Down-regulation of G protein-coupled receptor 137 by RNA interference inhibits cell growth of two hepatoma cell lines.

G protein-coupled receptors (GPCRs) are important signal transduction mediators and pharmacological therapeutic targets. G protein-coupled receptor 137 (GPR137) was initially reported as a novel orphan GPCR around 10 years ago. Some orphan GPCRs have been implicated in cancer cell proliferation and migration. The aim of this study is to investigate the role of GPR137 in hepatocellular carcinoma (HCC). GPR137 is widely expressed in several human HCC cell lines, as determined by real-time PCR. We then applied lentivirus mediated RNA interference (RNAi) to knock down GPR137 expression in two HCC cell lines HepG2 and Bel7404. Depletion of GPR137 remarkably inhibited cell proliferation and colony formation capacity. Knockdown of GPR137 in HepG2 cells led to cell cycle arrest at G0/G1 phase and G2/M phase, and induced cell apoptosis, as determined by flow cytometry analysis, which contributed to cell growth inhibition. Our findings suggested that GPR137 could facilitate HCC cell proliferation and thus promote hepatocarcinogenesis.

Suppression of CUGBP1 inhibits growth of hepatocellular carcinoma cells.

PURPOSE: The multifunctional RNA-binding protein, CUGBP1, regulates splicing, stability and translation of mRNAs. Previous studies have shown that CUGBP1 is expressed at high levels in the liver, although its role in hepatocellular carcinoma is unknown. Our aim was to determine if CUGBP1 could regulate hepatocellular carcinoma growth. METHODS: Expression levels of CUGBP1 were analyzed in 70 hepatic carcinoma and 20 normal hepatic tissue samples by immunohistochemistry (IHC). Using lentivirus-mediated short hairpin RNA (shRNA), CUGBP1 expression in human hepatocellular carcinoma HepG2 cells was knocked-down. The effect of CUGBP1 on hepatic cancer cell growth was investigated. RESULTS: CUGBP1 was expressed in 85.7% hepatocellular carcinoma specimens compared with 50% in normal liver specimens. CUGBP1 silencing remarkably decreased the proliferation of HepG2 cells, as determined by MTT assay. Flow cytometry analysis showed that knock-down of CUGBP1 led to G0/G1 phase cell cycle arrest, accompanied by sub-G1 accumulation. Moreover, depletion of CUGBP1 resulted in downregulation of cyclin B1 and upregulation of cyclin D1. CONCLUSION: These results suggest that CUGBP1 is essential for the growth of hepatocellular carcinoma cells. Knockdown of CUGBP1 might be a potential therapeutic approach for human hepatocellular carcinoma.

Serine protease HtrA1 expression in human hepatocellular carcinoma.

BACKGROUND: HtrA1, a serine protease, is down-regulated in various human solid tumors. Overexpression of HtrA1 in human cancer cells inhibits cell growth and proliferation in vitro and in vivo, suggesting its possible role as a tumor suppressor. METHODS: Immunohistochemistry was used to determine the expression of HtrA1 in 50 hepatocellular carcinoma specimens and adjacent liver tissues. The correlation between the expression of HtrA1 and the clinico-pathologic data were analyzed. RESULTS: The levels of HtrA1 were lower in tumor tissues than in their adjacent liver tissues. Moreover, an inverse relationship was found between HtrA1 expression and the differentiation of hepatocellular carcinoma. Loss of HtrA1 was more frequently found in tumors in Edmondson grade III-IV, especially in those with venous invasion, compared to tumors in Edmondson grade I-II. Most importantly, patients with higher HtrA1 expression had a better survival rate. CONCLUSION: All these data suggest an important role of HtrA1 in hepatocellular carcinoma development and progression, which may be a new target for its treatment.

IGF2BP3 and miR191-5p synergistically increase HCC cell invasiveness by altering ZO-1 expression.

Early studies have indicated that insulin-like growth factor II mRNA binding protein 3 (IGF2BP3/IMP3) may affect the progression of hepatocellular carcinoma (HCC); however, the detailed underlying mechanisms, particularly its linkage to tight junction protein-mediated cell invasion, remain unclear. The present study revealed that IGF2BP3 increased HCC cell invasiveness by suppressing zonula occludens-1 (ZO-1) expression, via direct binding to the 3' untranslated region (3'-UTR). Analysis of the molecular mechanisms demonstrated that IGF2BP3 binds to the overlapping targets of IGF2BP3-RNA cross-linkage and microRNA (miR)191-5p targeting sites, and promotes the formation of an miR191-5p-induced RNA-induced silencing complex. The knockdown of IGF2BP3 or the addition of a miR-191-5p inhibitor decreased cellular invasiveness and increased ZO-1 expression. Analysis of the human HCC database also confirmed the association between IGF2BP3 and HCC progression. Collectively, these preclinical findings suggest that IGF2BP3 increases HCC cell invasiveness by promoting the miR191-5p-induced suppression of ZO-1 signaling. This newly identified signaling effect on small molecule targeting may aid in the development of novel strategies with which to inhibit HCC progression more effectively.

lncRNA PCBP1-AS1 Aggravates the Progression of Hepatocellular Carcinoma via Regulating PCBP1/PRL-3/AKT Pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is a very belligerent primary liver tumor with high metastatic potential. Aberrant expression of lncRNAs drives tumorous invasion and metastasis. Whether lncRNAs engage mechanisms of liver cancer metastasis remains largely unexplored. PATIENTS AND METHODS: We collected HCC tissues from the tumors and their adjacent normal samples in the Chinese population and analyzed the levels of lncRNAs by microarray analysis. The gain- and loss-of-function analysis demonstrated that PCBP1-AS1 accelerated tumorous growth and metastasis in vivo and in vitro. Moreover, we used RNA-pulldown assay to show that PCBP1-AS1 physically interacted with polyC-RNA-binding protein 1 (PCBP1); meanwhile, PCBP1-AS1 was indeed detected in RIP with the PCBP1 antibody. Mechanistically, we first explored the relationship between PCBP1-AS1 and PCBP1 in HCC cell lines. RESULTS: Here we show that PCBP1-AS1, identified by microarray analysis on pre- and post-operative HCC plasma specimens, was highly expressed in human HCC, clinically verified as a prometastatic factor and markedly associated with poor prognosis in patients with hepatocellular carcinoma. PCBP1-AS1 was negatively related with PCBP1 at the messenger RNA and protein expression levels. PCBP1-AS1 triggered PRL-3 and AKT in HCC tumor cells. Additionally, the double knockout of PCBP1 and PCBP1-AS1 abolished the PCBP1-AS1-induced PRL-3-AKT signalling pathway activation. CONCLUSION: The upregulation of PCBP1-AS1 enhances proliferation and metastasis in HCC, thus regulating the PCBP1-PRL-3-AKT signalling pathway.