Mechanism Of High Glucose On The Progression Of Endometrial Cancer Via LncRNA GAS5

Objective:Endometrial carcinoma(EC)is one of the most common malignant tumors in women.Obesity,diabetes and hypertension are all high risk groups.In recent years the incidence of EC are increasing year by year,and the morbidity population is younger.The major strategy for EC treatment is surgical excision.However,the recurrence rate and mortality rate are still high after treatment,and the prognosis is poor.In clinical practice,we found that diabetes mellitus(DM)is a common complication of EC and long-term hyperglycemia increases the risk of occurrence and development of EC.LncRNA is a kind of non-coding RNA that is composed of more than 200 nucleotides.LncRNAs are abnormally expressed in a variety of tumors and play important roles in diverse biological processes.Furthermore,it has been proved to be an important gene expression regulator in the formation of human malignant tumors.The under expression of LncRNA growth arrest-specific transcript 5 has been found in multiple tumors,such as breast cancer,gastric cancer,cervical cancer.The overexpression GAS5 can inhibit cell proliferation,promote cell apoptosis and arrest cell cycle.However,the relationship between GAS5 and DM complicated with EC is largely unknown.The aim of this study is to the effect and mechanism of GAS5 in DM complicated with EC.Methods:1 、 According to the bioinformatics website,we predict the targets affecting EC survival.The clinical of information of the patients with EC which was treated in the Department of Anesthesiology,Tianjin Central hospital of Gynecology and Obstetrics in May 2017-February 2018 period were collected and counted.The patients with EC were divided into EC without DM group and DM with EC group.The clinical and pathological characteristics of the two groups of patients were analyzed.The expression of GAS5 and miR-222-3p in two groups of cancerous tissues were detected by qRT-PCR.Meanwhile,the relationship between the targets and clinicopathological characteristics and the relationship between targets were analyzed.2、HEC-1A and Ishikawa cells were cultured in vitro.Different concentrations of glucose(5.5mM,15 mM,20mM,25mM)were used to intervene EC cells for 48 hours.Next,the expression of GAS5 and miR-222-3p were detected in EC cells by qRT-PCR.Overexpression GAS5 vector and miR-222-3p inhibitor were transfected into HEC-1A and Ishikawa cells.CCK-8 assay was used to detect the cell proliferation.Cell clone formation assay was used to detect the clone forming ability.Cell invasion was detected by Transwell assay.3、Under different high glucose concentrations,the expression of P27 was detected by qRT-PCR.Then the Cytoplsmic and Nuclear RNA purification Kit was used to detect the subcellular localization of GAS5 in Ishikawa cells.Besides,the relationship between GAS5 and miR-222-3p and the relationship between miR-222-3p and P27(CDKN1B)were validated by Dual-Luciferase Reporter Assay System.When overexpression GAS5 and miR-222-3p mimics were co-transfected into Ishikawa cells,the expression of miR-222-3p and P27 were detected by qRT-PCR,and the expression of the target P27 protein was measured by Western Blot assay.Results:1、 We made use of bioinformatics and databases to candidate LncRNA GAS5 for further research.Compared with EC patients with high expression of GAS5,the prognosis of EC patients with low expression of GAS5 was poor and their survival was shortened(P=0.072).In the Clinical data analysis,tumor FIGO stage and grade was significantly higher in DM with EC group than EC without DM group.Furthermore,the lymph node metastasis was higher in EC with DM group(P<0.05).The expression of GAS5 was obviously decreased in EC tissues.The degree of GAS5 decrease was associated with tumor FIGO stage and grade,Diabetes and lymph node metastasis.Compared with patients with EC,the expression of GAS5 was much lower in tumor tissues in DM complicated with EC(P<0.05).The expression of miR-222-3p in EC patients with and without DM was higher than that in adjacent normal endometrial tissues.And it was significantly higher in DM complicated with EC group.The degree of miR-222-3p increase was related to FIGO stage and lymph nodemetastasis(P < 0.05).The expression of GAS5 was negatively correlated with miR-222-3p in both groups(P<0.05).2、Compared with normal endometrial cells,the expression of GAS5 was decreased significantly in HEC-1A and Ishikawa cells,but the expression of miR-222-3p was significantly increased(P<0.01).With the increase of high glucose concentration,the expression of GAS5 had shown a sharp decline,but the expression of miR-222-3p had a continuous rising trend(P<0.05).Overexpression of GAS5 plasmid was transfected into EC cells and expressed stably.Studies of cell biological behavior in vitro showed that high glucose(25mM)could stimulate EC cells proliferation,promote cloning formation and invasion.Overexpression of GAS5 or miR-222-3p inhibitor could reverse the proliferation,cloning formation and invasion of EC cells induced by high glucose(P<0.01).3、Compared with normal endometrial cells,the expression of P27 was decreased significantly in HEC-1A and Ishikawa cells(P<0.01).With the increase of high glucose concentration,the expression of P27 had shown a declining trend(P<0.05).Subcellular localization of GAS5 in Ishikawa cells confirmed that the expression of GAS5 in cytoplasm was higher than that in nucleus.Dual-Luciferase Reporter Assay showed that GAS5 and miR-222-3p could regulate each other directly,while P27(CDKN1B)could also bind to miR-222-3p.Compared with the control group,the expression of miR-222-3p was decreased and the expression of P27 was increased after overexpression of GAS5(P<0.01).MiR-222-3p mimics could decrease the expression of P27 mRNA and protein.When overexpression of GAS5 and miR-222-3p mimics were co-transfected into Ishikawa cells,the expression of P27 mRNA and protein was obviously decreased(P<0.01).Conclusion:1、Diabetes may promote the progression of endometrial cancer and influence the prognosis of patients by affecting the staging and grading of tumor and lymph node metastasis.LncRNA GAS5 could be used as a prognostic indicator for patients with EC.2、LncRNA GAS5 and miR-222-3p might play an important role in DM complicated with EC.Overexpression of GAS5 or miR-222-3p inhibitor can reverse theproliferation,cloning formation and invasion of endometrial cancer cells induced by high glucose.3 、 High glucose via LncRNA GAS5/miR-222-3p influences the proliferation and invasion of EC cells by regulating P27.