Lihong Ye

Suppression of tumorigenesis by human mesenchymal stem cells in a hepatoma model

Human mesenchymal stem cells (hMSCs) can home to tumor sites and inhibit the growth of tumor cells. Little is known about the underlying molecular mechanisms that link hMSCs to the targeted inhibition of tumor cells. In this study, we investigated the effects of hMSCs on two human hepatoma cell lines (H7402 and HepG2) using an animal transplantation model, a co-culture system and conditioned media from hMSCs. Animal transplantation studies showed that the latent time for tumor formation was prolonged and that the tumor size was smaller when SCID mice were injected with H7402 cells and an equal number of Z3 hMSCs. When co-cultured with Z3 cells, H7402 cell proliferation decreased, apoptosis increased, and the expression of Bcl-2, c-Myc, proliferating cell nuclear antigen (PCNA) and survivin was downregulated. After treatment with conditioned media derived from Z3 hMSC cultures, H4702 cells showed decreased colony-forming ability and decreased proliferation. Immunoblot analysis showed that β-catenin, Bcl-2, c-Myc, PCNA and survivin expression was downregulated in H7402 and HepG2 cells. Taken together, our findings demonstrate that hMSCs inhibit the malignant phenotypes of the H7402 and HepG2 human liver cancer cell lines, which include proliferation, colony-forming ability and oncogene expression both in vitro and in vivo. Furthermore, our studies provide evidence that the Wnt signaling pathway may have a role in hMSC-mediated targeting and tumor cell inhibition.

Elevation of Highly Up-regulated in Liver Cancer (HULC) by Hepatitis B Virus X Protein Promotes Hepatoma Cell Proliferation via Down-regulating p18

Background: The role of long noncoding RNA (lncRNA) highly up-regulated in liver cancer (HULC) in hepatocarcinogenesis mediated by hepatitis B virus X protein (HBx) remains unclear. Results: Up-regulation of HULC by HBx promotes hepatoma cell proliferation via down-regulating p18. Conclusion: HULC contributes to HBx-related hepatocarcinogenesis through suppressing p18. Significance: The finding provides insight into the roles of lncRNAs in HBx-associated hepatocarcinogenesis. Long noncoding RNAs (lncRNAs) play crucial roles in human cancers. It has been reported that lncRNA highly up-regulated in liver cancer (HULC) is dramatically up-regulated in hepatocellular carcinoma (HCC). Hepatitis B virus X protein (HBx) contributes importantly to the development of HCC. However, the function of HULC in HCC mediated by HBx remains unclear. Here, we report that HULC is involved in HBx-mediated hepatocarcinogenesis. We found that the expression levels of HULC were positively correlated with those of HBx in clinical HCC tissues. Moreover, we revealed that HBx up-regulated HULC in human immortalized normal liver L-O2 cells and hepatoma HepG2 cells. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay showed that HBx activated the HULC promoter via cAMP-responsive element-binding protein. We further demonstrated that HULC promoted cell proliferation by methyl thiazolyl tetrazolium, 5-ethynyl-2′-deoxyuridine, colony formation assay, and tumorigenicity assay. Next, we hypothesized that HULC might function through regulating a tumor suppressor gene p18 located near HULC in the same chromosome. We found that the mRNA levels of p18 were inversely correlated with those of HULC in the above clinical HCC specimens. Then, we validated that HULC down-regulated p18, which was involved in the HULC-enhanced cell proliferation in vitro and in vivo. Furthermore, we observed that knockdown of HULC could abolish the HBx-enhanced cell proliferation through up-regulating p18. Thus, we conclude that the up-regulated HULC by HBx promotes proliferation of hepatoma cells through suppressing p18. This finding provides new insight into the roles of lncRNAs in HBx-related hepatocarcinogenesis.

Dkk-1 secreted by mesenchymal stem cells inhibits growth of breast cancer cells via depression of Wnt signalling

We determined the molecular mechanism of inhibitory effect of human mesenchymal stem cells (hMSCs) on the growth of human MCF-7 breast cancer cells. Our finding showed that beta-catenin was down-regulated in MCF-7 cells by conditioned media from Z3 hMSCs, and the expression level of dickkopf-1 (Dkk-1) was higher in Z3 cells than that in MCF-7 cells. Neutralization of Dkk-1 and small interference RNA targeting Dkk-1 mRNA in Z3 cells attenuated the inhibitory effect of Z3 cells on MCF-7 cells. Overexpression of Dkk-1 in Z3 cells enhanced the inhibition. Therefore, Dkk-1 secreted by Z3 cells involves the inhibition via the Wnt pathway.

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.

Long noncoding RNA HULC modulates abnormal lipid metabolism in hepatoma cells through an miR-9-mediated RXRA signaling pathway.

HULC is a long noncoding RNA overexpressed in hepatocellular carcinoma (HCC), but its functional contributions in this setting have not been determined. In this study, we explored the hypothesis that HULC contributes to malignant development by supporting abnormal lipid metabolism in hepatoma cells. HULC modulated the deregulation of lipid metabolism in HCC by activating the acyl-CoA synthetase subunit ACSL1. Immunohistochemical analysis of tissue microarrays revealed that approximately 77% (180/233) of HCC tissues were positive for ACSL1. Moreover, HULC mRNA levels correlated positively with ACSL1 levels in 60 HCC cases according to real-time PCR analysis. Mechanistic investigations showed that HULC upregulated the transcriptional factor PPARA, which activated the ACSL1 promoter in hepatoma cells. HULC also suppressed miR-9 targeting of PPARA mRNA by eliciting methylation of CpG islands in the miR-9 promoter. We documented the ability of HULC to promote lipogenesis, thereby stimulating accumulation of intracellular triglycerides and cholesterol in vitro and in vivo. Strikingly, ACSL1 overexpression that generates cholesterol was sufficient to enhance the proliferation of hepatoma cells. Further, cholesterol addition was sufficient to upregulate HULC expression through a positive feedback loop involving the retinoid receptor RXRA, which activated the HULC promoter. Overall, we concluded that HULC functions as an oncogene in hepatoma cells, acting mechanistically by deregulating lipid metabolism through a signaling pathway involving miR-9, PPARA, and ACSL1 that is reinforced by a feed-forward pathway involving cholesterol and RXRA to drive HULC signaling.

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)

miR-520b Regulates Migration of Breast Cancer Cells by Targeting Hepatitis B X-interacting Protein and Interleukin-8

MicroRNAs play important roles in tumor metastasis. Recently, we reported that the level of miR-520b is inversely related to the metastatic potential of breast cancer cells. In this study, we investigated the role of miR-520b in breast cancer cell migration. We found that miR-520b suppressed the migration of breast cancer cells with high metastatic potential, including MDA-MB-231 and LM-MCF-7 cells, although the inhibition of miR-520b enhanced the migration of low metastatic potential MCF-7 cells. We further discovered that miR-520b directly targets the 3′-untranslated region (3′UTR) of either hepatitis B X-interacting protein (HBXIP) or interleukin-8 (IL-8), which has been reported to contribute to cell migration. Surprisingly, tissue array assays showed that 75% (38:49) and 94% (36:38) of breast cancer tissues and metastatic lymph tissues, respectively, were positive for HBXIP expression. Moreover, overexpression of HBXIP was able to promote the migration of MCF-7 cells. Interestingly, HBXIP was able to regulate IL-8 transcription by NF-κB, suggesting that the two target genes of miR-520b are functionally connected. In addition, we found that miR-520b could indirectly regulate IL-8 transcription by targeting HBXIP. Thus, we conclude that miR-520b is involved in regulating breast cancer cell migration by targeting HBXIP and IL-8 via a network in which HBXIP promotes migration by stimulating NF-κB-mediated IL-8 expression. These studies point to HBXIP as a potential therapeutic target for breast cancer.

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.

Long non-coding RNA HULC promotes tumor angiogenesis in liver cancer by up-regulating sphingosine kinase 1 (SPHK1)

Highly up-regulated in liver cancer (HULC) is a long non-coding RNA (lncRNA). We found that HULC up-regulated sphingosine kinase 1 (SPHK1), which is involved in tumor angiogenesis. Levels of HULC were positively correlated with levels of SPHK1 and its product, sphingosine-1-phosphate (S1P), in patients HCC samples. HULC increased SPHK1 in hepatoma cells. Chicken chorioallantoic membrane (CAM) assays revealed that si-SPHK1 remarkably blocked the HULC-enhanced angiogenesis. Mechanistically, HULC activated the promoter of SPHK1 in hepatoma cells through the transcription factor E2F1. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) further showed that E2F1 was capable of binding to the E2F1 element in the SPHK1 promoter. HULC increased the expression of E2F1 in hepatoma cells and levels of HULC were positively correlated with those of E2F1 in HCC tissues. Intriguingly, HULC sequestered miR-107, which targeted E2F1 mRNA 3′UTR, by complementary base pairing. Functionally, si-SPHK1 remarkably abolished the HULC-enhanced tumor angiogenesis in vitro and in vivo. Taken together, we conclude that HULC promotes tumor angiogenesis in liver cancer through miR-107/E2F1/SPHK1 signaling. Our finding provides new insights into the mechanism of tumor angiogenesis.

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.