Study On Expression Of Maternal Embryonic Leucine Zipper Kinase In Oral Squamous Cell Carcinoma And Epithelial Mesenchymal Transformation

PurposeThe aim of this study was to investigate the specific signaling pathway and molecular mechanism of Maternal embryonic leucine zipper kinase(MELK)mediating EMT in oral squamous cell carcinoma(OSCC)and to provide possible new targets and ideas for the diagnosis and treatment of OSCC.Methods1.Tissue samples of 62 patients with primary OSCC who underwent radical surgery in the Affiliated Stomatology Hospital of Guilin Medical University were randomly selected,and relevant clinicopathologic data of them were collected.The expressions of MELK and EMT-related proteins were analyzed by immunohistochemistry.Relative MELK m RNA levels were detected by RT-PCR in OSCC and paired normal tissues.The correlation between the expression of these proteins and the clinicopathological parameters of patients were analyzed using Graph Pad Prism 5.01 Statistical software package.2.OSCC cell lines with low MELK expression were constructed.The effects of MELK expression on the proliferation,invasion and metastasis ability of OSCC cells were analyzed by scratch and Transwell experiments.The CCK-8 method was used to detect cell viability.The effects of subcutaneous tumorigenesis in nude mice on the growth rate of OSCC tumors were also performed.3.Using high-throughput RNA sequencing data from the head and neck squamous cell carcinoma(HNSCC)queue in the cancer genome atlas program(TCGA)database,Kyoto encyclopedia of genes and genome(KEGG)pathway enrichment analysis was used to explore the possible signaling pathways of MELK in OSCC progression.q RT-PCR was used to detect the expression of MELK and EMT related genes.Western blot analysis was used to detect the expression of apoptosis related proteins,and immunofluorescence and confocal microscopy were used to observe the co-localization of EMT related proteins.Results1.MELK expression is up-regulated in OSCC and closely related to EMT.MELK is mainly expressed in the cytoplasm.Compared to normal oral mucosa,the expression level of MELK is significantly increased in OSCC,while in primary OSCC tissues with cervical lymph node metastasis,its expression is higher than in those without lymph node metastasis.Its high expression is dramatically correlated with poor pathological differentiation and lower five-year survival rate.In OSCC tissues,EMT process related proteins are highly expressed,and MELK expression is prominently correlated with the expression level of EMT markers.2.MELK gene knockdown inhibits the growth,the proliferation,invasion,and migration of OSCC cell.2.1 The highest expression levels of MELK in SCC9 and Fa Du cell lines were observed,and efficient sh RNA sequences were used for in vitro wound healing experiment and Transwell experiment in Fa Du and SCC9.Experiments have shown that the wound healing ability decreases sharply after 48 hours of MELK knockdown;Similar findings were also made in the Transwell experiment.2.2 Knocking down MELK could markedly reduce tumor growth and tumor volume in nude mice.The IHC staining results also support the alteration of EMT markers caused by MELK knockdown.3.Molecular mechanism of MELK regulating the EMT process of OSCC.3.1 The results of KEGG enrichment analysis showed that differentially expressed MELK related genes were mainly enriched in the Notch and Wnt signaling pathways.After MELK knockdown in Fa Du and SCC9 cells,the key components of the Notch/Wnt signaling pathway related genes,CTBP2 and MAPK9,were significantly reduced,while other regulatory factors did not show significant alterations.3.2 The morphological expression of cells was detected by immunofluorescence.The expression levels of EMT marker proteins after MELK knockdown were detected by Immunoblotting analysis.These results showed that the expression of EMT markers was significantly down-regulated after MELK-sh RNA transfection.Conclusion1.MELK is overexpressed in OSCC tissues and is associated with poor prognosis in OSCC patients.The higher the expression of MELK is,the worse prognosis is in OSCC patients.2.The expression levels of MELK is highly correlated with the expression levels of EMT-related proteins E-cadherin,Zeb2,Snail,Twist1,Vimentin,Slug,ESRP2 and N-cadherin in OSCC.3.MELK knockdown inhibited OSCC cell growth in vitro and tumorigenesis in vivo.4.MELK may participate in the EMT process of OSCC through regulating Notch and/or Wnt signaling pathways.