| Objective:To investigate the function of Non-muscle myosin heavy chain 9(MYH9)in esophageal squamous cell carcinoma cells(ESCC),and to identify the potential molecular mechanisms.Methods:The data of whole genome sequencing and exome sequencing of 104 ESCC patients were analyzed and MYH9 was identified to be a candidate gene associated with ESCC,the GEPIA 2 database was used for validation.The mutation of MYH9 in pan-cancer was analyzed by TCGA database.The immunohistochemical technique was used to detect the expression of MYH9 in 57 pairs of ESCC and its adjacent tissues in tissue microarray,and the clinical correlation was analyzed;Western blot and q PCR were performed to detect the endogenous expression in different ESCC cell lines and the interference efficiency of two MYH9-specific si RNAs and one non-specific sequence on MYH9;MTT and transwell assay were used to explore the proliferation and migration after MYH9 knockdown in ESCC cell lines.PCR-array was applied to detect the potential target genes and the signal pathways associated with MYH9.Tube formation was used to explore the angiogenesis activity after MYH9 knockdown in ESCC cell lines and q PCR was performed to verify the result of PCR-array analysis;the correlation between MYH9 and downstream genes was analyzed in TCGA database;GSCA database was applied to analyze the activation percentage of the downstream genes.Results:1.MYH9 was mutated with a mutation frequency of 2.88%(3/104)in ESCC by omics sequencing technology.The expression of MYH9 in esophageal cancer was significantly higher than that in normal tissues(p<0.05),and the expression increased with tumor progression in GEPIA 2 database(p<0.001).The TCGA database analysis showed that MYH9 was mutated in multiple common tumors and missense is the main mutation type.Moreover,we performed the subsistence analysis in the TCGA database and found the high MYH9 expression level was strongly associated with shortened survival of patients in cervical squamous carcinoma and head and neck squamous cancer(p<0.05).2.MYH9 was highly expressed in ESCC,whereas nearly negative in matched normal tissues and a significant statistical difference was found between the two groups(p<0.001).Moreover,the MYH9 expression level was related to lymph node metastasis(p=0.047),but it was not related to age,histological grading and clinical stage.3.A knockdown experiment was performed in the KYSE140 and KYSE180 with high endogenous expression of MYH9.The proliferation was detected by MTT assay and the growth curve showed that MYH9 had no significant effect on the proliferation of KYSE140 and KYSE180.Transwell assay showed that the cell migration and invasion activity were significantly inhibited.4.PCR-array showed that a total of 15 differentially expressed genes were identified(fold change ≥2.5),including 3 up-regulated genes ATP5A1,CASP9 and UQCRFS1;12down-regulated genes: FLT1,KDR,DSP,CFLAR,KRT14,LDHA,SNAI2,TEK,VEGFC,XIAP,ARNT and CDH2.These genes were mainly distributed in the pathways related to angiogenesis and epithelial-mesenchymal transition(EMT).5.Angiogenesis was inhibited after MYH9 knockdown in ESCC cells;MYH9knockdown reduced the expression of angiogenesis related markers FLT1,KDR,TEK and VEGFC.The expression of SNAI2,KRT14 and CDH2 markers associated with mesenchymal phenotypes were all decreased(p<0.05).A total of 549 cases of lung squamous cell carcinoma,307 cases of cervical squamous carcinoma and 565 cases of head and neck squamous cancer were analyzed in the TCGA database.It was found that MYH9 was significantly positively correlated with angiogenesis markers FLT1,KDR,TEK,and VEGFC;significantly positively correlated with EMT markers SNAI2,KRT14 and CDH2(p<0.001).EMT activation was the main pathway when were analyzed the downstream genes in GSCA database.Conclusion:1.MYH9 is often mutated in multiple common tumors;and the high expression of MYH9 in ESCC is associated with lymph node metastasis.2.MYH9 may promote the migration and invasion of ESCC via inducing angiogenesis and EMT. |
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