RNA-binding Protein HuR Is Essential For IL-1β-induced MRNA Stability Of Vascular Endothelial Growth Factor-C In Non-small Cell Lung Cancer Cells

BackgroundVascular endothelial growth factor (VEGF)-C, one of VEGF family members expressed in cancer cells as well as infiltrating inflammatory cells, is a potent secreted activator critical for tumor-induced lymphangiogenesis. Exaggerated expression of VEGF-C in primary tumors is implicated in the enhanced lymphatic invasion and metastasis, and may be an important prognostic factor in lung carcinomas. Like VEGF-A, the upregulation of VEGF-C expression is in response to hyponia, cytokines and proinflammatory cytokines such as interlukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α). Overexpressed HER-2/NEU induced the expression of VEGF-C in ovarian and breast cancer cells by p38 MAPK signal and transcriptional factor NF-κB activation cascade. In addition, demethylation of gene is also found to contribute to VEGF-C overexpression in gastric tumors and gastric cancer cells, suggesting transcriptional mechanism is involved in VEGF-C induction. However, the posttranscriptional regulation of VEGF-C is not yet understood. A better understanding of the molecular basis of VEGF-C expression in tumor will be needed to define the factors/mechanisms that regulate VEGF-C.Regulation of mRNA stability translational efficiency is an important component in the control of eukaryotic gene expression. The balance between transcript synthesis and its degradation determines the levels of individual mRNA. Unstable mRNA often contains adenylate-uridylate rich elements (AREs) within the 3'untranslated region (UTR) of mRNAs. The characteristic core sequence is the AUUUA motif, which is involved in the regulation of the mRNA turnover. It has been found that ARE-containing mRNAs encode a large number of proteins involving inflammation, cell proliferation and tumor progression. Dependent on the ARE size and AU content, ARE-containing mRNAs have been clustered into three classes. ARE-mediated mRNA turnover and translation is tightly controlled through complex interactions of RNA and RNA-binding proteins, such as human antigen R (HuR), AU binding factor1 (AUF-1), tristetraproline (TTP) and the members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins. HuR is a ubiquitously expressed RNA-biding factor belonging to the embryonic lethal abnormal vision (ELAV) family of mRNA binding proteins and stabilized ARE-containing mRNAs. In contrast, TTP and AUF-1 have been found to destabilize the transcripts that containing AREs in their 3'UTR. HuR consists of three RNA recognition motifs (RRMs), of which RRM1 acts a nucleo-cytoplasmic shuttling sequence and functions as an adapter protein in the nuclear export of mRNAs. Under various stress conditions including IL-1βor TNF-αstimulation, the predominantly nuclear HuR can translocate to the cytoplasm where it can stabilize the mRNAs and regulation translation.The elevated HuR protein expression has been found in high-grade brain tumors and colon tumors. Cytoplasmic HuR expression was closely related to poor outcomes and higher tumor grade in patients with breast, ovarian and gastric carcinomas. Recently, cytoplasmic immunoreactivity for HuR was found to be an independent factor of lymph node metastasis in non-BRCA1/2 breast cancer group. However, the underlying molecular mechanism has not been well characterized. It is reported that HuR plays a critical role in carcinogenesis and tumor metastasis through inducing angiogenic factors such as VEGF-A, IL-8 and COX-2, and cell cycle regulators such as cyclin A and cyclin B1, proliferation-related genes such as c-fos and c-myc. Their mRNAs exhibit short half-lives due to the presence of AREs in the 3'UTR. However, there are no reports about the association between HuR and VEGF-C in human cancers.Analysis of VEGF-C gene shows that 3'UTR of VEGF-C mRNA contains a high AU content, and contains several AUUUA repeats. The role of these motifs in the regulation of VEGF-C expression in cancer cells has not yet elucidated. Accordingly, the aim of this study was to explore the ability for HuR to interact with the VEGF-C 3'UTR by AREs and the potential to mediate posttranscriptional regulation of VEGF-C gene expression under IL-1βstimulation using lung cancer cell line as a model.ObjectiveTo analyze the expression of HuR and VEGF-C and their significance in NSCLC tumors and cells, and further the potential molecular mechanisms underlying proinflammatory cytokine increases the stability of VEGF-C mRNA via RNA-binding protein HuR.Methods1. HuR and VEGF-C expression levels in 81 NSCLC tissues and 15 control benign pulmonary lesion tissues were detected by immunohistochemistry method (SABC method).2. I1-βwas used to stimulate Lewis lung caicinoma (LLC) cells and the expression of cytoplasmic and secreted VEGF-C, and mRNA were determined by Western blotting, ELISA and quantitative RT-PCR assay, respectively.3. Actinomycin D (ActD) chase-phase experiments were performed to analyze the decay kinetics of VEGF-C mRNA in LLC cells with or without IL-1βstimulation.4. An interference plasmid, pGenesil-siHuR, for a short hairpin siRNA directed HuR was constructed using the pGenesil-1 vector, and the effect of HuR knockdown through transfection the pGenesil-siHuR to LLC cells on the induced stabilization and expression of VEGF-C was examined.5. A vector ovexpressing HuR pEGFP-HuR was constructed and transfected to LLC cells, and the effect of HuR overexpression on VEGF-C expression in vitro was investigated.6. Tranwell experiment and MTT assay was used to determine whether HuR overexpression affects cell migration of LLC cells and cell growth, respectively.7. The tumor models of LLC overexpressing HuR were established via subcutaneous injection of LLC cells in the dorsal site of the right ear of C57BL/6 mice, and the effect of HuR overexpression on VEGF-C expression, lymphangiogenesis, lymph node metastasis and lung metastasis in vivo was investigated.8. Immunofluorence assay and confocal microscopy was used to examine the nucleo-cytoplasmic shuttling of HuR in lung cancer cells in the present or absent of IL-1β.9. Luciferase report vectors were constructed containing the different 3'UTR fragments of mouse VEGF-C gene or its coding region as a control.10. Dual-Luciferase reporter assay system and RT-PCR were used to determine whether VEGF-C 3'UTR influences the luciferase activity and mRNA exression, and luciferase mRNA stability.Results1. In NSCLC tissues, positive rate of nuclear HuR, cytoplasmic HuR and VEGF-C was 45.7% (37/81), 82.7% (67/81) and 70.4% (57/81), respectively. There was a significant difference in positive expression of HuR or VEGF-C between NSCLC and lung benign lesion tissues (P < 0.01). A significant association between increased VEGF-C expression and lymph node metastasis (P < 0.01) as well as tumor stages (P < 0.01), but not sex, age, histological classification and differentiation stages (P > 0.05) was observed. The expression of cytoplasmic HuR was closely related to pTNM stages and lymph node metastasis (P < 0.05), and differentiation stages (P < 0.05), but not correlated with sex, age, histological classification and (P > 0.05). Furthermore, cytoplasmic immunoreactivity for HuR protein (P < 0.05) but not nuclear HuR expression (P > 0.05) was closely associated with high VEGF-C level in NSCLC.2. VEGF-C, but not VEGF-D was found to locate in lung cancer cells, as well as non-neoplastic cells inclusing macrophage cells. The expression of VEGF-C protein and mRNA in lung cancer cells increased by proinflammmatory cytokines IL-1βor TNF-αstimulation in a time- and dose-dependent manner.3. VEGF-C mRNA was more stabilized after IL-1βstimulation, which contributes to enhanced production of VEGF-C in LLC cells.4. The vector pGenesil-siHuR targeting HuR was successfully constructed. HuR knockdown blocked the induced stabilization and expression of VEGF-C by IL-1β.5. The vector overexpressing HuR, pEGFP-HuR was generated, and overexpression of HuR increased the expression of VEGF-C mRNA and protein in LLC cells.6. HuR overexpression increased cell migration and promoted cell proliferation.7. At 30 days after tumor inoculation, the presence of supracervical node and contralateral supracervical node metastases in control mice was 100% and 83.3%, respectively, compared with 100% and 66.7% in HuR-overexpresseded mice. In addition, the metastatic ratio of brachial nodes in HuR-overexpresseded tumors (100%) was significantly higher than that in control tumors (33.3%). Furthermore, overexpressed HuR enhanced the numbers of lung metastastic nodules from (6.5±2.1) to (10.7±2.4). Immunochemical analysis for LYVE-1 expression showed that LMVD in control tumors was (19.7±2.4) compared with (13.2±2.8) in HuR-overexpresseded tumors.8. Western blotting and PCR-PCR showed that expressin level of VEGF-C mRNA and protein in HuR-overexpressed LLC tumors was higher than that in control tumors. 9. Untreated LLC cells contained low HuR protein levels in the cytoplasm, which is consistant with the weak fluorescence in the perinuclear region observed by confocal microscopy. Furthermore, stimulation with IL-1βcaused a time-dependent increase in the level of cytoplasmic HuR with a maximal effect seen at 24 h, suggesting that IL-1βincreased cytoplasmic accumulation of HuR in lung cancer cells.10. The p38 MAPK specific inhibitor SB352038 inhibited IL-1β-induced HuR cytoplasmic shuttling and VEGF-C expression, and reduced the stability of VEGF-C mRNA.11. Different constructs were generated by cloning of VEGF-C CR or VEGF-C 3'UTR fragments into the pGL3-Promoter reporter plasmid in the XbaI site located in the downstream of lucigerase gene.12. The 3'UTR of VEGF-C mRNA confered IL-1β-induced mRNA stability.ConclusionsHuR plays a major role in posttranscriptional regulation of IL-1β-induced VEGF-C expression and tumor lymphangiogenesis by enhancing VEGF-C mRNA stability. These effects are coupled to HuR's elevated presence in the cytoplasm through the p38 MAPK pathway and its interaction with 3'UTR of VEGF-C mRNA.