| Objective: Endometrial cancer(EC)is one of the three major gynecological malignancies.Risk factors for EC include elevated estrogen levels(caused by obesity,diabetes,and high-fat diet),premature menarche,no delivery,no passing late,Lynch syndrome,?55 years of age,and use of tamoxifen.Emerging evidence shows that the dysregulation of long non-coding RNA is involved in the occurrence and development of a variety of tumors.LncRNA MONC is abnormally expressed in head and neck squamous cell carcinoma,lung cancer,colorectal cancer and acute megakaryocyte leukemia,but the biological function and potential regulation mechanism of MONC in endometrial cancer stem cells and endometrial cancer cells have not been studied.Cancer stem cells are a type of cells that have the unique characteristics of self-renewal and the ability to differentiate into a heterogeneous lineage of tumor cells.Cancer stem cells can initiate tumor formation,promote the proliferation of tumor cells and differentiate into differentiated tumor cells,and play a vital role in the occurrence,development,metastasis,recurrence and drug resistance of malignant tumors.In this study,we aimed to explore the tumor suppressor effect and mechanism of MONC in regulating endometrial cancer stem cells and endometrial cancer cells.Methods: 1?QRT-PCR was used to detect the RNA expression levels of MONC,miR-636 and GLCE in tissues,and to analyze the relationship between their expression levels and clinicopathological parameters.Pearson's coefficient analyzes the correlation between MONC and miR-636.Western Blot detects the protein expression level of GLCE in tissues.2?Predict the binding site between MONC and miR-636 by RNAhybrid,the dual luciferase reporter gene was used to detect the binding site between MONC and miR-636.Flow cytometry was used to obtain endometrial cancer stem cells from Ishikawa cells.Transfect endometrial cancer stem cells,Ishikawa and HEC-1A cells with overexpression and silencing MONC lentivirus,select stable transfected cell lines,and verify the transfection efficiency by qRT-PCR.Endometrial cancer stem cells,Ishikawa and HEC-1A cells were transfected with miR-636 Agomir and Antagomir,and the transfection efficiency was verified by qRT-PCR.The expression of miR-636 after overexpression or silence of MONC and the expression of MONC after overexpression or silence of miR-636 were detected by qRT-PCR.The spheroid formation experiment detects the effect of MONC in endometrial cancer stem cells on the speed of spheroid formation.CCK-8 assay detects the effect of MONC on the proliferation of endometrial cancer stem cells,Ishikawa and HEC-1A cells.The cell invasion assay detects the effect of MONC on the invasion of endometrial cancer stem cells,Ishikawa and HEC-1A cells.The cell cycle experiment detects the effect of MONC on inducing endometrial cancer stem cells,Ishikawa and HEC-1A cell cycle arrest.The cell apoptosis test detects the effect of MONC on the apoptosis of endometrial cancer stem cells,Ishikawa and HEC-1A cells.3?Predict the binding site between miR-636 and GLCE by miRDB,the dual luciferase reporter gene was used to detect the binding site between miR-636 and GLCE.Transfect miR-636 antagomir into endometrial cancer stem cells,Ishikawa cells and HEC-1A cells that stably overexpress MONC,and transfect miR-636 agomir into endometrial cancer stem cells that stably knock down MONC,Ishikawa and HEC-1A cells.The spheroid formation experiment detects the inhibitory effect of miR-636knockdown-mediated MONC on the rate of spheroid formation in endometrial cancer stem cells.CCK-8 assay detects the effect of miR-636 knockdown-mediated MONC on the inhibition of endometrial cancer stem cells,Ishikawa and HEC-1A cells proliferation.The cell invasion assay was used to detect the effect of miR-636 knockdown on the inhibition of endometrial cancer stem cells,Ishikawa and HEC-1A cell invasion.The cell cycle experiment detects the effect of miR-636 knockdown-mediated MONC knockdown on the cell cycle arrest of endometrial cancer stem cells,Ishikawa and HEC-1A.Apoptosis experiments were used to detect the effect of miR-636 knockdown on the enhancement of endometrial cancer stem cells,Ishikawa and HEC-1A cell apoptosis.Transfect endometrial cancer stem cells,Ishikawa and HEC-1A cells with overexpression and silencing GLCE plasmid,and use the same method to detect the effect of transfection on the speed of endometrial cancer stem cell spheroid formation,as well as on endometrial cancer stem cells and The effect of endometrial cancer cell proliferation,invasion,cycle and apoptosis.Western blot was used to detect the effect of MONC combined with miR-636 on GLCE protein expression and EMT related index protein expression.The effect of MONC combined with miR-636 on the tumor size and GLCE of the transplanted tumor in nude mice was tested by the nude mouse transplanted tumor model.Results: 1?LncRNA MONC is low expressed in endometrial cancer,and the expression is related to the depth of invasion and FIGO staging.miR-636 is high expressed in endometrial cancer,and the expression is related to the depth of invasion and FIGO staging.The expression of miR-636 is negatively correlated with the expression of MONC.GLCE is low expressed in endometrial cancer,and the expression is related to differentiation,depth of invasion and FIGO staging.(All P<0.05).2?The bioinformatics database(RNAhybrid)predicted that there is a binding site between MONC and miR-636,and the dual luciferase reporter gene verified the binding site between MONC and miR-636.Dual fluorescence in situ hybridization experiments showed that MONC and miR-636 were relatively co-localized in the cytoplasm of Ishikawa cells.qRT-PCR showed that the expression of miR-636 decreased in the MONC overexpression group,while the expression of miR-636 increased in the MONC knockdown group,and the expression of MONC decreased in the miR-636 agomir group,while the expression of MONC increased in the miR-636 antagomir group.CCK-8 assay,sphere formation experiment,transwell invasion experiment,cell cycle experiment and apoptosis experiment showed that MONC inhibited the malignant biological behavior of endometrial cancer stem cells and endometrial cancer cells.(All P<0.05).3?The bioinformatics database(miRDB)predicted that there is a binding site between GLCE and miR-636,and the dual luciferase reporter gene experiment verified the binding site between MONC and miR-636.Westernblot showed that MONC rescued the promotion of AntagomiR-636 on GLCE protein expression.CCK-8 assay,sphere formation experiments,Transwell invasion experiments,cell cycle experiments and apoptosis experiments showed that knockdown of miR-636 mediates the tumor suppressor effect of MONC overexpression in ECSCs and ECCs.CCK-8 assay,sphere formation experiment,transwell invasion experiment,cell cycle experiment and apoptosis experiment showed that MONC inhibited the malignant biological behavior of endometrial cancer stem cells and endometrial cancer cells.Western blot experiments showed that MONC combined with miR-636 inhibited the EMT process of tumors.In addition,we have verified the tumor suppressor effect of MONC in nude mice,and rescue experiments showed that miR-636 can rescue the tumor suppressor effect of overexpression of MONC.(All P<0.05).Conclusion: LncRNA MONC is low expression in endometrial cancer and its overexpression can inhibit the development of malignant biological behaviors of endometrial cancer stem cells and endometrial cancer cells.MONC inhibits the malignant biological behavior of endometrial cancer stem cells and endometrial cancer cells by regulating the miR-636/GLCE axis.The MONC/miR-636/GLCE axis may provide a new therapeutic strategy for the treatment of human endometrial cancer. |
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