Experimental Study On The Inhibition Of Migration And Invasion Of Oral Squamous Cell Carcinoma Cells By Overexpression Of NDR1 Protein Kinase

【Objective】 To investigate the expression level of NDR1 in oral squamous cell carcinoma（OSCC）tissues and multiple oral squamous cell cancer cell lines,to explore the effects of NDR1-overexpression on the proliferation,migration and invasion ability in OSCC cell lines,and to further explore the possible molecular biological mechanism of its inhibition of migration and invasion ability in OSCC.【Methods】 Real-time quantitative reverse transcription polymerase chain reaction（q RT-PCR）was used to detect the expression level of NDR1 in oral squamous cell carcinoma tissues,control tissues,4 oral squamous cell carcinoma lines（SCC9,SCC15,SCC25 and CAL27）and normal epithelial cell lines（HOK）.Firstly,we used high expression plasmid to raise the NDR1 expression level in oral squamous cell carcinoma cell line SCC9,while we built highly express NDR1 adeno-associated virus to increase the NDR1 expression level in oral squamous cell carcinoma cell line CAL27.Then after validated its high efficiency by western blot,scratch assay and transwell chamber assay were further used to detect the differences in cell migration and invasion ability between the high expression NDR1 group and the control group;The OSCC cells proliferation ability between the two groups was detected by Ed U proliferation assay.After detecting the position relationship between NDR1 kinase and centrosomes in SCC9 and CAL27 cells by double immunofluorescence staining,immunofluorescence microscopy was used to detect the proportion of cell centrosome reorientation,so as to verify whether NDR1 kinase affects the migration and invasion ability of OSCC cells by changing the transfer rate of cell centrosomes.【Results】 q RT-PCR results showed that the m RNA expression of NDR1 in oral squamous cell carcinoma was significantly lower than that in paracarcinoma tissues（P<0.001）.The m RNA expression level of NDR1 in the four oral cancer cell lines SCC9,SCC15,SCC25 and CAL27 was significantly lower than that in the normal oral keratinocyte line HOK（SCC9,P<0.001;SCC15,P<0.001;SCC25,P<0.001;CAL27,P<0.01）.Furthermore,SCC9 cells were transferred by high expression plasmid,and CAL27 was infected by high expression of adeno-associated virus.The expression level of NDR1 protein in the high expression NDR1 group was significantly higher than that in the control group by western blot.Scratch assay results showed that SCC9 and CAL27 cells with high expression of NDR1 had significantly reduced migration compared to the control group（SCC9,P<0.001;CAL27,P<0.001）;Transwell migration assay results also indicated that SCC9 and CAL27 cells with high expression of NDR1 were significantly less migratory than the control group（SCC9,P<0.01;CAL27,P<0.01）.Transwell invasion assay showed that SCC9 and CAL27 cells with high expression of NDR1 were significantly less invasive than those in the control group（SCC9,P<0.05;CAL27,P<0.01）.The proliferation ability of SCC9 and CAL27 cells with high expression of NDR1 was significantly reduced compared to the control group by the Ed U proliferation assay（SCC9,P<0.001;CAL27,P<0.01）.By double immunofluorescence staining,NDR1 kinase was aggregated in the centrosomes.To research the centrosome transfer rate of cells,the immunofluorescence results showed that the proportion of centrosome reorientation of SCC9 and CAL27 cells with high expression of NDR1 was significantly lower than that of the control group（SCC9,P<0.05;CAL27,P<0.0001）.【Conclusion】The m RNA expression level of NDR1 was decreased in both OSCC patients tissues and OSCC cells.After NDR1 overexpressed in vitro,the migration and invasion ability of oral squamous cell carcinoma were significantly decreased,and the proliferation ability was significantly reduced.NDR1 may inhibit the migration and invasion ability of OSCC cells by regulating specific signaling molecular pathways to reduce the centrosome reorientation.As a potential molecular target,NDR1 may play a role in inhibiting the progression of oral squamous cell carcinoma.