Study On The Mechanism Of UBE2C Upregulation And Its Effect On Regulating Cell Migration And Invasion Through P53 In Endometrial Cancer

Part 1.The expression and clinical significance of UBE2 C in endometrial cancerObjective: To investigate the expression of UBE2 C in endometrial cancer tissues,and to study its relationship with the clinicopathological characteristics and prognosis of endometrial cancer.Methods: The expression levels of UBE2 C in a normal endometrium and endometrial cancer tissues were analyzed by real-time fluorescent quantitative polymerase chain reaction(q RT-PCR)and western blot.The data of 543 endometrial cancer samples and 23 normal samples from the Cancer Genome Atlas(TCGA)database was used to verify the expression of UBE2 C,and explore the relationship between UBE2 C expression and tumor recurrence,pathological grading,FIGO stage,subtype and prognosis.Resluts: 1.The m RNA and protein levels of UBE2 C in endometrial cancer tissues were significantly higher than those in normal endometrial tissues.2.TCGA data verified that UBE2 C was significantly up-regulated in endometrial cancer tissues,high UBE2 C expression was more common in patients with tumor recurrence,higher pathological grade,higher FIGO stage,highly malignant pathological type,and the overall survival of patients with high expression of UBE2 C was lower than patients with low UBE2 C expression.Conclusion: UBE2 C was highly expressed in endometrial cancer,and it was related to the poor prognosis of patients,suggesting that UBE2 C may play an important role in the development of endometrial cancer.Part 2.UBE2 C promotes the proliferation,migration and invasion of endometrial cancer cellsObjective: To investigate the effect of UBE2 C on the biological behavior of endometrial cancer cells.Methods: The expression levels of UBE2 C in four endometrial cancer cell lines(Ishikawa,RL95-2,HEC-1B,KLE)were analyzed by western blot.Short hairpin RNA(sh UBE2C)and overexpression plasmids targeting UBE2 C were constructed,and were used to transfect Ishikawa and KLE cells respectively;the expression level of UBE2 C and EMT-related markers after cell transfection was detected by q RT-PCR and western blot.CCK8 test was used to assess the ability of cell proliferation.Transwell assay was applied to assess the ability of cell migration and invasion.Results: 1.UBE2 C expression was higher in Ishikawa and RL95-2 cells than KLE and HEC-1B cells.3.Compared with the control group,the proliferation,migration and invasion of Ishikawa cells in the sh UBE2 C group were significantly inhibited,the expression of E-cadherin increased,and the expression of Vimentin decreased;in contrast,UBE2 C overexpression enhanced the ability of cell proliferation,migration and invasion,and resulted in the downregulation of E-cadherin and upregulation of vimentin in KLE cells.Conclusion: UBE2 C promoted endometrial cancer cell proliferation,migration,invasion and EMT in vitro.Part 3.UBE2 C promotes EC migration and invasion by down-regulating p53Objective: To explore the role of p53 in UBE2C-induced EC cell migration,invasion and EMT and the regulatory mechanism of UBE2 C on p53.Methods: The expression level of UBE2 C and p53 after UBE2 C knockdown and overexpression in Ishikawa and KLE cells were detected by q RT-PCR and western blot.Cycloheximide(CHX)and proteasome inhibitor MG132 were used to treat endometrial cancer cells,and western blot was used to detect the expression of p53.Co-immunoprecipitation technology was used to detect ubiquitination of p53 in Ishikawa and KLE cells after UBE2 C downregulation and overexpression.Transwell chambers and western blot were used to examine the migration,invasion and the expression of E-cadherin and Vimentin after UBE2 C knockdown and overexpression along with p53 knockdown and overexpression in Ishikawa and KLE cells.Results: 1.The p53 protein expression was upregulated by UBE2 C knockdown in Ishikawa cells,but downregulated by UBE2 C overexpression in KLE cells.However,the p53 m RNA level was not influenced after UBE2 C knockdown or overexpression.2.p53 ubiquitination was enhanced by UBE2 C overexpression in KLE cells and diminished by UBE2 C knockdown in Ishikawa cells.The stability of p53 protein increased after UBE2 C knockdown,but UBE2 C overexpression decreased the stability of p53 protein,and this process can be reversed by the proteasome inhibitor MG132.3.p53 knockdown partially rescued the migration and invasion of UBE2C-knockdown Ishikawa cells,whereas p53 overexpression partly inhibited the increased migration and invasion in UBE2 C overexpressing KLE cells.E-cadherin upregulation and vimentin downregulation caused by UBE2 C knockdown were partially rescued by transfection with p53-sh RNA.Conversely,ectopic p53 expression significantly inhibited E-cadherin downregulation and vimentin upregulation in UBE2C-expressing cells.Conclusion: UBE2 C may promote ubiquitination-dependent degradation of p53,and promote EC migration and invasion,at least partially,via p53.Part 4.The regulatory mechanism of estrogen on UBE2CObjective: To explore the regulatory relationship between estrogen and UBE2 C and the regulatory mechanism of estrogen on UBE2 C.Methods: The expression levels of UBE2 C and ERα in four endometrial cancer cell lines and endometrial cancer specimens were analyzed by western blot and immunohistochemistry,respectively.Ishikawa,RL95-2 and HEC-1B cells were treated with estrogen at different concentrations,western blot and q RT-PCR were used to detect the expression of UBE2C;Ishikawa and RL95-2 cells were stimulated with estrogen after pretreating cells with the estrogen receptor inhibitor ICI182780,and the protein expression of UBE2 C was detected by western blot.Luciferase reporter was used to analyze the activation effect of estrogen on UBE2 C promoter.Transwell and western blot experiments were applied to analyze the influence of sh UBE2 C on estrogen-induced EC migration,invasion,p53,E-cadherin and Vimentin expression.Five-week-old female nude mice were selected to be injected subcutaneously with negative or UBE2C-silenced Ishikawa cells,followed by subcutaneous injection of estrogen once a week.Mice were sacrificed 24 days later.The tumors were removed,weighed,and tissues were analyzed the expression of E-cadherin and Vimentin by immunohistochemistry.Results: 1.Among the four endometrial cancer cell lines(correlation coefficient r =-0.9609,P = 0.0391)and endometrial cancer tissue specimens(correlation coefficient r =-0.6640,P = 0.0363),the UBE2 C and ERα The expression levels were positively correlated.2.After estrogen treatment,the UBE2 C expression of ERα-positive Ishikawa and RL95-2 cells increased in a concentration-dependent manner,and the estrogen receptor inhibitor ICI182780 could block the effect of estrogen on upregulating UBE2 C.3.Whereas,there was no significant change of UBE2C expression in the ERα-negative HEC-1B cells after estrogen treatment.Morever,when ESR1 was overexpressed in HEC-1B cells,UBE2 C expression levels were significantly increased after treatment with estrogen.4.An ESR1 binding site(-620 bp--601 bp)was predicted in the UBE2 C promoter region using JASPAR database.The luciferase report showed that estrogen enhanced the transcriptional activity of UBE2 C through this site.5.Estrogen promoted the migration and invasion of EC cells,downregulated the protein expression of E-cadherin and p53,and upregulated the protein expression of Vimentin.After downregulating UBE2 C,these effects of estrogen were reversed.6.Compared with the control,the weight of transplanted tumor and Vimentin expression increased,E-cadherin expression decreased in the group of estrogen treatment,while in the sh UBE2 C group,tumor weight and Vimentin expression decreased,E-cadherin expression increased.Compared with the estrogen-treated group,tumor weight and Vimentin expression decreased,E-cadherin expression increased in the estrogen + sh UBE2 C group.Conclusion: ERα and UBE2 C expression were positively correlated in endometrial cancer tissues and cell lines,and estrogen upregulated the expression of UBE2 C by promoting the binding of ERα to the UBE2 C promoter region.Downregulating UBE2 C inhibited estrogen-induced migration,invasion,and EMT of endometrial cancer cells in vivo and in vitro.