| Objective: Head and neck squamous cell carcinoma(HNSCC)is the sixth most common type of human cancer and is the most common type of lethal malignancy in head and neck region.One of the leading causes of poor prognosis of HNSCC patients is its feature of loco-regional invasion and metasitasis to cervical lymph nodes.Therefore,it is important to clarify the molecular mechanism underneath and identify clinical intervening treatment for the targeted therapies of HNSCC.SP1 is a zinc finger transcription factor that is elevated in many human cancers including breast cancer,gastric cancer,pancreatic cancer,lung cancer and thyroid cancer,and the levels of SP1 is associated with patients’ clinical stage,tumor metastasis and prognosis.SP1 can modulate cell proliferation,invasion,metastasis,apoptosis and agiogenesis via p53,VEGF,PDGF,MDC1,MMP-9 and many other molecules.Micro RNAs are small non-coding RNAs at 21-25 nt that regulate downstream gene expression at a post-transcriptional level by binding to the 3’ untranslated regions(UTR)of target m RNA transcripts and causing translational repression or degradation.MiR-92 b is a member of miR-17-92 cluster and has been reported to be involved in the progress of human cancer.For example,in oral squamous cell carcinoma miR-92 b could facilitate cell survival and inhibit apoptosis via NLK.Chemokine receptor CCR7 is a member of G-protein-coupled receptor family.It participates in many biological processes by interacting with its two ligands-CCL19 and CCL21.In our previous studies in HNSCC,we found that CCR7 affected the migration and invasion through regulating PI3K/Akt/m TOR,Cdc42,Rac,p70s6 k,MAPK family,pyk2,STAT3 and many other down-stream molecules.In this study we focused on the biological function of miR-92 b in the process of HNSCC progression,the interaction between miR-92 b and transcription factor SP1,and the regulation of CCR7 from SP1/miR-92 b signaling axis,to complement the molecular mechanism of HNSCC invasion and metastasis and provide basis theory of HNSCC targeted treatment.Methods: 1.Real-time PCR was used to evaluate the levels of miR-92 b,SP1 and CCR7 m RNA in cells and tissues.After transfecting miR-92 b mimics,miR-92 b inhibitor,SP1 siRNA and SP1 siRNA+miR-92 b inhibitor plasmid into cells,Real-time PCR was used to detect miR-92 b,SP1 and CCR7 m RNA levels in every group.2.Western blot was used to evaluate SP1 and CCR7 protein expression.After transfecting miR-92 b mimics,miR-92 b inhibitor,SP1 siRNA and SP1 siRNA+miR-92 b inhibitor plasmid into cells,Western blot was used to detect SP1 and CCR7 protein levels in every group.3.Analysis of HNSCC patients’ data in TCGA database was used to evaluate the levels of miR-92 b in the adjacent tissue,primary focus and metastasis of HNSCC patients,the relationship between SP1 and miR-92 b levels,and between CCR7 and SP1 levels.4.Transwell migration assay and wound-healing assay was used to evaluate the migratory abilities of HNSCC cells.5.Transwell invasion assay was used to evaluate the invasiveness of HNSCC cells.6.CCK-8 assay was used to evaluate the cell proliferation.7.Dual-luciferase reporter assay was used to evaluate the direct binding of miR-92 b and CCR7 m RNA 3’-UTR.8.Lentivector was constructed to knockdown SP1 expression in HNSCC cells.In vivo tumorigenesis in nude mice was used to evaluate the effect of SP1 knocking down on tumor growth and volume.9.Immunohistochemistry was used to examine the SP1 and CCR7 protein levels in xenograft tumor tissues.Results: 1.Expression level of miR-92 b in HNSCC cells Through Microarray screening we identified miR-92 b as one highly expressed micro RNA in HNSCC cells with higher migratory in invasive ability.Real-time PCR verified this differential expression.By analyzing HNSCC patients’ data in TCGA database,we found the expression of miR-92 b in HNSCC primary focus was higher than adjacent non-cancerous tissue,and in metastasis tissue even higher than primary tumor.2.MiR-92 b is related to the clinical stage and prognosis of HNSCC patients.By analyzing TCGA data,we found that miR-92 b expression was higher in stage III&IV patients than in stage I&II patients,and higher expressed in N+ patients than in N0 patients.HNSCC patients with high miR-92 b expression were expected to have lower survival possibilites than patients with low miR-92 b expression.Also miR-92 b is related to patients’ N stage.N stage and high level of miR-92 b are risk factors associated with death of HNSCC patients.3.MiR-92 b affects migration,invasion and proliferation of HNSCC cells After transfecting miR-92 b mimics and inhibitor into HNSCC cells,miR-92 b levels were accordingly up-regulated or down-regulated.Transwell migration assay and wound-healing assay demonstrated that high expression of miR-92 b resulted in a significant increase in cell migration,and low expression of miR-92 b led to a decrease in cell migration.Transwell invasion assay verified that high expression of miR-92 b caused an increase in cell invasion while knocking down miR-92 b caused a decrease in cell invasion.CCK-8 assay showed that up-regulation of miR-92 b caused an increase in cell proliferation,and down-regulation of miR-92 b caused a decrease of cell proliferation.4.MiR-92 b regulates SP1 expression By analyzing TCGA data,we found that the expression levels of SP1 increased with the increase of miR-92 b levels in HNSCC samples.After up-regulating the expression of miR-92 b in HNSCC cells,there was an increase in both SP1 m RNA and protein levels.While down-regulation of miR-92 b expression caused a decrease of both SP1 m RNA and protein levels.5.SP1 regulates CCR7 expression By analyzing TCGA data,we found that the expression levels of CCR7 increased with the increase of SP1 levels in HNSCC samples.After transfecting SP1 siRNA into HNSCC cells,there was a decrease in both CCR7 m RNA and protein levels.6.MiR-92 b regulates CCR7 expression Up-regulation of miR-92 b in HNSCC cells caused a decrease of CCR7 m RNA level,and down-regulation of miR-92 b caused an increase of CCR7 m RNA.Luciferase reporter assay verified that miR-92 b could directly regulate CCR7 expression by binding to its 3’-UTR region.This regulation is less obvious in the protein level.When simultaneously down-regulating both SP1 and miR-92 b levels in HNSCC cells,the down-regulation of CCR7 was attuned.7.Verifying SP1/miR-92b/CCR7 signaling axis in animal expriments Constructed SP1 knocking down lentivector was infected to PCI-37 B cells.Nude mice were divided into blank control group,SP1 control group and SP1 knock down group.The tumor growth speed and volume was significantly decreased in SP1 knock down group.In the specimen of SP1 knock down group,there were reduced levels of both miR-92 b and CCR7 protein.Conclusion: MiR-92 b could promote migration,invasion and proliferation of HNSCC cells,and is related to patients’ clinical characteristics,stages and prognosis.Transcription factor SP1 forms a positive feedback loop with miR-92 b in HNSCC cells.SP1/miR-92 b regulatory loop generally activates CCR7 expression with a major promotion from SP1 and a minor inhibition from miR-92 b. |
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