Shaobing Cheng

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.

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.

Over Expression of Long Non-Coding RNA PANDA Promotes Hepatocellular Carcinoma by Inhibiting Senescence Associated Inflammatory Factor IL8

It has been reported that long non-coding RNA PANDA was disregulated in varieties types of tumor, but its expression level and biological role in hepatocellular carcinoma (HCC) remains contradictory. We detected PANDA expression in two independent cohorts (48 HCC patients following liver transplantation and 84 HCC patients following liver resection), and found that PANDA was down-regulated in HCC. Thereafter we explored its function in cancer biology by inversing its low expression. Surprisingly, overexpression of PANDA promoted HCC proliferation and carcinogenesis in vitro and in vivo. Mechanistically, PANDA repressed transcriptional activity of senescence associated inflammatory factor IL8, which leaded to inhibition of cellular senescence. Therefore, our research help to better understand the complex role of PANDA in HCC, and suggest more thoughtful strategies should be applied before it can be treated as a potential therapeutic target.

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.

Downregulation of Peptidylprolyl isomerase A promotes cell death and enhances doxorubicin-induced apoptosis in hepatocellular carcinoma

Peptidylprolyl isomerase A (PPIA) is a peptidyl-prolyl cis-trans isomerase that is known to play a critical role in the development of many human cancers. However, the precise biological function of PPIA in hepatocellular carcinoma (HCC) remains largely unclear. In this study, lentiviral overexpression vectors and small interfering RNA knockdown methods were employed to investigate the biological effects of PPIA in HCC. PPIA levels in HCC tissues and peritumoral tissues were detected by real-time Polymerase Chain Reaction (RT-PCR), Western blotting, and immunohistochemistry. Our results indicate that PPIA levels were significantly higher in the HCC tissues compared to the matched peritumoral tissues. Moreover, PPIA expression was significantly associated with tumor size in these tissues. Interestingly, serum PPIA (sPPIA) levels were significantly higher in healthy controls compared to the HCC patients. Knockdown or overexpression of PPIA was shown to downregulate and upregulate cell growth, respectively. Moreover, PPIA siRNA knockdown appears to promote doxorubicin-induced apoptosis in HCC cells, altering the expression of downstream apoptotic factors. In summary, our results indicate that PPIA may play a pivotal role in HCC by regulating cell growth and could serve as a novel marker and therapeutic molecular target for HCC patients.

TAZ regulates cell proliferation and sensitivity to vitamin D3 in intrahepatic cholangiocarcinoma

The transcriptional coactivator with PDZ binding motif (TAZ) is reported as one of the nuclear effectors of Hippo-related pathways. TAZ is found overexpressed in many primary tumors and could regulate many biological processes. However, little is known about the role of TAZ in Intrahepatic Cholangiocarcinoma (ICC). In this study, we found that TAZ is expressed more in ICC tissues than in peritumoral tissue, and a robust expression of TAZ is correlated with a lower overall survival rate of ICC patients after hepatectomy. TAZ knockdown results in an increase in cell apoptosis, a promotion of cell-cycle arrest and a decrease in tumor size and weight in vivo through an increased expression of p53. Vitamin D3 can also inhibit cell proliferation by promoting p53 expression in ICC cells. A reduction in TAZ can also enhance the sensitivity of tumor cells to vitamin D by regulating the p53/CYP24A1 pathway. In conclusion, TAZ is associated with the proliferation and drug-resistance of ICC cells, and could be a novel therapeutic target for the treatment of ICC.

Genome-wide CRISPR screen reveals SGOL1 as a druggable target of sorafenib-treated hepatocellular carcinoma

The genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen is a powerful tool used to identify therapeutic targets that can be harnessed for cancer treatment. This study aimed to assess the efficacy of genome-wide CRISPR screening to identify druggable genes associated with sorafenib-treated hepatocellular carcinoma (HCC). A genome-scale CRISPR knockout (GeCKO v2) library containing 123,411 single guide RNAs (sgRNAs) was used to identify loss-of-function mutations conferring sorafenib resistance upon HCC cells. Resistance gene screens identified SGOL1 as an indicator of prognosis of patients treated with sorafenib. Of the 19,050 genes tested, the knockout screen identified inhibition of SGOL1 expression as the most-effective genetic suppressor of sorafenib activity. Analysis of the survival of 210 patients with HCC after hepatic resection revealed that high SGOL1 expression shortened overall survival (P = 0.021). Further, matched pairs analysis of the TCGA database revealed that SGOL1 is differentially expressed. When we used a lentivirus Cas9 vector to determine the effect of targeting SGOL1 with a specific sgRNA in HCC cells, we found that SGOL1 expression was efficiently inhibited and that loss of SGOL1 was associated with sorafenib resistance. Further, loss of SGOL1 from HCC cell decreased the cytotoxicity of sorafenib in vivo. We conclude that the CRISPR screen is a powerful tool for therapeutic target analysis of sorafenib treatment and that SGOL1 serves as a druggable target for HCC treated with sorafenib and an indicator of prognosis.