Effect Of MELK Gene On Migration And Invasion Of Nasopharyngeal Carcinoma Cells

Objective:Nasopharyngeal carcinoma(NPC)is a common malignant tumor in southern China.Its incidence is related to factors such as genetics,diet,environment and EBV virus infection.The metastasis and invasion of tumor cells will promote the malignant phenotype,which is also the main reason for the poor clinical efficacy.MELK is highly expressed in a variety of tumor tissues,participates in the occurrence and development of tumors through multiple biological processes,and is associated with poor prognosis,suggesting that MELK is a potential therapeutic target of tumors.EMT program activation is the initial step of tumor cell metastasis.Reversing EMT program can inhibit tumor metastasis by inducing and promoting tumor cell invasion and proliferation.However,the role of MELK in NPC metastasis is not clear.Therefore,clarify the role of MELK in NPC metastasis and provide a theoretical basis for targeted treatment of NPC.Methods:1、Analyze the gene chip of geo database,select the patient tissue chip related to NPC expression,obtain the differentially expressed genes(DEGs)in the data set,determine the target genes,verify the expression of MELK,predict the relevant enrichment pathways,and explore the impact of its high expression on the prognosis of patients.Firstly,four data sets related to NPC expression were retrieved and downloaded from GEO database,including GSE13597,GSE12452,GSE53819 and GSE646344.The DEGs in each data set were analyzed by GEO2 R,and the DEGs expressed in different data sets were analyzed and summarized by the "Robust Rank Aggreg" toolkit in R software to determine the target gene MELK,verify whether the MELK expression in the four data sets is different between NPC patients’ tumor tissues and normal tissues,and predict the functional enrichment pathway related to MELK by GSEA gene functional enrichment.GEPIA and Progno Scan databases were used to retrieve the effects of MELK high expression and low expression on the prognosis of patients.2、To detect the expression of MELK mRNA and protein in four NPC cells,then construct MELK knockdown and overexpression cell models,and explore the effects of MELK expression on the biological function of NPC cells.qRT-PCR and Western blot were used to detect the mRNA and protein expression of MELK in four NPC cell lines(5-8F,HNE2,c666-1 and CNE1).MELK knockdown was performed on cells with high MELK expression,and cells with relatively low MELK expression were overexpressed.MELK interference and overexpression vectors were constructed according to the expression,and the corresponding NPC cells were transfected to obtain the NPC cell model with stable Melk knockdown and overexpression.Similarly,through verification,the expression efficiency of MELK mRNA and protein in knockdown and overexpression cell lines was detected.Then explore the effects of different expression states of MELK on cell function.Cell experiments include scratch,migration and invasion,and explore the effect of MELK on NPC cell migration and invasion.3、To explore whether MELK is involved in NPC cell metastasis.Western Blot was used to detect the protein expression levels of EMT related marker molecules ecadherin,Vimentin,Snail,Slug,MMP2,MMP7 and MMP9 in nasopharyngeal carcinoma cell lines with MELK knockdown and overexpression.Cytofunctional studies verified the effect of MELK expression on the migration and invasion of NPC cells.4、To explore the effect of MELK knockdown on tumor growth and metastasis.MELK knockdown cells were used to construct the nude mouse model of metastatic tumor,and the tumor size and metastasis site were observed.Whether MELK was involved in the EMT process was verified in vivo,and the expression of MELK and EMT labeled molecules e-cadherin and Snail protein in mouse metastatic tumor tissues was detected by Western blot assay.Results:1、Expression of MELK in NPC tissues and its effect on prognosisDifferential gene expression analysis was performed on 4 sets of gene expression data sets(GSE13597,GSE12452,GSE53819 and GSE646344),and the results showed that in 4 sets of nasopharyngeal carcinoma gene expression data sets,Up-regulated genes included MMP1,TNFAIP6,PTGS3,MMP3,LHX2,STAR,CXCL10,CXCL11,FN1,CCL8,IL13RA2,MMP12,GAD1,HOXA10,IFIT1,MELK,Down-regulated genes included LTF,DNALI1,SCGB1A1,MSMB,TPPP3,C2Oorf85,ADH1 C,SNTN,MUC16,TSPAN1,TMC5,SPAG6,PIP,FAM81 B and PROM1.In this study,MELK was selected as the target gene to verify the expression of MELK in NPC and normal nasopharynx in NPC related data sets.Four NPC tissues showed significantly higher expression of MELK than normal nasopharynx.We enriched MELK with GSEA gene functions,including activation of E2 F,MYC V1/V2,G2 M,EMT,unfolded protein reaction,angiogenesis,MTORC1,oxidative phosphorylation,glycolysis,DNA repair,UV reaction,mitotic spindle,NF-k B and spermatogenesis pathway.Inhibition of estrogen receptor,peroxisome,heme metabolism,acid metabolism,KRAS,and myogenesis pathways to explore the prognosis of patients with different expression status of MELK.The GEPIA and Progno Scan data only classified MELK as head and neck tumors.A sequence search of the databases showed that MELK was highly expressed in tissues including head and neck tumors.In addition,the OS,DFS and RFS time of patients with high expression were significantly shorter than those with low expression.In the above results,MELK was highly expressed in NPC in different data,and was associated with the shortening of OS,DFS and RFS time in head and neck tumor patients,suggesting that MELK can be used as a potential therapeutic target and prognostic biomarker for NPC.2、Expression and biological function of MELK in NPC cells(5-8F,CNE1,C666-1and HNE-2)To verify whether MELK was expressed in NPC cells,the mRNA and protein expressions of MELK in 4 NPC cells(5-8F,CNE1,C666-1 and HNE-2)were detected by qRT-PCR and Western Blot assay using immortalized nasopharyngeal cell NP69 as control.qRT-PCR and Western Blot results were consistent,MELK mRNA and protein were expressed in 5-8F,CNE1,C666-1 and HNE-2 cells,especially in 5-8F cells,followed by C666-1,HNE-2 and CNE1 cells.To explore the effects of MELK on the biological functions of NPC cells,5-8F cells with relatively high MELK expression were selected for MELK interference expression,and C666-1 cells with relatively low MELK expression were overexpressed for MELK,so as to obtain cell models with different expression states of MELK(5-8F,C666-1).Functional experiments were performed on 5-8F and C666-1 cells,including scratch,migration and invasion experiments.The results showed that the healing area of 5-8F cells with MELK knockdown was significantly reduced compared with that of NC group,while the healing area of C666-1 cells with MELK overexpression was significantly increased compared with that of Vector group,suggesting that MELK promoted cell healing.Transwell cell migration and invasion experiment also showed consistent results,the number of migration and invasion cells in 5-8F cells with MELK knockdown significantly decreased compared with NC group,and the number of migration and invasion cells in C666-1 cells with MELK overexpression significantly increased compared with Vector group.These results suggest that MELK promotes the migration and metastasis of nasopharyngeal carcinoma cells in vitro.3、MELK plays a role in NPC by participating in the EMT processTo confirm the correlation between MELK and EMT,we detected the expression levels of EMT related marker proteins in MELK knockdown cells(5-8F)and overexpressed cells(C666-1).Western Blot results showed that e-cadherin was upregulated in MELK knockdown cells(5-8F).Vimentin,Snail and Slug were downregulated.Epithelial E-cadherin was down-regulated and vimentin,Snail,and Slug were up-regulated in overexpressed cells(C666-1).In addition,overexpressed cells(C666-1)combined with EMT inhibitors reversed this effect.Matrix metalloproteinase is a marker molecule of EMT.Western Blot results showed that MMP-2,MMP-7 and MMP-9 expressions were down-regulated in 5-8F cells with MELK knockdown compared with NC group.The expressions of MMP-2,MMP-7 and MMP-9 in MELK overexpressed C666-1 cells were upregulated compared with those in Vector group.These results suggest that MELK promotes the EMT process.To further explore MELK promoting NPC progression through the EMT pathway,we conducted cell function experiments on MELK overexpressed cells(C666-1)combined with EMT inhibitors.Cell scratch results showed that EMT inhibitor reduced the healing area compared with the control group,and reversed MELK overexpression to promote scratch healing.The results of cell migration and invasion experiments showed that the number of migrating and invading cells decreased after EMT inhibitor compared with the control group,and the promoting effect of MELK overexpression on cell migration and invasion was reversed.These results suggest that MELK induces EMT activation to promote the migration and invasion of NPC cells in vitro.4、MELK is involved in vivo lung metastasis of nasopharyngeal carcinomaTo investigate the role of MELK in nasopharyngeal carcinoma metastasis in vivo,we constructed a nude mouse metastatic tumor model using MELK knockdown cells(5-8F).The results showed that the number of pulmonary metastatic nodules in MELK knockdown group was lower than that in control group.Western Blot was used to detect the expression levels of MELK and EMT labeled molecular proteins(E-cadherin and Snail)in lung metastatic tumor tissues,and it was found that the expression levels of MELK and E-cadherin in tumor tissues were significantly down-regulated after low deletion of MELK.These results suggest that targeted MELK expression can inhibit lung metastasis in nasopharyngeal carcinoma through the EMT pathway.Conclusion:1.MELK was highly expressed in four NPC datasets,and the OS,DFS and RFS time of patients with head and neck tumor with high MELK expression and low MELK expression were significantly shortened.MELK can be used as a biomarker of NPC.2.MELK is highly expressed in NPC cell lines and promotes the migration and invasion of NPC cells in vitro.3.MELK promotes the exmigration and invasion of NPC cells through EMTrelated pathways.4.Knockdown MELK can inhibit in vivo metastasis of nasopharyngeal carcinoma cells.