Zhen Lv

Long Non-Coding RNA HOTAIR Promotes Cell Migration and Invasion via Down-Regulation of RNA Binding Motif Protein 38 in Hepatocellular Carcinoma Cells

Long non-coding RNA HOTAIR exerts regulatory functions in various biological processes in cancer cells, such as proliferation, apoptosis, mobility, and invasion. We previously found that HOX transcript antisense RNA (HOTAIR) is a negative prognostic factor and exhibits oncogenic activity in hepatocellular carcinoma (HCC). In this study, we aimed to investigate the role and molecular mechanism of HOTAIR in promoting HCC cell migration and invasion. Firstly, we profiled its gene expression pattern by microarray analysis of HOTAIR loss in Bel-7402 HCC cell line. The results showed that 129 genes were significantly down-regulated, while 167 genes were significantly up-regulated (fold change >2, p < 0.05). Bioinformatics analysis indicated that RNA binding proteins were involved in this biological process. HOTAIR suppression using RNAi strategy with HepG2 and Bel-7402 cells increased the mRNA and protein expression levels of RNA binding motif protein 38 (RBM38). Moreover, the expression levels of RBM38 in HCC specimens were significantly lower than paired adjacent noncancerous tissues. In addition, knockdown of HOTAIR resulted in a decrease of cell migration and invasion, which could be specifically rescued by down-regulation of RBM38. Taken together, HOTAIR could promote migration and invasion of HCC cells by inhibiting RBM38, which indicated critical roles of HOTAIR and RBM38 in HCC progression.

NDRG1 as a biomarker for metastasis, recurrence and of poor prognosis in hepatocellular carcinoma

N-myc downstream-regulated gene 1 (NDRG1) has been reported to be a multifunctional protein associated with carcinogenesis, however, the cellular function of NDRG1 remains elusive in human cancers. Here, our proteomics profile analysis of HCC tissues with different metastatic capabilities revealed that NDRG1 was correlated with metastasis and recurrence in HCC patients after liver transplantation (LT). Immunohistochemical staining of 143 HCC patients after LT showed that NDRG1-positive expression had poor prognosis, either for shorter disease-free survival or overall survival (P < 0.001), compared with NDRG1-negative expression. Multivariate analysis confirmed NDRG1 as an independent prognostic value (P < 0.001). In addition, in vitro experiments HCC cells with small interfering RNA against NDRG1 significantly suppressed its proliferation, colony formation, invasion and migration ability. Microarray analysis revealed that NDRG1 modulated the expression of genes associated with transmembrane transporter activity, chemoattractant activity, immune response, cell adhesion and cell proliferation process. Taken together, these results suggested that NDRG1 was an important molecule in controlling HCC metastasis and thus suggested as a novel biomarker for predicting HCC recurrence after LT.

Long non‑coding RNA PVT1 is associated with tumor progression and predicts recurrence in hepatocellular carcinoma patients

PVT1, which maps to chromosome 8q24, is a copy number amplification-associated long non-coding RNA. Overexpression of PVT1 is a powerful predictor of tumor progression and patient survival in a diverse range of cancer types. However, the association between PVT1 and hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to examine the expression pattern of PVT1, and its clinical significance in HCC. Between 2003 and 2012, reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of PVT1 in two independent cohorts: Cohort one, 58 HCC resection samples; and cohort 2, 214 HCC transplant samples. Additionally, the correlation between PVT1 expression levels and clinical parameters and outcomes was analyzed. The relative expression levels of PVT1 were significantly higher in cancerous tissues compared with the corresponding non-cancerous tissues (cohort one, P=0.0016; cohort two, P=0.0274). Furthermore, overexpression of PVT1 was associated with a higher serum α-fetoprotein expression level (P=0.011) and a higher recurrence rate (P=0.004). Kaplan-Meier analysis indicated that the patients with high PVT1 expression exhibited poor recurrence-free survival (P=0.021), and multivariate analysis demonstrated that high levels of PVT1 expression are an independent predictor for HCC recurrence (P=0.042; hazard ratio, 1.653). Thus, the high expression levels of PVT1 in HCC may serve as a novel biomarker for predicting tumor recurrence in HCC patients, and as a potential therapeutic target.

MiR-126-3p suppresses tumor metastasis and angiogenesis of hepatocellular carcinoma by targeting LRP6 and PIK3R2

BackgroundThe deregulation of microRNAs has been reported to play a pivotal role in hepatocellular carcinoma (HCC). MiR-126-3p has been reported to be associated with poor prognosis in HCC. However the underlying mechanism of miR-126-3p in HCC remains unclear.MethodsThe expression levels of miR-126-3p in HCC tissues and cells were detected by RT-PCR. Transwell assay and capillary tube formation assay were applied to assess the metastasis and angiogenesis in vitro. Nude mice subcutaneous tumor model was used to perform in vivo study. Dual- luciferase reporter assay was conducted to confirm the direct binding of miR-126-3p and target genes. The changes of biomarker protein levels were examined by western blot and Immunohistochemistry.ResultsWe observed that the miR-126-3p expression levels in HCC tissues and cells were significantly down-regulated. Through gain- and loss- of function studies, we showed that miR-126-3p dramatically inhibited HCC cells from migrating and invading extracellular matrix gel and suppressed capillary tube formation of endothelial cells in vitro. Furthermore, overexpression of miR-126-3p significantly reduced the volume of tumor and microvessel density in vivo. LRP6 and PIK3R2 were identified as targets of miR-126-3p. Silencing LRP6 and PIK3R2 had similar effects of miR-126-3p restoration on metastasis and angiogenesis individually in HCC cells. Furthermore, the miR-126-3p level was inversely correlated with LRP6 and PIK3R2 in HCC tissues. In addition, the rescue experiments indicated that the metastasis and angiogenesis functions of miR-126-3p were mediated by LRP6 and PIK3R2.ConclusionOur results demonstrates that deregulation of miR-126-3p contributes to metastasis and angiogenesis in HCC. The restoration of miR-126-3p expression may be a promising strategy for HCC therapy.

MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling.

Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

The Up-Regulation of Histone Deacetylase 8 Promotes Proliferation and Inhibits Apoptosis in Hepatocellular Carcinoma

BackgroundHistone deacetylase 8 (HDAC8), a member of class I HDACs, has been reported to be involved in transcriptional regulation, cell cycle progression, and developmental events, and several studies have shown that HDAC8 plays a critical role in tumorigenesis. However, the expression level and the potential role of HDAC8 in hepatocellular carcinoma (HCC) remain unclear.AimThe purpose of this study was to investigate protein expression of HDAC8 in HCC tissues and the effects of HDAC8 knockdown on the proliferation and apoptosis of liver cancer cells, and to explore the possible mechanisms.MethodsFirst, we used quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemical staining, and western blot to examine the mRNA and protein expression of HDAC8 in HCC cell lines and tissues. Then, we assessed the correlation between clinicopathological parameters and the protein expression of HDAC8. Furthermore, we employed the interfering RNA method to explore the potential role of HDAC8 in HCC progression in vitro.ResultsOur results showed that expression of HDAC8 was significantly up-regulated both in HCC cell lines and tumor tissues compared to human normal liver cell line LO2 and corresponding non-tumor tissues. Moreover, we found that HDAC8 knockdown could dramatically inhibit HCC cell proliferation and enhance the apoptosis rate in vitro. Western blot revealed that intrinsic apoptotic pathway proteins, including BAX, BAD, and BAK, were elevated after HDAC8 knockdown. The cleavage of caspase-3 and PARP, which are downstream of intrinsic apoptotic pathway, were also enhanced. In addition, suppression of HDAC8 also elevated the expression of p53 and acetylation of p53 at Lys382, whereas the acetylation of p53 at Lys373 did not change.ConclusionsOur study revealed that HDAC8 was overexpressed in HCC. HDAC8 knockdown suppresses tumor growth and enhances apoptosis in HCC via elevating the expression of p53 and acetylation of p53 at Lys382. HDAC8 might serve as a potential therapeutic target in HCC.

Prohibitin-2 promotes hepatocellular carcinoma malignancy progression in hypoxia based on a label-free quantitative proteomics strategy

The rapid growth of hepatocellular carcinoma (HCC) leading to tumor hypoxia is a common pathological phenomenon. Meanwhile, tumor hypoxia can promote a change in the biological properties of tumor cells. It may enhance the survival of tumor cells under stress conditions, resulting in resistance to apoptosis and angiogenesis. The moleculars that could modulate the malignant phenotypes of HCC cells remain largely unknown. Based on label‐free quantitative proteomic data, we found a significant upregulation of prohibitin‐2 (PHB2) in HCC tissues. Treatment of hepatoma cells with small interfering RNAs against PHB2 suppressed cell growth and colony formation, led to G1 phase arrest and sensitized HCC cells to apoptosis. Moreover, inhibition of PHB2 expression dramatically repressed the ability of HCC cells to adapt to hypoxic microenvironments and resist chemotherapy‐induced apoptosis. Thus, PHB2 in HCC supports the development and progression of hepatocellular malignancy to hypoxia, and implicates the potential antagonist function of PHB2 in transarterial chemoembolization treatment.

Interleukin-35: the future of hyperimmune-related diseases?

Interleukin (IL-35) is a newly identified heterodimeric cytokine belonging to the IL-12 family. It contains Epstein-Barr virus-induced gene 3 subunit and IL-27 p35 subunit. Although its receptor and signaling pathway are not clear, we presumed that its receptor is composed by two chains that might be similar to those receptors of IL-12, IL-23, and IL-27. We also believe that the signal transducer activator of transcription family members is involved in its signaling pathway. It was reported that IL-35 could suppress Teff cell proliferation and Th17 development. It was considered to have a potential therapeutic effect against immune diseases. In our perspective, the finding of IL-35 is of great significance, since it can regulate T cells, which is an important therapeutic target of immunological disorders. IL-35 would promote the development of different kinds of vaccines, even vaccine for special cancer, and be promising to cure autoimmune and inflammatory diseases.

Tumor–stroma ratio is a prognostic factor for survival in hepatocellular carcinoma patients after liver resection or transplantation

BACKGROUND The stromal compartment in several organs seems to play an important role in the initiation, growth, and progression of certain neoplasms. The tumor-stroma ratio (TSR) has been found to be an independent factor for prognosis of several types of carcinomas, but the effect of the TSR on hepatocellular carcinoma (HCC) has not been explored yet. The objective of the study is to evaluate the prognostic importance of TSR in HCC patients after liver resection or transplantation. METHODS A total of 300 patients with HCC who underwent liver resection or transplantation were included in this study. TSR was assessed on hematoxylin and eosin-stained sections by 2 independent investigators. Patients were divided into 2 groups: a stroma-rich group (stroma ≥ 50%) and a stroma-poor group (stroma < 50%). Chi-square test, Kaplan-Meier method, and Cox univariable and multivariable regression were used in to analyze the data. RESULTS Among the post liver resection patients, the TSR was associated with overall survival (OS) in univariate and multivariate analyses (hazard ratio [HR], 4.35 [95% CI, 2.54-7.47] and HR, 2.55 [95% CI, 1.44-4.52], respectively). Among the post liver transplant patients, the TSR was also associated with OS in univariate and multivariate analyses (HR, 2.92 [95% CI, 1.63-5.23] and HR, 2.76 [95% CI, 1.47-4.85], respectively), and TSR with recurrence-free survival (RFS) in univariate and multivariate analyses (HR, 2.63 [95% CI, 1.42-4.86] and HR, 1.93 [95% CI, 1.03-3.62], respectively). Patients with stroma-poor tumor and who were within the Milan criteria or the UCSF criteria had a better in OS and RFS. CONCLUSION We show for the first time that TSR is an independent prognostic factor for HCC patients after liver resection or transplantation. TSR may enable better identification of patients at risk for recurrence in HCC patients after curative treatment and may aid in patient management and development of individualized medicine for treatment of HCC.

Romidepsin induces G2/M phase arrest via Erk/cdc25C/cdc2/cyclinB pathway and apoptosis induction through JNK/c-Jun/caspase3 pathway in hepatocellular carcinoma cells

Graphical abstract The pathways for Romidepsin‐induced apoptosis and cell cycle arrest in HCC cells. Figure. No Caption available. ABSTRACT The aim of the study is to demonstrate the effect of Romidepsin in hepatocellular carcinoma (HCC) by inducing G2/M phase arrest via Erk/cdc25C/cdc2/cyclinB pathway and apoptosis through JNK/c‐Jun/caspase3 pathway in vitro and in vivo. Human HCC cell lines were cultured with Romidepsin and DMSO (negative control) and 5‐fluorouracil (positive control). Then the cells’ viability and apoptosis were determined by cell proliferation assay and flow cytometry. Protein concentrations and expression changes were measured by Western blot. Subsequently, Huh7 cells were subcutaneously inoculated into the nude mice, which were employed to further probe the tumor‐suppressive effect of Romidepsin in vivo. Romidepsin treatment led to a time‐ and dose‐dependent induction of cell cycle arrest in the G2/M phase and apoptosis. G2/M phase arrest inhibited the proliferation of HCC cells by alterations in p21/cdc25C/cdc2/cyclinB proteins. Increased concentrations of Erk and JNK phosphorylations were observed in a dose‐dependent manner in the Romidepsin group, but p38 phosphorylation was not affected. G2/M phase arrest and the apoptosis of HCC cells induced by Romidepsin were mediated by the activation of Erk/MAPK pathways and JNK/MAPK pathways. The tumor size was significantly larger in the negative control group compared to Romidepsin group and no significant loss in body weight was observed in the Romidepsin group. Our findings offer proof‐of‐concept for use of Romidepsin as a novel class of chemotherapy in the treatment of HCC.