The Mechanisms Of MiR-99a In The Malignant Biological Phenotype Of Endometrial Cancer Via Regulating Phenotype Of Tams

Objective:To explore the mechanism of miR-99a in the malignant biological phenotype of endometrial cancer via macrophages polarization,so as to lay an experimental and theoretical basis for in-depth study on the development,progression and biologic treatment of endometrial cancer.Methods:First,the expression of CD68 and CD31 in endometrial cancer was detected by immunohistochemistry.CD68~+TAMs in tumor stroma were isolated by MACS,and the expression of miRNAs in TAMs of endometrial cancer with different myometrial invasion depths were compared via miRNAs gene microarray technique.The supernatant of human endometrial cancer cells was used to induce human monocyte U937to differentiate into M2-type macrophages,namely tumor-associated macrophages(TAMs).The synthetic miR-99a mimic fragment was transfected into TAMs.Then,RT-PCR was used to detected the expression of miR-99a,CD68,CD163 and CD206,and ELISA was used to measure the secretion of IL-12,IL-4 and IL-10.Finally,TAMs overexpressing miR-99a were co-cultured with endometrial cancer cells.Transwell assays was used to detect the recruitment of TAMs overexpressing miR-99a.CCK-8,Matrigel invasion assays and Tube formation assay were used to detect the effects of the malignant phenotype of endometrial cancer.Western blot was used to detect the expression of mTOR and its downstream proteins in TAMs overexpressing miR-99a.Results:The high expression of CD68 in endometrial cancer means the TAMs infiltration was positively correlated with tumor myometrial invasion and angiogenesis.The expression of miR-99a in TAMs of>1/2muscle layer patients decreased significantly compared with that in TAMs of>1/2 muscle layer patients.The supernatant of tumor cells successfully induced monocytes to differentiate into M2-type TAMs.After TAMs overexpressing miR-99a,the expression of CD68 and CD163 decreased compared with the control group,but there was no statistical difference in the expression of CD206.The secretion of IL-12 increased while the secretion of IL-4 and IL-10 decreased,suggesting that macrophages were inhibited to M2-type polarization.The number of transmembrane TAMs overexpressing miR-99a was significantly lower than that of the control group.The overexpression of miR-99a in TAMs attenuated endometrial cancer cell proliferation,invasion and angiogenesis.The expression of mTOR and its downstream proteins decreased in TAMs overexpressing miR-99a.Conclusion:miR-99a can reverse the polarization of monocytes to M2 phenotype and inhibit the progression of endometrial cancer.