| Background and ObjectivesOral squamous cell carcinoma(OSCC)is a common malignant tumor of the head and neck and the main causes of patient mortality due to OSCC are early neck metastasis,post-treatment recurrence and distant metastasis.Although its treatment methods have been improved in recent years,such as surgery,adjuvant chemotherapy and radiotherapy,the five-year survival rate of patients is still not effectively ameliorated.Therefore,it is of great clinical significance to explore the molecular mechanism of OSCC occurrence and development and seek more effective treatment methods.However,due to the complexity of OSCC pathogenic factors and tumor microenvironment,the molecular mechanisms involved in regulating the malignant biological behaviors of OSCC tumor proliferation,migration and apoptosis in the process of disease occurrence and development have not been fully elucidated.Studies indicate that NOTUM,as a kind of α/β hydrolase,is involved in the progression of various diseases,such as periodontitis,osteoporosis,and neurological diseases.In recent years,the correlation between NOTUM and cancer has gradually become a research hotspot.Researches have shown that NOTUM is highly expressed in liver cancer and colon cancer,which can promote the proliferation and migration of tumor cells.However,the regulatory role played by NOTUM with respect to OSCC has not been reported.Therefore,work aimed to explore the expression level of NOTUM in OSCC tissues and cells and its regulatory effect on the biological behavior of OSCC cells,and to clarify the possible mechanism of its action,hoping to provide an effective target for OSCC tumor therapy.Materials and Methods1.Detection of NOTUM expression in oral squamous cell carcinoma tissues and cells(1)The TCGA-HNSC database was downloaded from the UCSC Xena database,and 305 OSCC tissues samples and 30 control samples were screened for bioinformatics analysis to explore the difference in NOTUM expression between OSCC tissues and control samples.The expression of NOTUM,PCNA and Ki-67 in OSCC tissues and normal tissues were detected by immunohistochemical staining.(2)The expression of NOTUM in OSCC cell lines(Cal-27,Scc-15)and the normal epithelial keratinocyte cell line HOK were detected by Western blot,qRT-PCR and ELISA,and the expression of NOTUM in OSCC cell lines(Cal-27,Scc-15)was assessed by cell immunofluorescence.2.Evaluation of the regulatory effect of NOTUM on the biological behavior of OSCC cells(1)Construction and identification of NOTUM overexpression plasmid and knockdown interfering RNAThe transfection efficiency of OSCC cell lines(Cal-27 and Scc-15)was detected by cytofluorescence,and the NOTUM overexpression plasmid and knockdown interfering RNA were identified and screened by qRT-PCR to confirm whether NOTUM was overexpressed or knocked down in OSCC cells.(2)Effects of NOTUM on the proliferation,migration and apoptosis of OSCC cells by in vitro and in vivo experimentsThe effects of human recombinant protein NOTUM,NOTUM overexpression plasmid and Notum knockdown interfering RNA on the proliferation of OSCC cells were detected by CCK8,Western blot,qRT-PCR and clone formation in vitro.The effects of human recombinant protein NOTUM,NOTUM overexpression plasmid and Notum knockdown interfering RNA on the migration of OSCC cells were detected by scratch healing assay,transwell migration assay and qRT-PCR.The effects of NOTUM knockdown on apoptosis of OSCC cells was detected by Western blot,qRT-PCR and flow cytometry.The effect of NOTUM knockdown on tumor growth was detected by nude mice tumorigenesis assay.3.Exploration of the mechanism of NOTUM in regulating the biological behavior of OSCC cells(1)Bioinformatics analysis and screening of NOTUM-related signaling pathways in OSCC tissuesThe related signaling pathways and interacting proteins of NOTUM in OSCC tissues were screened by gene set enrichment analysis(GSEA)and protein-protein interaction(PPI).(2)Examining the effects of NOTUM knockdown of the Sonic hedgehog signaling pathwayWestern blot and qRT-PCR were used to detect the effect of NOTUM knockdown on Sonic hedgehog signaling pathway.Immunohistochemical staining was used to detect the expression of Shh,a ligand of sonic hedgehog signaling pathway,in human OSCC tissues and tumor tissues paraffin sections of nude mice.(3)To detect the effect of Shh knockdown on NOTUM-promoted proliferation and migration of OSCC cellsThe interfering RNA of Shh knockdown was constructed,qRT-PCR was employed to identify and screen Shh interfering RNA.qRT-PCR was performed to detect the effect of Shh knockdown on the promotion of OSCC cells proliferation and migration by human recombinant protein NOTUM.(4)To detect the coupling effect of GSK3β phosphorylation on Sonic hedgehog signaling pathway and Wnt/β-catenin signaling pathway in NOTUM regulation of OSCC cellsqRT-PCR was used to detect the correlation between NOTUM and β-catenin,an important target of Wnt signaling pathway in OSCC tissues;Immunohistochemical staining was used to detect the expression of β-catenin in OSCC tissues and the paraffin section of tumorigenic tumor tissue of nude mice in vivo.Western blot was used to detect the effect of Shh knockdown on Wnt/β-catenin signaling pathway in the regulation of OSCC cell proliferation and migration by exogenous human recombinant protein NOTUM.By exogenous addition of p-GSK3β agonist Licl,qRT-PCR was used to detect the effect of GSK3β phosphorylation on PCNA and MMP2 mRNA levels when OSCC cells was knocked NOTUM.Western blot was used to detect the effect of GSK3β phosphorylation on Wnt/β-catenin signaling pathway during NOTUM knockdown inhibition of OSCC cells proliferation and migration.Results1.NOTUM is highly expressed in oral squamous cell carcinoma tissues and cellsBioinformatics results showed that NOTUM was highly expressed in OSCC tissues compared with oral health tissues.Immunohistochemical staining results showed that NOTUM and proliferation markers(Ki-67 and PCNA)were highly expressed in OSCC tissues compared with oral healthy tissues.Compared with normal epithelial keratinocyte line(HOK),NOTUM was highly expressed in OSCC cells.2.NOTUM promotes OSCC cells proliferation,migration and inhibits its apoptosisCell experiments showed that human recombinant protein NOTUM promoted the proliferation and migration of OSCC cells in a dose-dependent(0,1,2 and 4 μg/mL)and time-dependent(0,24,48 and 72 hours)manner.Overexpression of NOTUM can significantly promotes OSCC cell proliferation and migration.Knockdown of NOTUM can promote the apoptosis of OSCC cells.The results of tumorigenic experiments in nude mice showed that knockdown of NOTUM can significantly inhibit the growth of subcutaneous tumors in nude mice.3.NOTUM promotes the proliferation and migration of OSCC cells through the Shh/p-GSK3β/β-catenin signaling pathwayThe results of GSEA and PPI analysis showed that NOTUM was related to the hedgehog signaling pathway in OSCC,especially the ligand Shh of the hedgehog signaling pathway.qRT-PCR results showed that NOTUM knockdown decreased mRNA levels of Sonic hedgehog signaling pathway related factors Ptch1,Gli1 and Shh;Western blot showed decreased Shh protein levels.In order to further verify the effect and molecular mechanism of Shh on NOTUM on the proliferation and migration of OSCC cells,the small interfering RNA of Shh was constructed in this study,qRT-PCR results showed that Shh knockdown could inhibit the effect of human recombinant protein NOTUM on the proliferation and migration of OSCC cells.Western blot results showed that the phosphorylation level of Wnt/β-catenin signaling pathway-related protein GSK3β and the expression of β-catenin protein were inhibited after Shh knockdown;qRT-PCR and immunohistochemical staining results showed that β-catenin was highly expressed in OSCC tissue.To further define the coupling role of GSK3β phosphorylation level of Sonic hedgehog signaling pathway and Wnt/β-catenin signaling in the regulation of OSCC cell processes by NOTUM,Licl,an agonist of p-GSK3β,was added in this part,and the Western blot results showed that the increase of GSK3βphosphorylation level could rescue the inhibition of NOTUM knockdown on the proliferation and migration of OSCC cells,and activated the expression of β-catenin.Conclusions1.NOTUM is highly expressed in OSCC tissues and cells.2.Human recombinant proteins NOTUM and NOTUM overexpression promote OSCC cell proliferation and migration,NOTUM knockdown inhibites OSCC tumor growth and promotes OSCC cell apoptosis.3.As a secreted protein,NOTUM regulates the biological behavior of OSCC through the Shh/p-GSK3β/β-catenin signaling pathway,and it is expected to be an effective therapeutic target for OSCC. |
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