| BackgroundEndometrial cancer(endometrial cancer,EC)is one of the most common malignant tumors of the female reproductive system.With the change of Chinese people's lifestyle and habits,the morbidity and mortality of endometrial cancer are also increasing year by year,seriously threatening the health of Chinese women.Most early endometrial cancer patients can be successfully treated by surgery or chemotherapy and other treatment methods,but for advanced patients,the treatment methods and prognosis are still not ideal.At present,the pathogenesis of endometrial cancer is still not clear,and there is a lack of detection indicators for early diagnosis,recurrence and metastasis.Melanoma nuclear protein 18(Melanoma nuclear protein 18,Mel-18)is one of the core members of the polycomb group genes(PcG)family and plays an important role in embryonic development,cell proliferation.?apoptosis and self-renewal and other processes.Previous studies have shown that the abnormal expression of Mel-18 protein is closely related to the occurrence and development of various human malignant tumors.However,the expression of Mel-18 in endometrial cancer and its effect on the biological behavior of endometrial cancer cells and its potential mechanism have not been reported.Purpose1.In this study,we examined the expression of Mel-18 in endometrial cancer tissues and normal endometrial tissues and analyzed its correlation with the clinicopathological characteristics.2.To further validate the function of Mel-18 in vitro,investigate the influence of abnormal expression of Mel-18 on the biological behavior of endometrial cancer cell lines and its potential mechanism.It provides a new direction and theoretical basis for the study of endometrial cancer and Mel-18.Methods1.Total 45 human endometrial tissue specimens were collected,including 30 cases of endometrial cancer tissue,include type I:23 cases;type II:7 cases;15 cases of normal endometrial tissue.Immunohistochemical staining(IHC)and reverse transcription-quantitative polymerase chain reaction(RT-qPCR)assays were performed to detect the expression of Mel-18 in endometrial cancer tissues and normal endometrial tissues.The correlation between Mel-18 expression and the clinical and pathological characteristics of patients was analyzed.2.Endometrial cancer cell line RL95-2 was selected to construct Mel-18 overexpressed and low-expressed cell lines by transfected with Mel-18 overexpressed adenovirus and Mel-18siRNA.After cell transfection,the expressions of Mel-18 mRNA and protein in cells were detected by RT-qPCR and Western blot assays.3.MTT assay was performed to detect the effect of Mel-18 on cell proliferation ability.4.Plate cloning assay was conducted to detect the effect of Mel-18 on cell cloning ability.5.Transwell chamber experiment was conducted to detect the effect of Mel-18 on cell migration ability.6.The effect of Mel-18 expression on cell cycle was detected by flow cytometry.7.Western blot assay was used to detect the effect of Mel-18 on PI3K/Akt/mTOR signal transduction pathway and other related proteins expression.Results1.Expression of Mel-18 in endometrial cancer tissues and normal endometrial tissues.IHC and RT-qPCR results showed that Mel-18 was significantly highly expressed in endometrial cancer tissues and low expression in normal endometrial tissues(P<0.05).There was no significant correlation between the high expression of Mel-18 in endometrial cancer tissues and the clinical and pathological characteristics of endometrial cancer patients,such as staging,grading,age and tumor size(P>0.05).2.Construction of Mel-18 over-expressed and low-expressed cell lines.Cells transfected with Mel-18 over-expressed adenovirus and Mel-18-siRNA can effectively up-regulate or down-regulate the expression of Mel-18mRNA and protein in RL95-2 cells(P<0.05).3.The effect of over-expression of Mel-18 on the biological behavior of endometrial cancer cells.After over-expression of Mel-18 in cells,the proliferation,cloning and migration abilities of cells were increased,promoted the progress of cell cycle,and the proportion of G2/S cells in the cell cycle was increased(P<0.05).4.The effect of down-expression of Mel-18 on the biological behavior of endometrial cancer cells.Knocked down the expression of Mel-18 in cells by transfected with Mel-18siRNA,cell proliferation,cloning and migration abilities were significantly inhibited,cell cycle was arrested in the G1 phase,the proportion of G2/S cells was decreased(P<0.05).5.The effect of Mel-18 expression on PI3K/Akt/mTOR signal pathway.Upregulation of Mel-18 increased the expression of phosphorylated Akt and mTOR in cells,the expression of c-myc?Bcl-2 proteins were also increased.On the contrary,when the expression of Mel-18 was decreased in cells,the phosphorylation level of the pathway proteins was decreased,the expression of c-myc and Bcl-2 proteins were also inhibited(P<0.05).Conclusion1.Mel-18 is highly expressed in endometrial cancer tissues.Mel-18 may play an oncogenic role in the occurrence and development of endometrial cancer.2.The abnormal expression of Mel-18 affects the biological behavior of endometrial cancer cells.Mel-18 can promote cell proliferation,cycle progression and migration abilities by positively regulate the activation of PI3K/Akt/mTOR signal transduction pathway. |
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