The Biological Role And Clinical Significance Of HnRNP AB In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies worldwide. The prognosis of HCC remains dismal due to the high rate of tumor recurrence and metastasis after curative resection. The molecular pathogenesis and complicated signal transduction pathways involved in HCC are not fully understood. Although several molecular markers that may define the risk of recurrence and the metastatic potential of HCC have been proposed, none have been approved for routine clinical use. Considering the high rate of frequent tumor recurrence and metastasis after curative resection, it is critical to understand the mechanisms behind metastasis and identify new targets for therapy.Heterogeneous ribonucleoproteins (hnRNPs) are a set of nuclear proteins that bind to nascent transcripts produced by RNA polymerase II and have a wide range of roles in DNA repair, telomere biogenesis, cell signaling, and regulating gene expression at both transcriptional and translational levels. Emerging evidence suggests that some hnRNPs (including hnRNP A2B1, hnRNP M4, and hnRNP K) are essential factors in tumor development and progression.HnRNP AB is ubiquitously expressed and involved in fundamental processes such as RNA metabolism, transcription factor activity and translational control. HnRNP AB contains2evolutionarily conserved RNA recognition motifs that mediate DNA/RNA binding and nuclear shuttling domain, and mediates the stability of proinflammatory mRNAs by binding to the AU-rich element. It is involved in nuclear-cytoplasmic transport of mRNA as a complex with actin. HnRNP AB has also been described as a transcriptional regulator named by CBF-A in rat, regulating the Ha-ras response element and the spi2gene. We have found that hnRNPA1, another subfamily hnRNP AB, has the potential to be a novel marker for outcome of HCC patients in a previous study. HnRNP AB is highly conserved and in rat it is referred to as CBF-A, recently identified as EMT inducer, which engages the genes encoding the EMT proteome by formation of the CBF-A/KAP-1/FTS-1 complex.A few reports have directly addressed the biological role of hnRNP AB in human tumorigenesis and tumor progression. The elevated levels of this hnRNP in some mammary tumors and solid tumor metastasis together with reports of EMT with metastatic conversion and its role in the transactivation of the rat Ha-ras oncogene strongly suggests a possible role implicated in tumorigenesis and tumor progression. Thus, the prognostic value of hnRNP AB in HCC, and its role in HCC carcinogenesis and metastasis, needs to be determined.In our previous study, we found that hnRNP AB is highly expressed in HCC tissues compared with peritumoral tissues, and this result suggested that hnRNP AB may play an important role in HCC. Here, we establish that hnRNP AB level is elevated in high-metastatic HCC cells and patients with HCC recurrence, and correlate with reduced expression of E-cadherin and poor patient survival. Enforced expression of hnRNP AB in noninvasive HCC cells induced an epithelial mesenchymal transition (EMT) accompanied by enhanced metastatic potential. HnRNP AB directly activates sanil1implicated in downregulation of epithelial and activation of mesenchymal genes. Importantly, we showed high expression of hnRNP AB/Kap1together was characterized by accumulation of Snail in HCC tissues.Taken together, our current findings support a key role of hnRNP AB/snail1/E-cadherin axis in the regulation of malignant HCC cancer phenotype. Hence, translational regulation by hnRNP AB is a restriction point enabling coordinated expression of a network of EMT-inducing transcription factors, and may be a good target candidate for suppressing EMT under pathologic circumstances. Part One HnRNP AB overexpression is correlated significantly with HCC metastasis.Objective:To identify the hnRNP AB expression in HCC cell lines with different metastatic potential and HCC samples, and the relationship between hnRNP AB expression and HCC metastasis.Methods:HnRNP AB expression in HCC samples and HCC cell lines was analyzed by qRT-PCR, western blot and immunohistochemical (IHC) staining. And based on knockdown or overexpression experiments in vitro and in vivo, we assessed the role of hnRNP AB in HCC metastasis.Results:We found that hnRNP AB was significantly overexpressed in tumors when compared to corresponding peritumoral tissues (p<0.01). Samples from patients with tumor recurrences (57/94) had higher levels of hnRNP AB than those from patients without recurrences (37/94; p<0.01). qRT-PCR showed that hnRNP AB expression was significantly increased in the highly metastatic cell lines (MHCC97H and HCCLM3) relative to the poorly metastatic HCC cell lines (HepG2and PLC/PRF/5;p<0.01). Stable overexpression of hnRNP AB in HepG2cells and successful shRNA-mediated knockdown of hnRNP AB expression in HCCLM3cells were confirmed by qRT-PCR and western blot analyses (p<0.01). In vitro invasion assays showed that the number of invasive hnRNP AB shRNA-treated HCCLM3cells was significantly decreased when compared with the control and less metastatic lesions were found in lung compare with untreated group in vivo (p<0.001), while the number of invasive hnRNP AB cDNA-transfected HepG2cells was significantly higher than that of the control cells and more metastatic lesions were found in lung compare with untreated group in vivo (p<0.001).Conclusion:These data indicates that hnRNP AB expression is positively correlated with the metastatic potential of HCC cells, and hnRNP AB is necessary to confer this metastatic phenotype in HCC. Part Two HnRNP AB overexpression is correlated significantly with EMT and induces EMT in vitro.Objective:To study the mechanism of hnRNP AB in HCC metastasis.Methods:The expression of E-cadherin, N-cadherin and vimentin were detected by qRT-PCR and western blot in HCC cell lines and HCC samples. Then, the relationships between hnRNP AB and EMT makers were assessed.Results:The elevated level of hnRNP AB in HCC cell lines was correlated with the loss of E-cadherin and induction of N-cadherin, and vimentin at both mRNA and protein levels. We found an interesting phenomenon that the HepG2cells after transfected with hnRNP AB cDNA were observed to exhibit mesenchymal or a spindle-like morphology, and to express high levels of N-cadherin and low levels of E-cadherin, indicating that these cells were undergoing the EMT process. Substantial reduction of hnRNP AB protein level in HCCLM3was observed after transfection shRNA-hnRNP AB, with a concomitant reduction of mesenchymal markers (N-cadherin and vimentin), and up-regulation of epithelial marker (E-cadherin). This inverse association between hnRNP AB and E-cadherin expression in HCC samples was also confirmed by qRT-PCR, immunoblotting and IHC. In addition, high level expression of hnRNP AB was also associated with an elevated level of the mesenchymal marker, vimentin; and hnRNPA1has no relation with EMT hallmarks.Conclusion:Such a specific expression pattern suggests a role for hnRNP AB in EMT induction of HCC. Part Three HnRNP AB regulated snail expression to inhibit E-cadherin in HCC. Objective:To study the mechanism of hnRNP AB involved in EMT.Methods:We performed a Chip combined with a microarray (Chip on chip) in HCCLM3cell to screem hnRNP AB target genes and corfirmed by Chip-PCR. And then, we assessed the relationships between hnRNP AB and target genes in HCC cell lines and HCC samples.Results:Using Chip-chip analysis, we found that the snail gene was prone to be actually binded by hnRNP AB, while there was no significantly different affinity with E-cadherin and vimentin in promoter; and then validate by chip-PCR in HCCLM3. The snail up-regulation in HCC cells correlated with hnRNP AB expression as measured by qRT-PCR and western blots. Immunoblotting showed that the cellular level of snail was significantly increased after up-regulation of hnRNP AB in the low metastastic HCC cells. And knock-down of hnRNP AB in the high metastastic HCC cells resulted in downregulation of snail expression. We silenced snail expression in HepG2-hnRNP AB cells (high hnRNP AB and low E-cadherin expression) by using snail-specific shRNA. Inhibition of snail expression resulted in increased E-cadherin expression compared with HepG2-hnRNP AB cells, despite stable hnRNP AB overexpression. To further validate, we overexpressed snail cDNA in HCCLM3-shRNA cells (hnRNP AB expression is stably repressed, snail expression is low, and E-cadherin expression is high). Forced snail expression in HCCLM3-shRNA cells resulted in decreased E-cadherin expression compared with HCCLM3-shRNA-hnRNP AB cells, despite the repression of hnRNP AB expression. The positive relationship between hnRNP AB and snail was also confirmed in HCC samples, and the causal role of hnRNP AB expression snail-dependent changes in E-cadherin expression was found in the xenografts by IHC.Conclusion:HnRNP AB mediates snail1-dependent regulation of E-Cadherin expression, and subsequent EMT. Part Four High expression of hnRNP AB/Kap1together promotes metastasis of HCC cells through up-regulation of snail and with poor prognosis in HCC patients.Objective:To further understand the molecular mechanism by which hnRNP AB regulated transcription of the snail gene, and to investigate the clinical significance in hepatocellular carcinoma.Methods:We carried out co-immunoprecipitation with the extracts made from HCCLM3cells using hnRNP AB antibody, and associated proteins were identified by mass spectrometry and was identified by immunoblotting.323cases of HCC tissue were accumulated and made into tissue microarrays. Expression of hnRNP AB/Kap1proteins was detected by IHC staining, and then the clinical significance of hnRNP AB/Kap1expression was analyzed by SPSS16.0.Results:We identified kap1as molecular partners for hnRNP AB in HCCLM3cells and HepG2cells as well. We showed high expression of hnRNP AB/kap1together was characterized by accumulation of snail1in HCC cells. Multivariate analyses revealed that hnRNP AB alone or in combination with kap1were independent prognostic indicators for overall survival and time to recurrence.Conclusion:The expression levels of hnRNP AB alone or in combination with kap1in HCC patients are important because they provide not only a predictor for HCC prognosis but also a therapeutic target for future studies. Novelty:1. Our findings firstly support a key role of hnRNP AB/snail/E-cadherin axis in the regulation of malignant HCC cancer phenotype.2. The translational regulation by hnRNP AB is a restriction point enabling coordinated expression of a network of EMT-inducing transcription factors, and may be a good target candidate for suppressing EMT under pathologic circumstances.Potential merits for clinical application:HnRNP AB/Kap1together may be a potential and valuable biomarker and therapeutic target in HCC.