Study On The Mechanism Of CKMT1B Regulating The Function Of Endometrial Cancer Cells Through Akt/mTOR Pathway

Background:Uterine corpus endometrial carcinoma(UCEC)has a high mortality rate in the world.There are many pathogenic factors of the disease,such as obesity,aging,early menarche,late menopause,nulliparous women and postmenopausal estrogen therapy.Although many cases of endometrial cancer were diagnosed in the early stage,but a small number of patients with poor prognosis,and have a high recurrence and metastasis rate.It is very important to find the biomarkers related to apoptosis to indicate the prognosis of endometrial cancer.After transcriptome microarray screening,CKMT1 B gene was found to be differentially expressed in endometrial cancer and normal tissues.The gene is a mitochondrial creatine kinase,which can promote the growth of tumor cells to resist apoptosis.The increased expression of CKMT1 B not only occurs in liver malignant lesions,but also in gastric cancer,lung cancer and other tumors with poor prognosis.Recently,CKMT1 B has been found to be an independent prognostic indicator.Therefore,the specific mechanism of CKMT1 B in the pathogenesis and progression of endometrial cancer is worthy of further study.Research objective:1.To investigate the expression of CKMT1 B in normal endometrial tissues,cancer tissues and cells.2.The purpose of this study was to investigate the effects of si RNA interferes with CKMT1 B expression and construct recombinant adenovirus ad CKMT1 B to make CKMT1 B overexpression on the proliferation,apoptosis,migration and invasion of endometrial carcinoma,and to explore the regulatory mechanism of CKMT1 B gene in Ishikawa cells.Research methods:1.Using R language and bioinformatics methods to obtain clinical endometrial cancer samples from GEO database for statistical analysis.2.The expression of CKMT1 B in normal endometrium and cancer tissues was compared by immunohistochemistry(IHC),RT-qPCR and Western blotting.3.After CKMT1 B down-regulation and overexpression,cell invasion was detected by Transwell chamber,and cell migration was evaluated by cell scratch test.4.CCK8 assay,cell clone formation assay and cell immunofluorescence assay(IF)were used to detect the growth and proliferation of cell after CKMT1 B down-regulation and overexpression.5.Flow cytometry(FCM)was used to detect the effect of CKMT1 B expression on apoptosis rate.Two kits were used to detect the expression of ATP and lactate dehydrogenase(LDH)before and after transfection.6.RT-qPCR and Western blotting were used to detect the expression of the characteristic markers of cell proliferation,migration,apoptosis and invasion after CKMT1 B down-regulation and overexpression,and to detect the changes of AKT / mTOR signaling pathway proteins.Research conclusion:1.CKMT1 B is highly expressed in endometrial carcinoma and is associated with poor prognosis.2.Down regulation of CKMT1 B can inhibit the proliferation,migration and invasion of Ishikawa cells,and promote the apoptosis of Ishikawa cells.3.Down regulation of CKMT1 B can inhibit AKT / mTOR pathway,affect ATP production and the activity of LDH.4.Overexpression of CKMT1 B can affect the proliferation,migration and invasion of Ishikawa cells,and activate AKT / mTOR pathway.