| BackgroundEndometrial carcinoma(EC),also known as uterine cancer,often occurs in perimenopausal and postmenopausal women.Adenocarcinoma is the most common type of endometrial cancer in clinical diagnosis and treatment.It is widely known as endometrioid adenocarcinoma.In recent years,epidemiological studies on endometrial cancer about abroad and China have been conducted that the incidence of endometrial cancer has exceeded the cervical lesions and is the number one in female genital tract malignant tumors in European and American countries and Mainland China.With the continuous detection of young female cases,the age of patients is becoming more and more young.According to the pathological characteristics and clinical diagnosis and treatment characteristics of endometrial cancer,EC can be roughly divided into two subtypes:I endometrioid adenocarcinoma(EEC)is estrogen sensitive,and the lesions mainly originate from proliferative endometrial tissue.The main pathogenesis is related to the long-term stimulation of high level estrogen in the circulatory system of female patients without the antagonism of progesterone.Compared with type II,EEC is commonly seenperimenopausal women and young women.The main pathological types include high and moderately differentiated endometrial adenocarcinoma,a small amount of mucinous adenocarcinoma and some poorly differentiated adenocarcinomas.Its molecular mechanism involves genes mutations such as PTEN,K-ras and CTNNB1 and associated with microsatellite instability(MSI)caused by DNA mismatch repair genes.The degree of differentiation of I type tumor cells is often better,the metastasis and invasion ability of tumor cells is weak,and the progression of patients is slow,and the non tumor survival time of I patients is longer.Type II EC,also known as non endometrioid endometrial carcinoma,accounts for about 1/5 non estrogen dependence of the total number of endometrium cancers,and the lesions are common in the endometrium of the postmenopausal atrophy.The main pathological types of Type II include plasma breast cancer liquid endometrial adenocarcinoma,transparent cell carcinoma and adenosscale squamous cell carcinoma,which are not associated with the level of female progestin,occurs in postmenopausal women.The clinical diagnosis of type ? patients is mostly in the late clinical stage,and the patient's multiple organs have been metastased.About 1/2 of the patients relapsed within five years after the operation,and eventually died.Changes in the molecular biological phenotype of type ? endometrial carcinoma are caused by mutations in TP53 and P16,as well as Her2/neu and Nrf2 genes.Tumor markers have a very important value in the clinical diagnosis of tumor screening,monitoring and evaluation of the disease,and the prognosis of patients.In recent years,many biochemical markers,such as CA125 and HE4,have been widely used in the clinical diagnosis of endometrial cancer and the detection of patients' prognosis.Benign diseases of endometrium epithelia,such as meningitis and adenomyosis,also cause abnormal elevation of CA125,which is often lack of specificity in the diagnosis of endometrial carcinoma.With the guidance of immunohistochemistry,the molecular typing of endometrial carcinoma can better reflect the biological behavior of the tumor,and can promote the individualization and precision of the treatment,such as PTEN and TP53.At present,the clinical blood biochemical indexes and pathological immuno histochemical indexes are far from meeting the clinical needs.It is very urgent to find new markers of tumor invasion and metastasis,to further improve the clinical diagnosis of endometrial carcinoma,to improve the treatment of patients and to improve the quality of life of the patients.RbAp48(retinoblastoma binding protein 4)is a specific nuclear expression protein associated with a variety of tumors.RbAp48 as a variety part of chromatin remodeling and nucleosome assembly,modification of complex components,can directly regulate the occurrence and development of tumor through altering the conformational of chromosome and histone such as E2F,CREB,NF-?B etc.It has been reported that RbAp48 protein is closely related to the occurrence of a variety of tumors,such as breast cancer and liver cancer,but it has not yet been reported about endometrial lesions to date.Wnt/?-catenin signaling plays an important role at the early stage of human embryonic organ differentiation,cell migration and tissue repair and regeneration.According to the literature data,the occurrence of endometrial carcinoma is closely related to the abnormal activation of the classic Wnt/?-catenin signaling pathway.The mutation of PTEN gene of patients with endometrioid adenocarcinoma can lead to the weakening of the synthesis of the ?-catenin degradation complex(GSK-3 beta/APC,etc.)and the accumulation of ?-catenin protein in the cytoplasm enters the nucleus?In addition to PTEN gene mutation inactivation factor,endometrial adenocarcinoma patients often have the CTNNB1 gene mutation encoding ?-catenin protein.The ?-catenin protein is dissociated from E-cadherin and bound to the TCF/LEF protein family in the nucleus.The target genes downstream of the classic Wnt/?-catenin signaling pathway,such as c-myc,cyclinDl,and the transcriptional regulators of the upstream of EMT,have been overexpressed,inducing the unlimited proliferation and dedifferentiation of normal cells and the change of cell morphology,which leads to the cell production and migration of the tumor.The latest research data show that estrogen may act as an inducer of gene mutation and can also affect the abnormal activation of the Wnt/?-catenin signaling pathway.ObjectiveTo explore the relationship between RbAp48 protein and Wnt/beta-catenin signaling pathway,and whether RbAp48 can regulate the proliferation,migration and invasion of endometrial adenocarcinoma cells.by regulating the Wnt/beta-catenin signaling pathway.Methods1.immunohistochemical staining(IHC)was used to detect the expression of RbAp48 and beta-catenin in endometrial carcinoma and matched adjacent tissues,and the relationship between them2.Establishment of a low expression of RbAp48 in KLE and Ishikwa cell lines by the lentiviral vector3.Using transwell and Matrigel matrix to detected the change of migration and invasion in KLE and Ishikwa cell lines of low expression of RbAp48 by the lentiviral vector.4.Using CCK-8,clone formation,EdU,FACS to detected the change of migration and invasion in KLE and Ishikwa cell lines of low expression of RbAp48 by the lentiviral vector5.Western Blot and RT-qPCR were performed to investigate the role of RbAp48 protein in Wnt/?-catenin signaling pathway.6.Western Blot and RT-qPCR were performed to investigate the effects of RbAp48 on the epithelial mesenchymal transition process(EMT)of endometrial carcinoma(E-cadherin,Snail,Twist,N-cadherin and Vimenti)7.Constructing a nude mouse model with low expression of RbAp48 to simulate the development of endometrial carcinoma in vivo,and to further verify the role of RbAp48 in the proliferation,migration and invasion of endometrial carcinoma.Results1.The result showed that Compared with the para cancerous tissue,the expression of RbAp48 and ?-catenin in the endometrium cancer tissues is significantly enhanced(P<0.05).There was a significant inverse correlation between RbAp48/?-catenin levels and differentiation of tumor.A significant positive correlation was observed between RbAp48 and?-catenin(P<0.01).2.The expression of RbAp48 was significantly down-regulation infected by the lentiviral vector.3.The results of trans well migration/colony formation/EdU/CCk8/FACS show that RbAp48 can promoted the migration/proliferation/invasion of endometrial carcinoma cells(P<0.05).4.RbAp48 down-regulation inhibited the expression of ?-catenin,c-myc,cyclinDl,in at mRNA level and protein level,the Wnt/?-catenin signaling pathway was inhibited(P<0.05).5.RbAp48 down-regulation increased the expression of E-cadherin(epithelial phenotype)inhibited the expression of Twist,N-cadherin,Snail,Vimentin(mesenchymal phenotype),RbAp48 down-regulation inhibited the epithelial mesenchymal transition process(EMT)of endometrial carcinoma(P<0.05).6.tumor xenografts in nude mice:the average tumor volume and growth speed of RbAp48 down-regulation were lower than Con groupe,RbAp48 down-regulation inhibited the proliferation of endometrial cancer cells in vivo.(P<0.05).Conclusion1.Down regulation of RbAp48 inhibits the proliferation of endometrial adenocarcinoma cells,and its specific molecular mechanism may be related to activation of the classical Wnt/beta-catenin signaling pathway.2.The down-regulation of RbAp48 reverses the transformation of epithelial mesenchymal transition in endometrial carcinoma cells and inhibits metastasis and invasion of endometrial adenocarcinoma cells,and its specific molecular mechanism may be related to the downregulation of Snail and twist downstream of the classic Wnt/beta-catenin signaling pathway. |
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