Downstream targets of heterogeneous nuclear ribonucleoprotein A2 mediate cell proliferation.
ABSTRACT Over-expression of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is regarded as an early marker for several cancers. This protein is associated with proto-oncogenes and tumor suppressor genes and has itself been described as a proto-oncogene. Our earlier experiments drew a connection between hnRNP A2/B1 levels and cell proliferation and raised the possibility that this protein contributes to the uncontrolled cell division that characterizes cancer. Limited knowledge of the downstream targets of hnRNP A2/B1 has, however, precluded a clear understanding of their roles in cancer cell growth. To define the pathways in which this protein acts we have now carried out microarray experiments with total RNA from Colo16 epithelial cells transfected with an shRNA that markedly suppresses hnRNP A2/B1 expression. The microarray data identified 123 genes, among 22 283 human gene probe sets, with altered expression levels in hnRNP A2/B1-depleted cells. Ontological analysis showed that many of these downstream targets are involved in regulation of the cell cycle and cell proliferation and that this group of proteins is significantly over-represented amongst the affected proteins. The changes detected in the microarray experiments were confirmed by real-time PCR for a subset of proliferation-related genes. Immunoprecipitation-RT-PCR demonstrated that hnRNP A2/B1 formed complexes with the transcripts of many of the verified downstream genes, suggesting that hnRNP A2/B1 contributes to the regulation of these genes. These results reinforce the conclusion that hnRNP A2/B1 is associated with cellular processes that affect the cell cycle and proliferation.
Article: Up-regulation and subcellular localization of hnRNP A2/B1 in the development of hepatocellular carcinoma.[show abstract] [hide abstract]
ABSTRACT: Hepatocellular carcinoma (HCC) is one of the world's leading causes of death among cancer patients. It is important to find a new biomarker that diagnoses HCC and monitors its treatment. In our previous work, we screened a single-chain antibody (scFv) N14, which could specifically recognize human HepG2 HCC cells but not human non-cancerous liver LO2 cells. However, the antigen it recognized in the cells remained unknown. Recombinant scFv N14 antibody was expressed as an active antibody. Using this antibody with a combination of immunological and proteomic approaches, we identified the antigen of scFv N14 antibody as the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). The expression of hnRNP A2/B1 in HCC cells was then investigated by semi-quantitative RT-PCR and immunohistochemistry. We found that the up-regulation of hnRNP A2/B1 was measured at both transcriptional and translational levels in rat HCC cells but not in rat hepatic cells. We also found that in various human hepatic tissues, hnRNP A2/B1 was highly expressed in both human hepatitis virus positive liver tissues and human HCC tissues but not in normal liver tissues. Interestingly, we observed that the localization of hnRNP A2/B1 in HCC cells was altered during the development of HCC. In human hepatitis virus infected tissues hnRNP A2/B1 resides exclusively in the nuclei of hepatocytes. However, when the HCC progressed from a well differentiated to a poorly differentiated stage, hnRNP A2/B1 was increasingly localized in the cytoplasm. In contrast, the HCC tissues with hnRNP A2/B1 highly expressed in the nucleus decreased. This work is the first to show that hnRNP A2/B1 is the antigen specifically recognized by the scFv N14 antibody in HCC cells. The over-expression of hnRNP A2/B1 was confirmed in cultured human and rat HCC cell lines, human virus related hepatitis liver tissues and human HCC tissues. The increased localization of hnRNP A2/B1 in the cytoplasm of HCC cells was revealed during the dedifferentiation of hepatocellular carcinoma. Therefore, we suggest that the increased expression and cytoplasmic localization of hnRNP A2/B1 can be used as a diagnostic biomarker to assess the risk of human liver cancer.BMC Cancer 01/2010; 10:356. · 3.01 Impact Factor
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ABSTRACT: Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been reported to be overexpressed in lung cancer and in other cancers such as breast, pancreas, and liver. However, a mechanism linking hnRNP A2/B1 overexpression and progression to cancer has not yet been definitively established. To elucidate this mechanism, we have silenced hnRNPA2/B1 mRNA in non-small-cell lung cancer cell lines A549, H1703, and H358. These cell lines present different levels of expression of epithelial-to-mesenchymal transition (EMT) markers such as E-cadherin, fibronectin, and vimentin. Microarray expression analysis was performed to evaluate the effect of silencing hnRNP A2/B1 in A549 cells. We identified a list of target genes, affected by silencing of hnRNP A2/B1, that are involved in regulation of migration, proliferation, survival, and apoptosis. Silencing hnRNP A2/B1 induced formation of cell clusters and increased proliferation. In the anchorage-independent assay, silencing hnRNP A2/B1 increased colony formation by 794% in A549 and 174% in H1703 compared with a 25% increase in proliferation, in both cell lines, in a two-dimensional proliferation assay. Silencing hnRNP A2/B1 decreased migration in intermediate cell line A549 and mesenchymal cell line H1703; however, no changes in proliferation were observed in epithelial cell line H358. Silencing hnRNP A2/B1 in A549 and H1703 cells correlated with an increase of E-cadherin expression and downregulation of the E-cadherin inhibitors Twist1 and Snai1. These data suggest that expression of hnRNP A2/B1 may play a role in EMT, in nonepithelial lung cancer cell lines A549 and H1703, through the regulation of E-cadherin expression.Cancer Research 09/2010; 70(18):7137-47. · 7.86 Impact Factor