Chengli Du

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.

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.

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.

BAG3 regulates epithelial–mesenchymal transition and angiogenesis in human hepatocellular carcinoma

Bcl2-associated athanogene 3 (BAG3) protein is a co-chaperone of heat-shock protein (Hsp) 70 and may regulate major physiological and pathophysiological processes. However, few reports have examined the role of BAG3 in human hepatocellular carcinoma (HCC). In this study, we show that BAG3 regulates epithelial–mesenchymal transition (EMT) and angiogenesis in HCC. BAG3 was overexpressed in HCC tissues and cell lines. BAG3 knockdown resulted in reduction in migration and invasion of HCC cells, which was linked to reversion of EMT by increasing E-cadherin expression and decreasing N-cadherin, vimentin and slug expression, as well as suppressing matrix metalloproteinase 2 (MMP-2) expression. In a xenograft tumorigenicity model, BAG3 knockdown effectively inhibited tumor growth and metastasis through reduction in CD34 and VEGF expression and reversal of the EMT pathway. In conclusion, BAG3 is associated with the invasiveness and angiogenesis in HCC, and the BAG3 gene may be a novel therapeutic approach against HCC.

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.

Downregulation of HDAC6 promotes angiogenesis in hepatocellular carcinoma cells and predicts poor prognosis in liver transplantation patients.

HDAC6, a member of histone deacetylation family, is reported to play critical roles in transcription regulation, cell cycle progression, and cancer development. However, the expression status and significance of HDAC6 in hepatocellular carcinoma (HCC) is still controversial, and little is known about the role of HDAC6 in HCC angiogenesis and the correlation between expression of HDAC6 and prognosis of HCC patients with liver transplantation (LT). Our experiments showed HDAC6 was significantly downregulated in HCC tissues (P = 0.025), and low expression of HDAC6 was found to be closely associated with recurrence (P = 0.006), and could predict poor recurrence‐free survival (P = 0.047) for HCC patients with LT. Moreover, knockdown of HDAC6 could promote HUVEC migration, proliferation, and tube formation in vitro, and suppress HCC cell apoptosis, and promote HCC cell proliferation in hypoxia. Remarkably, knockdown of HDAC6 could significantly up‐regulate the expression of HIF‐1α and VEGFA in vivo and in vitro, which facilitated HIF‐1α mediated angiogenesis in HCC. Further study showed that HDAC6 was down‐regulated under hypoxia in a time dependent manner. Hence, the present findings suggested a role for suppression of HDAC6 in promoting the angiogenesis in HCC by HIF‐1α/VEGFA axis. HDAC6 may serve as a recurrence predictive factor for HCC after LT and pharmacological or genetic activation of HDAC6 could be a novel anti‐angiogenesis approach for HCC therapy.

KCTD11 inhibits growth and metastasis of hepatocellular carcinoma through activating Hippo signaling

A lack of effective prognostic biomarkers and molecular targets is a serious problem in hepatocellular carcinoma. KCTD11, reported as a tumor suppressor, are still not well understood. In this study, KCTD11 was found low-expressed in HCC tissues and cell lines. The HCC patients with low expression of KCTD11 suggested shorter overall survival. We found KCTD11 inhibiting cell proliferation in vitro and tumor growth in vivo, by activating p21 and repressing cycle related proteins. KCTD11 also inhibited cell adhesion by decreasing CTGF and CLDN1. We found CTGF binding COL3A1 in HCCLM3, which might lead to reduction of COL3A1 expression. KCTD11 also inhibited cell migration and invasion in HCC, by repressing MMPs and EMT. We found the tumor suppression function of KCTD11 was at least partly through activating Hippo pathway in HCC. Base on the enhanced Hippo pathway, KCTD11 could activate p21 by stabilizing p53 or promoting the MST1/ GSK3β/p21 signaling in HCC. Overall, these results suggest that KCTD11 works as a tumor suppressor and owns prognostic and therapeutic potentials in HCC.

BAG3 and HIF-1α Coexpression Detected by Immunohistochemistry Correlated with Prognosis in Hepatocellular Carcinoma after Liver Transplantation

Objective. The objective is to determine the effects of BAG3 and HIF-1α expression on the prognosis of HCC patients after liver transplantation. Methods. Samples from 31 patients with HCC receiving liver transplantation were collected for this study. The immunohistochemistry was used to detect the expression of BAG3 and HIF-1α of HCC samples. Results. According to the immunohistochemistry results, BAG3 and HIF-1α staining were significantly associated with tumor TNM stage (P = 0.004, P = 0.012). A significant association between high BAG3/HIF-1α levels and a shorter overall survival was detected, so as the combined BAG3 and HIF-1α analysis. Conclusion. The results suggested that the expression level of BAG3 and HIF-1α is efficient prognostic parameters in patients with HCC after liver transplantation.

Expression and Critical Role of Interleukin Enhancer Binding Factor 2 in Hepatocellular Carcinoma

Interleukin enhancer binding factor 2 (ILF2), a transcription factor, regulates cell growth by inhibiting the stabilization of mRNA. Currently, its role has gained recognition as a factor in the tumorigenic process. However, until now, little has been known about the detailed role ILF2 plays in hepatocellular carcinoma (HCC). In this study, we investigated the expression levels of ILF2 in HCC tissue with Western blot and immunohistochemical assays. To examine the effect of ILF2 on liver cancer cell growth and apoptosis, small interfering RNAs (siRNAs) targeting ILF2 were recombined to create lentiviral overexpression vectors. Our results showed higher expression levels of ILF2 mRNA and ILF2 protein in HCC tissue compared with matched peritumoral tissue. Expression of ILF2 may regulate cell growth and apoptosis in liver cancer cells via regulation of B-cell lymphoma 2 (Bcl-2), Bcl-2 related ovarian killer (Bok), Bcl-2-associated X protein (BAX), and cellular inhibitor of apoptosis 1 (cIAP1). Moreover, we inoculated nude mice with liver cancer cells to investigate the effect of ILF2 on tumorigenesis in vivo. As expected, a rapid growth was observed in cancer cells inoculated with a lentiviral vector coding Flag-ILF2 (Lenti-ILF2) compared with the control cells. Hence, these results promote a better understanding of ILF2’s potential role as a therapeutic target in HCC.