The Study Of Mechanism Of Primary Cilia-related Gene KIF3A On Oral Squamous Cell Carcinoma Through Hedgehog

Objective: To investigate the mechanism of primary cilia-associated gene KIF3 A on oral squamous cell carcinoma through Hedgehog.Methods: Human tongue squamous cell carcinoma cells TCA8113,CAL27 were cultured as the research object.Pre-experiment was divided into KIF3A-si RNA transfection group for 12 hours,KIF3A-si RNA transfection group for 24 hours,negative control group for 12 hours,negative control group for 24 hours and blank control group.The expression level of KIF3 A protein in each group was detected by Western Blot.MTT assay was used to observe the proliferation of TCA8113,CAL27 in each group.The migration of TCA8113,CAL27 in each group was observed by Wound healing assay.The primary cilia of CAL27 cells were detected under the fluorescence microscope by immunofluorescence staining.The expression level of KIF3 A,Shh,Gli1 protein in each group was detected by Western Blot.The relative expression levels of KIF3 A,Shh,Gli1 m RNA in each group were detected by Real-time q PCR.Results: 1.Immunofluorescence staining results: The CAL27 cells were starved for24 hours,and the Arl13 b protein was mainly concentrated in the axon of primary cilia observed under fluorescence microscope.The slender cilia were consistent with the morphological characteristics of primary cilia,which proved the existence of primary cilia in CAL27 cells.2.MTT assay results: The proliferation rate of TCA8113 and CAL27 cells was significantly lower than that of the control group after transfection for 24 hours and culture for 24,48 and 72 hours respectively(P<0.01).3.Wound healing assay: TCA8113 and CAL27 cells were transfected for 24 hours,and cultured for 24 hours after scratch.Compared with the control group after 0,24,48 and 72 hours,there was no significant difference in cell migration ability in scratches among all groups after 24 hours(P>0.05).After 48 hours and 72 hours,cell migration ability in scratch was significantly inhibited compared with blank control group,with statistical significance(P<0.01).4.Western Blot results:(1)After transfection of TCA8113 cells 12 and 24 hours,the expression level of KIF3 A protein showed no obvious downward trend after 12 h transfection which compared with control group and negative control group.But the expression of protein decreased significantly after 24 h transfection(P<0.05).(2)After transfection of TCA8113 and CAL27 cells 24 h,the protein expression levels of KIF3 A,Shh and Gli1 decreased significantly(P<0.05).5.Real-time q PCR experiment results: After transfection of TCA8113 cells 24 h,the m RNA expression levels of KIF3 A,Shh and Gli1 decreased significantly,showing statistical significance(P<0.01).After transfection of CAL27 cells 24 h,the m RNA expression levels of KIF3 A,Shh and Gli1 decreased significantly,showing statistical significance(P<0.05).Conclusion:1.Primary cilia are exist in oral squamous cell carcinoma.2.In oral squamous cell carcinoma,the primary cilium-related gene KIF3 A may affect the proliferation and migration by regulating the Hedgehog.