Study On The Mechanism Of Human Astroglioma With Diseased Progression Regulated By Angiogenin And Its Interaction With FHL3

Angiogenin(Ang) is the first human tumor-derived protein with in vivoangiogenic activity. The Ang expression is up-regulated in numerous tumors, but nota tumor-specific product. Ang has been conceived to play a more important role incell survival, growth and proliferation than the mediation of angiogenesis. Angactivates cell signaling pathway through the putative receptor on endothelial cells andpromotes cell proliferation, and it also prevents stress-induced cell death through boththe Bcl-2and NF-κb pathways. Direct nuclear functions of Ang are required forAng-induced cell proliferation and angiogenesis.Glioma is the most frequent type in primary tumors of central nervous systemand brain astrocytoma is the most frequent (75%) one within the variousneurogliomas, among which glioblastoma multiforme (GBM) is the most malignantbrain glioma subtype. Although at present there have been a lot of treatment methods,the prognosis is still very poor and the life quality of patients was seriously influenced.Ang is detectable in different kinds of intracranial tumours with the lowest amount inlow grade astrocytomas and contributes to the malignant transformation of gliomas.We detected the expression of Ang in different grade astrocytoma tissues by real-timequantitative PCR, the results showed that the expression of Ang was positivecorrelated with the malignant grades. U87MG cell is a polymorphic glioblastoma cellstrain. MTT assay showed that Ang could promote U87MG cell proliferation andup-regulate the expression of anti-apoptotic protein Bcl-xL and oncogene c-myc.The expression of various intracellular signaling moleculas can accelerate ordelay the progress of the disease by mediating the related signaling pathways duringevolution process of gliomas. NF-κB is activated and involved in cell survival,proliferation and migration in GBM. The phosphorylation of ERK1/2and p38wassignificantly enhanced with the increasing of tumor grade, suggesting that these twosignaling pathways are activated in astroglioma. For further investigate therelationship between Ang-induced cell proliferation and the activated signaling pathways in GBM, U87MG cells were stimulated with different concentrations ofAng. The results showed that Ang activated p38and NF-κB pathways withoutinfluencing ERK1/2phosphorylation. Blocking the p38and NF-κB pathways, onlyNF-κB was required for Ang-induced cell proliferation. Meanwhile, Ang wastranslocated to the nucleus of U87MG cells and inhibition of the translocation alsosuppressed Ang-induced cell proliferation partly. The results suggest that Ang couldpromote U87MG cell proliferation by mediating signaling pathways and its nucleusfunction.HeLa cells were chosen for comparing the Ang's mechanisms of action inastroglioma. Ang could promote HeLa cell proliferation and translocate in the nucleusthat are consistent with the biological effect of Ang in U87MG cell. However,whether it involes the mediation of signaling pathways is not clear. Our researchesshowed that Ang could promote HeLa cell proliferation by activating ERK pathway.Besides, the function of Ang in malignant tumor of blood is not yet clarified.Malignant lymphoma is the incidence of the fastest-growing malignant tumor,60%ofwhich are B non-Hodgkin's lymphoma (B-NHL). Our data showed that there was nodifference of Ang expression in peripheral blood betweent B-NHL patients and healthadults. However, high concentration of Ang inhibited Ramos cell proliferation and theactivation of related pathways. The results are not only to suggest that Ang mediatesdifferent signaling pathways in different cells tumors, but also to support effectivecomparison and analysis for the molecular regulation mechanism of astroglioma byAngFor better clarifying the association between Ang expression and evlutionprocess of astroglioma, the interaction between Ang and FHL3was confirmed withGST pull-down and CoIP assays in our study. FHL3(four and a half LIM domainsprotein3) is a member of LIM protein superfamily, which could interacts withproteins through LIM domains and inhibits tumor cell proliferation bySmads-mediated signaling pathway. Moreover, FHL3interacts with ERK2and playsan important role in regulating ERK pathway. The expression of FHL3and themalignant grades of astrogliomas were negatively correlated. FHL3in highconcentration inhibited U87MG cell proliferation, but the phosphorylated p38andIκBα were unchanged in knockdown-FHL3expression cells. Down-expression ofFHL3promoted Ang-induced activation of signaling molecules, the nucleartranslocation of Ang and Ang-stimulated U87MG cell proliferation. The results suggest that FHL3could mediate Ang-induced U87MG cell growth by influencingAng-regulated signaling pathway and nuclear translocation of Ang. However, inHeLa cells, down-regulation of FHL3expression inhibited Ang-mediatedphosphorylation of ERK1/2, nuclear translocation of Ang, Ang-promotedtranscription stimulatory activity of Ang-binding elements (ABE, ctctctctctctctctccctc)and Ang-induced cell proliferation. The results suggest that FHL3could also mediateAng-induced HeLa cell growth by influencing Ang-induced signaling pathway andnuclear translocation of Ang in adverse way. The results above also suggest thatFHL3mediates different Ang-induced functions in different tumor cells.FHL3is involved in the mediation of Smad pathway, but the phosphorylation ofSmad2was undetected without stimulation in U87MG. Thus, TGFβ1which is anactivator of Smad pathway could support more clues for studying the regulatorymechanisms of Ang. Surprisingly, TGFβ1inhibited the expression of Ang,phosphorylation of ERK1/2and Ang-induced p38phosphorylation. The resultssuggest Ang has no direct effect on Smad signling pathway but the relationshipbetween Ang and TGFβ1may lay basis on further study of regulatory mechanisms ofAng. In HeLa cells, Ang inhibited phosphorylation of Smad2and TGFβ1-inducedphosphorylation of Smad2, but TGFβ1unchanged Ang expression. These provide anexplanation for Ang-promoted HeLa cell proliferation.MicroRNAs (miRNAs) were chosen for further investigate the possiblemolecular mechanism of astrocytoma regulated by Ang, because Ang has been foundto exhibit ribonucleolytic and transcription factor activity. MicroRNAs (miRNAs) areendogenous~22nt non-coding small RNAs which associates with target mRNAsthrough miRNA: mRNA base-pairing interactions, most often within3′UTRs.MiRNAs are involved in the regulation of gene expression through the targeting ofmRNAs during cell proliferation, apoptosis, differentiation, metabolism and tumormetastasis by binding to their targets with imperfect or perfect complementarity, thetarget mRNA undergoes accelerated turnover and/or translational repression.MiRNAs play a role in regulating endothelial cells, especially in angiogenesis.Screening miRNAs that may regulate Ang expression by bioinformatics, theexpression of Ang was detected in U87MG cells by qPCR. HeLa cells, HepG2cellsand HUVEc were chosen for comparison. The results demonstrate that miR409-3pcould inhibit the expression of Ang at mRNA and protein levels only in HUVEc.On the other hand, Ang has been found to exhibit ribonucleolytic activity, thus can catalyze the cleavage of both28S and18S ribosomal RNA. At the same time,Ang is able to bind to DNA in the cell nuclear and to stimulate rRNA transcription.An angiogenin-binding DNA sequence has been identified from non-transcriptionregion of rRNA gene and designated as angiogenin-binding element (ABE). ABEbinds to angiogenin specifically and exhibits angiogenin-dependent promoter activityin a luciferase reporter system, indicating that Ang could regulate expression of Angas transcription factor. The elements containing7CT repeats located in the5'-flanking region within upstream2000bp of the gene were chosen by BLAST andamplified from HeLa cDNA. The transcription-stimulatory activity of the promoterelements of melanocortin3recepto(rM3CR)was enhanced by stimiulating with Ang.The results showed that Ang may regulate the expression of M3CR. MiRNAs wereextracted from Ang-stimulated cells and might be regulated by Ang, which weredetected in U87MG cells, HeLa cells and HUVEc. Only the expression of miR17-3pwas significantly inhibited in the three cells. The down-regulation of miR17-3p inastroglioma may provide new clues to further investigate the evolution process ofastroglioma regulated by Ang.Overall,the interaction of Ang with FHL3and the feedback regulation betweenAng and miRNAs could provide not only the new theories and clues to explore theanti-apoptosis mechanism of astrocytoma, but also a new strategy for the clinicaltherapy against cancer, while a new target for the development of anti-cancer newdrugs.