The Studies On The Effect Of 14-3-3? And LKB1 Complex In Oral Squamous Carcinoma Apoptosis

Objectives:Oral squamous cell carcinoma(OSCC)is one of the most common carcinoma in the oromaxillo-facial region.However,the five-year survival rate of OSCC is only 30%-45%.In the thesis,we focus on the studies of the expression of apoptosis related protein 14-3-3? and LKB1 in OSCC,the relationship between 14-3-3? and LKB1 in the regulation of apoptosis and growth of OSCC and the molecular basis for 14-3-3? and LKB1 binding by crystallography.And further,we intend to verify the effects of the binding of 14-3-3? and LKB1 on OSCC growth and apoptosis by designing mutation according to the crystal structure.Through our research,we plan to set the structural basis for the treatment of OSCC by biological targeted management,and expect to support with a new strategy for OSCC therapy to complement current combinative treatment.Method:1.Measuring the expression variance of 14-3-3? in primary oral epithelial cells and oral squamous cell carcinoma cell line CAL27 at m RNA and protein level by Real-time PCR and immunopreciptation assay.Using si RNA,designing plasmids of 14-3-3? over-expression and knocking down and detected 14-3-3? effects on CAL27 proliferation and apoptosis by FACS.2.Detecting the interaction between 14-3-3? and LKB1 in oral squamous cell carcinoma by co-immunoprecipitaion assay.And meantime,got purified protein complex of 14-3-3?-LKB1 by means of protein purification and solved the crystal structure of 14-3-3?-LKB1 by crystallography to further confirm the binding and revealed the moleculer mechanism and physiclogical importance of the binding.3.Designed mutations for the key binding sites of 14-3-3? and LKB1 complex;transfected the plasmids of the mutations to CAL27 cell line;detected the disassociation of the complex by co-immunoprecipitaion and verified the effects of LKB1 mutation on CAL27 growth and apoptosis by FACS.Results:1.Both 14-3-3? transcription and expression levels were higher in CAL27 cell line compared with that of primary oral epithelial cells.Transfected the plasmid of 14-3-3? overexpression into CAL27,and by FACS we finded the proliferation of CAL27 accelerates comparing with control cells.Transfected the si RNA targeting 14-3-3? into CAL27,and by FACS we find the apoptosis of CAL27 was dramatically increased.2.We finded strong interaction of 14-3-3? and LKB1 in CAL27 cell line by co-immunoprecipitation,and the interaction was rather weak in primary oral epithelial cells.And the crystal structure of 14-3-3?-LKB1 complex revealed that 14-3-3? interacts with LKB1 in vitro,and the binding site of LKB1 was 334-340.The site of 336 of LKB1 was the key-binding site for 14-3-3?.3.According to the crystal structure of 14-3-3?-LKB1 complex,we mutated the glutamine of 336 to alanine,and transfected the plasmid into tumor cells.We finded 14-3-3? can not interact with LKB1 by FACS,and the apoptosis of CAL27 cells occurs.