USP7 Stabilizes LETM1 Promotes Follicular Thyroid Carcinoma Proliferation,Migration,and Invasion

Objective:As the most common endocrine malignancy,Thyroid cancer accounts for approximately 1-3%of all new cancer cases and occurs predominantly in women.The incidence of thyroid cancer has steadily increased over the past decades,with a particularly marked rise since the early 1990s,but its mortality rate has remained relatively stable.The main reason for the increase in thyroid cancer is the increase in the incidence of papillary thyroid carcinoma(PTC),but in fact all forms of thyroid cancer have increased in incidence.Of these,follicular thyroid cancer(FTC)is a well-differentiated malignancy that accounts for about 10-15%of all thyroid cancers and has a 5-year survival rate of about 90%and a 10-year survival rate of about 80%.Since follicular thyroid cancer is difficult to diagnose accurately and often leads to overdiagnosis,appropriate molecular diagnostic tools are increasingly becoming a future trend and provide an excellent diagnostic idea.The aim of this experiment was to investigate the mechanism of LETM1 in follicular thyroid carcinoma and the relevant role of related expression in the development of metastasis and invasion of the thyroid gland.Methods:In vitro,FTC-133 and FTC-238 follicular thyroid cancer cells were selected as the subjects.Firstly,we used Western blot analysis experiments to verify the protein expression of LETM1 in FTC-133 and FTC-238 cells compared with normal thyroid cells,followed by knockdown/overexpression of LETM1 protein by transfection assay.Then,after knockdown/overexpression of LETM1 protein by transfection assay,the corresponding cloning assay,transwell and scratch assay were performed to verify the effect of LETM1 expression on the proliferation,invasion and metastasis of thyroid follicular carcinoma cells,and then western blot assay was applied to detect the changes of corresponding pathway proteins(PI3K,AKT,p-AKT)after knockdown/overexpression of LETM1.The possible regulatory relationship between USP7 and LETM1 was verified by knocking down USP7 and detecting the changes of LETM1 by western blot assay and observing the related indexes after immunoprecipitation assay and corresponding protein inhibitor treatment;at the tissue level,immunohistochemical experiments were performed on postoperative pathological sections of follicular thyroid cancer collected from Dalian Medical University to further confirm The expression of LETM1 in follicular thyroid cancer tissues was further confirmed by immunohistochemical assays on postoperative pathological sections collected from Dalian Medical University.In vivo,12 nude mice were randomly divided into two groups by rearing 4 weeks old:control group and si-LETM1 group with LETM1 knockdown.Nude mice were subjected to tumorigenesis experiment at 5 weeks,FTC-133 cells were selected,Then counting and quantifying FTC-133 approximately 1×10~6 pieces mixed with 0.1 m L of phosphate buffered saline and a mixture of 100u cancer cells and PBS was injected into the axilla of each mouse,and the tumor size was subsequently observed daily,and the tumor was executed and photographed after about 4 weeks.Results:In vitro,immunohistochemical results of postoperative histopathological sections showed that LETM1 was highly expressed in cancerous tissues compared with paracancerous tissues;western blot assay showed that LEMT1 was highly expressed in FTC-133,FTC-238compared with normal thyroid cells.In in vitro experiments,after knocking down LETM1,the proliferation of FTC-133,FTC-238 cells was significantly reduced in the si-LETM1 group compared with the si-NC group and increased in the Oe-LETM1 group compared with the Oe-NC group in clone formation assays.The invasiveness of the cells in the si-LETM1 group was significantly reduced and that in the Oe-LETM1 group was significantly increased.In the scratch assay cells in the si-LETM1 group showed a significant decrease in cell metastasis compared to cells in the si-NC group,and cells in the Oe-LETM1 group showed a significant increase in cell metastasis compared to cells in the Oe-NC group.After knockdown/overexpression of LETM1,the related pathway proteins were examined by western blot assay,and the results showed that PI3K and p-AKT protein expression decreased after knockdown of LETM1 and increased after overexpression of LETM1,and AKT protein was basically unchanged.After knockdown of USP7,LETM1 expression decreased,and immunoprecipitation experiments showed binding between USP7 and LETM1.Application of the corresponding protein inhibitors showed that LETM1 expression decreased after USP7knockdown and shortened the protein half-life of LETM1,so USP7 may be a deubiquitinating enzyme of LETM1 and negatively regulates the degradation of LETM1.In vivo,experiments revealed that the volume size weight of tumors in the si-LETM1group of mice was significantly reduced compared to the control si-NC group.Conclusion:LETM1 is highly expressed in thyroid follicular carcinoma tissues and cells;USP7 can bind and stabilize the level of LETM1 and affect the proliferation,migration,and invasion of thyroid follicular carcinoma through PI3K/AKT pathway.