The Function And Mechanism Of MiR-152 Targeting CDC25B In Endometrial Cancer

Objective:To explore the expression of miR-152 in endometrial cancer tissues and cells and its function,and further explore the possible mechanism by screening and identifying its target genes,to provide theoretical basis for the pathogenesis and molecular typing of endometrial cancer and explore new directions for early diagnosis and targeted therapy of endometrial cancer.Method:Part One:The expression of miR-152 in endometrial cancer tissue and its correlation with different clinicopathological features: Tumor tissue specimens from 35 cases of endometrial cancer were selected as the study group,20 cases of patients who underwent total hysterectomy due to uterine fibroids during the same period were selected as control group.The expression level of miR-152 in two groups was detected by q RT-PCR(Quantitative Real-time Polymerase Chain Reaction).In addition,we also contrasted the expression of miR-152 in groups with different clinicopathological features.Part Two:The effects of miR-152 on the biological function of endometrial cancer cell lines: The expression of miR-152 in different human endometrial cancer cell lines was detected by q RT-PCR.Cells with a significant decrease in relative expression were selected and transfected with miR-152 mimics to overexpress miR-152 in endometrial cancer cells.The proliferation and cell cycle regulation of endometrial cancer cell lines after transfection of miR-152 mimics were detected by MTT,Brd-U and flow cytometry.Part Three: The regulation mechanism of miR-152 on the proliferation and cell cycle of endometrial cancer cells:Bioinformatics software was used to predict target gene that might be regulated by miR-152;This was verified by the use of luciferase reporter assay;The expression of CDC25B(cell division cycle protein25B)in human endometrial cancer cell after transfection of miR-152 mimics was detected by Western blot.The expression of CDC25 B in human endometrial cancer cell after transfection of si-CDC25 B was detected by Western blot;The proliferation and cell cycle regulation of endometrial cancer cells after transfection of si-CDC25 B were detected by MTT,Brd-U and flow cytometry.miR-152 mimics and pc DNA3.1-CDC25 B or pc DNA3.1-empty vectors were co-transfected into KLE cells,and cell proliferation and cell cycle regulation were detected by MTT,Brd-U and flowcytometry,respectively,to determine whether CDC25 B affected the inhibitory effect of miR-152.Result:Part One : The expression level of miR-152 in endometrial cancer tissue samples was statistically lower than that in normal tissue samples(P <0.05);According to FIGO staging,the expression level of miR-152 in patients with stage III + IV was statistically lower than that in stage Ⅰ + Ⅱ;The expression of miR-152 in the patients with vascular tumor plugs was statistically lower than that without vascular tumor plugs;The expression of miR-152 in the patients with muscle infiltration depth≥1/2 was statistically lower than that with muscle invasion depth < 1/2;The expression of miR-152 in the patient of PR-negative was statistically lower than the patients of PR-positive(P <0.05).The expression level of miR-152 has no statistical difference between the patients of different pathological types 、 ER-positive or negative、the athological grade in endometrioid adenocarcinoma.(P> 0.05).Part Two:In human endometrial cancer cell lines HEC-1B,Ishikawa,KLE and RL-952,the expressions of miR-152 in HEC-1B and KLE cells decreased statistically;Human endometrial cancer cells HCC-1B and KLE cells were transfected with miR-152 mimics,the expression level of miR-152 was statistically increased,the cell proliferation was decreased,and the cell percentage of G2/M phase was increased.Part Three:Targetscan analysis predicted that miR-152 binding sites were present in the 3’UTR of CDC25 B.The luciferase reporter assay showed that the relative luciferase activity of KLE cells co-transfected with CDC25B-WT/CDC25B-MUT and miRNA-152 mimics was decreased compared with the control group.The expression of CDC25 B was decreased in KLE cells transfected with miR-152 mimics.Compared with the control group,after transfection of si-CDC25 B the expression of CDC25 B in human endometrial cancer cells in KLE and HEC-1B was statistically decreased,and the cell proliferation ability was decreased;The percentage of cells in G2/M phase was increased;Compared with the control group,KLE cells co-transfected with miR-152 mimics and pc DNA3.1-CDC25 B showed increased cell proliferation and decreased G2/M phase percentage.Conclusion:(1)The expression of miR-152 in endometrial cancer tissue samples is lower than that in normal endometrial cancer tissue samples;The expression of miR-152 in endometrial cancer tissue samples maybe related to the clinicopathological stage,vascular space invasion,depth of muscular layer invasion and progesteronereceptor expression.(2)Overexpression of miR-152 inhibits the proliferation of human endometrial cancer cell lines KLE and HEC-1B,and induce G2/M phase arrest,suggesting that miR-152 had an inhibitory effect on the proliferation endometrial cancer cell.(3)Downregulation of CDC25 B inhibits the proliferation of human endometrial cancer cell lines KLE and HEC-1B,and induce G2/M phase arrest,which is similar to the effection of overexpression of miR-152;Compared to control,co-transfection of miR-152 mimic and pc DNA3.1-CDC25 B,the proliferation ability of KLE cell is increased,while cell G2 / M phase arrest decreased,suggesting that miR-152 inhibits the proliferation of human endometrial cancer cells by inhibiting the expression of CDC25 B.To sum up,miR-152 targeting CDC25 B plays an important role in the occurrence and development of endometrial cancer.