Study On The Regulatory Mechanism Of S100A8 Gene In Laryngeal Carcinoma

Laryngeal carcinoma is one of the most common head and neck cancer.Its morbidity has shown an increasing tendency in recent years.Accumulating evidence suggests that inflammation plays an important role in the development and advancement of carcinomas.Our clinical analysis also reveals that the patients suffering from laryngeal cancer often had a history of chronic irrtation and inflammation.In our previous study, we demonstrated that S100A8 mRNA expression level is inconsistent with the protein one by using CGH,cDNA array,RT-PCR,Western blot,immunohistochemistry,which implies that there might be aberrance in post-transcriptional regulation of S100A8. MicroRNAs(miRNAs)are a set of non-coding post-transeriptial molecules of 18～23 nt.They are processed from short stem-loop precursors that can be encoded in genomes of plans,animals and viruses.According to the current understanding, microRNA is firstly transcribed as long primary microRNA,which is processed into 70～90 nt microRNA precursor(pre-miRNA)by Dicer.Then miRNA combines with several proteins,such as Argonaute,to form miRISC(miRNA induced silencing complex).Under the direction of miRNA,miRISC interacts with the 3'-untranslated regions(3'-UTRs)of the target mRNA in a perfect or imperfect base pairing manner, which determines that a single miRNA can target numerous mRNAs and regulate these genes in the post-transcriptional level.A computational analysis indicates that more than one-third of protein-encoding genes are regulated by miRNAs.miRNAs are functioning as oncogenes or tumor suppressor genes involved in the regulation of a variety of biological processes including developmental timing,signal transduction, apoptosis,cell proliferation and tumorigenesis.In one word,miRNAs play an important role in both physilogical and pathological process.As for the curial role of protein-protein interaction in human process,which involved in many regulation network,such as duplication,transcription,translation,splicing,secretion,cell cycle control,signal transduction and cell metabolism.Study on protein-protein interaction is also an important means for searching down-stream regulation genes.Our previous study revealed that S100A8 gene located down-stream of P65 gene and up-stream of BCL-2 gene,plays a pivotal role in the genesis of LSCC probably through NF-Kappa B pathway by using RNA interference.However,the exact molecular mechanism of S100A8 in the NF-Kappa B pathway is still unclear.At presence,studies on neither the regulation of S100A8 by microRNAs nor the detailed mechanism of S100A8 gene in the NF-Kappa B pathway are reported.In this study,we will find and study the regulatory microRNAs in up-stream of S100A8 gene and further explore the functional mechanism of S100A8 gene in NF-Kappa B pathway through the identification and study of S100A8-interacted proteins in laryngeal carcinoma,which will not only describe the regulation mechanism of S100A8 in LSCC,but also provide useful targets for gene diagnosis and therapy of laryngeal carcinoma based on microRNAs and interaction proteins associated competitive inhibitors.Materials and MethodsPossible microRNAs targeting S100A8 3'UTR were predicted via bioinformatics software.Expression of the predicted microRNAs was detected by End-point PCR and real-time PCR.With constructing wide type and mutated S100A8 3'UTR Luciferase expression plasmids,co-transfection technology was used to evaluate the post-transcriptional regulation of microRNAs on S100A8 and the expression of S100A8 in Hep2 cells undergoing co-transfection was detected by Real-time PCR and Western blot assay.Meanwhile,we analyzed the biological effect of the microRNA on Hep2 cells by observing the morphological alteration and detecting the invasion ability in vitro.Hep2 ceils were used in protein-protein interaction study,we detected and identified S100A8 interaction proteins by immunoprecipitation and mass spectrum, while the confirmation work was done by co-immunoprecipitation and immunocytochemistry assays.RNA interference was further used to reveal the regulation mechanism between interaction proteins.ResultsPrediction of targeting microRNA on S100A8 mRNAThe S100A8 mRNA was predicted to be a target of miR-24 after computational analysis using two different programs(Miranda and RNA22).With RNA22,the program returned a hit between the 341 bp sequences of S100A8 and miR-24. Homologous analysis showed that miR-24 had a high homology of 95%to rat.Result of miR-24 expression in LSCCUsing U6-snRNA as an internal control,we detected the expression of miR-24 in seven cell lines and laryngeal cancerous tissues by miRNA End-point PCR and qRT-PCR.Compared to that in GES-1 cell line,significantly low levels of miR-24 were detected in cancer cell lines including Hep2,HeLa,Bel,SGC7901,BGC823 and HEK293(P＜0.05).Meanwhile,majority of laryngeal cancerous tissues from 10 patients(7/10,70%)showed a lower miR-24 expression than their corresponding normal tissues.Student's t-tests showed significant differences(P＜0.05).Post-transcriptional regulation of miR-24 on S100A8 in Hep2 cellsWe successfully constructed the wide type and mutated S100A8 3'UTR Luciferase plasmids confirmed by sequencing.Transient transfection of Hep2 and HEK293 cells with the wild type 3'UTR reporter construct and pre-miR-24 led to a significant decrease in reporter activity compared to the controls.While,anti-miR-24 inhibitor can effectively reverse the reporter activity in both cell lines.Furthermore,the protein level of S100A8 was significantly down-regulated in Hep2 cells followed by pre-miR-24 transfection(P＜0.05),while mRNA level remained almost no change(P＞0.05).Effect of miR-24 on Hep2 cell invasion abilityA significantly morphological change and decreased invasion ability of Hep2 cells were observed in the presence of exogenous miR-24(P＜0.05).Detection and identification of novel protein-binding partners for S100A8 in LSCC cell linesWe successfully detected and identified four novel protein-binding partners for S100A8 in Hep2 cells by immunoprecipitation and mass spectrum assays,which were hypothetical protein LOC80154,MHC classⅠHLA-B,similar to T-box 1 isoform C and sarcolemmal associated protein 1,respectively.The results of co-immunoprecipitation and Immunocytochemistry confirmed the binding ability between S100A8 and HLA-B.Effect of RNA interference on HLA-B geneThe expression of HLA-B gene was rectified by that ofβ-actin.RT-PCR results showed that the interference effect at 7^thday was most obvious and there was significant difference in the mRNA leves of HLA-B between interference group and negative or blank control group.However,no significance was found between the later two groups(＜0.05).Biological effects of HLA-B on Hep2 cellsThe biological effects detected by flow cytometry and Transwell showed that the apoptotic rate in siHLA-B transfection group significantly increased compared to either non-transfection or transfection control group(P＜0.05).Meanwhile,cells were arrested in S phase and cell proliferation ability also increased in siHLA-B transfection group.The invasion capability of Hep2 cells in siHLA-B transfection group at day 7 was much stronger than that in either non-transfection or transfection control group(P＜0.05).S100A8 gene expression after RNA interference of HLA-BThe results of both RT-PCR and Western blot indicated that S100A8 expressed in all of the three groups including siHLA-B transfection,non-transfection and control siRNA transfection groups,but was significantly down-regulated in HLA-B siRNA transfection group(P＜0.05).Conclusion(1)miR-24 functionally targets and negatively regulates S100A8 gene expression by binding to the specific motif of S100A8 3'UTR.(2)miR-24 may alter the cell phenotype and inhibit the invasion ability of Hep2 cells probably by down-regulating S100A8 gene expression.(3)Four novel protein-binding partners for S100A8 identified in LSCC Hep2 cell line are hypothetical protein LOC80154,MHC classⅠHLA-B,similar to T-box 1 isform C and sarcolemmal associated protein 1,respectively.(4)The aberrant expression of HLA-B is associated with the biological effect of LSCC and regulate S100A8 gene expression partly through P65/HLA-B/S100A8/BCL-2/Caspase-9/(-3)path way.