NF-κB were downregulated in the treated cells by reporter gene assay and quantitative real-time PCR in a dose-dependent manner. At the protein level, NF-κB and p-IκBα decreased in the treated cells by Western blot analysis.Conclusion:Conditioned media from hMSCs are able to inhibit the proliferation of tumor cells. NF-κB downregulation is one of reasons for the depression of tumor cell proliferation mediated by hMSCs.

miRNA-520b and miR-520e sensitize breast cancer cells to complement attack via directly targeting 3'UTR of CD46.

MicroRNAs (miRNAs), non-coding RNAs that function as post-transcriptional gene regulators, play a pivotal role in cancer development. In the present study, we elucidated the roles of miR-520b and miR-520e in breast cancer cells. We examined the expression levels of miR-520b and miR-520e in the immortalized breast cell line, HBL-100, and in three breast cancer cell lines: MCF-7, LM-MCF-7 and MDA-MB-231. We show the expression levels of miR-520b and miR-520e in the breast cancer cell lines were lower than that in the HBL-100 cells. Furthermore, the breast cancer cell lines showed less sensitivity to complement-dependent cytotoxicity (CDC). We found that overexpression of miR-520b and miR-520e increases the sensitivity of the breast cancer cells to CDC, whereas further suppression of miR-520b and miR-520e decreases the sensitivity of the breast cancer cells to CDC. We then demonstrate that miR-520b and miR-520e are able to directly target the 3′untranslated regions (3′UTR) of the membrane-bound complement regulatory protein CD46; suggesting that miR-520b and miR-520e down-regulate CD46 at post-transcriptional level. Enzyme-linked immunosorbent assay (ELISA) showed that overexpression of miR-520b and miR-520e results in the increased expression of C3b, which is mediated by downregulated CD46. These results suggest that miRNA-520b and miR-520e mediated down-regulation of CD46 induces opsonization of cancer cells via an alternative pathway resulting in complement activation. Thus, we conclude that miR-520b and miR-520e contribute to CDC in breast cancer cells via directly targeting the 3′UTR of CD46.

Hepatitis B virus X protein upregulates Lin28A/Lin28B through Sp-1/c-Myc to enhance the proliferation of hepatoma cells.

Hepatitis B virus X protein (HBx) plays critical roles in the pathogenesis of hepatocellular carcinoma (HCC). Here, we were interested in knowing whether the oncogene Lin28A and its homolog Lin28B are involved in the hepatocarcinogenesis mediated by HBx. We showed that the expression levels of Lin28A and Lin28B were increased in clinical HCC tissues, HepG2.2.15 cell line and liver tissues of p21-HBx transgenic mice. Interestingly, the expression levels of HBx were positively associated with those of Lin28A/Lin28B in clinical HCC tissues. Moreover, the overexpression of HBx resulted in the upregulation of Lin28A/Lin28B in hepatoma HepG2/H7402 cell lines by transient transfection, suggesting that HBx was able to upregulate Lin28A and Lin28B. Then, we examined the mechanism by which HBx upregulated Lin28A and Lin28B. We identified that the promoter region of Lin28A regulated by HBx was located at nt −235/−66 that contained Sp-1 binding element. Co-immunoprecipitation showed that HBx was able to interact with Sp-1 in HepG2-X cells. Moreover, chromatin immunoprecipitation (ChIP) demonstrated that HBx could bind to the promoter of Lin28A, which failed to work when Sp-1 was silenced. Electrophoretic mobility shift assay (EMSA) further identified that HBx was able to interact with Sp-1 element in Lin28A promoter via transcription factor Sp-1. In addition, we found that c-Myc was involved in the activation of Lin28B mediated by HBx. In function, Lin28A/Lin28B played important roles in HBx-enhanced proliferation of hepatoma cells in vitro and in vivo. In conclusion, HBx activates Lin28A/Lin28B through Sp-1/c-Myc in hepatoma cells. Lin28A/Lin28B serves as key driver genes in HBx-induced hepatocarcinogenesis.

Hepatitis B virus X protein activates CD59 involving DNA binding and let-7i in protection of hepatoma and hepatic cells from complement attack

Emerging evidence has shown that hepatitis B virus (HBV) X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma. Complement regulatory proteins including CD46, CD55 and CD59 contribute to escape of tumor cells from complement-dependent cytotoxicity (CDC). However, little is known about the potential role of HBx in anti-CDC activity during hepatocarcinogenesis. In the present study, we for the first time report that HBx decreases the sensitivity of hepatoma and hepatic cells to CDC. Coincidentally, we demonstrated that HBx increased the promoter activity of CD59, as well as their messenger RNA and protein levels. Moreover, flow cytometry showed the increased expression level of CD59 protein on the surface of HBx-positive cells. Of interest, we found that HBx up-regulated CD59 by binding with cAMP response element-binding to the promoter region of the CD59 gene using chromatin immunoprecipitation assay. In addition, we showed that HBx up-regulated CD59 by let-7i at post-transcriptional regulation level. Our data showed that the deposition of C5b-9 were decreased on the cell surface in HepG2-X cells relative to HepG2 cells, suggesting that increased CD59 mediated by HBx prevents the formation of functional membrane attack complex. Furthermore, we demonstrated that down-regulation of CD59 was sufficient to abolish the resistance capability of CDC in HBx-positive cells by RNA interference (siRNA) in vitro and in vivo. Thus, we conclude that HBx contributes to cells resistance to CDC through CD59. Therapeutically, CD59 may serve as a target in HBV-associated hepatoma patients.

HBXIP upregulates CD46, CD55 and CD59 through ERK1/2/NF-κB signaling to protect breast cancer cells from complement attack.

Hepatitis B X‐interacting protein (HBXIP) is able to enhance migration of breast cancer cells. However, the role of HBXIP in regulation of complement‐dependent cytotoxicity (CDC) in breast cancer is not understood. Here, we report that HBXIP contributes to protecting breast cancer cells from CDC by upregulating membrane‐bound complement regulatory protein (mCRPs), including CD46, CD55 and CD59. We found that HBXIP upregulated mCRPs through activating p‐ERK1/2/NF‐κB. Interestingly, the knockdown of CD59 was able to block the HBXIP‐enhanced breast tumor growth in animal. Thus, we conclude that HBXIP upregulates CD46, CD55 and CD59 through p‐ERK1/2/NF‐κB signaling to protect breast cancer from CDC.