肿瘤相关巨噬细胞相关性miR-99a对子宫内膜癌细胞生长和侵袭的调控作用

Effects of tumor-associated macrophage-related miR-99a on the cell growth and invasion of endometrial cancer cells

背景与目的:肿瘤相关巨噬细胞(tumor-associated macrophage,TAM)浸润与肿瘤进展密切相关,但作用机制尚不明确,因此,探索miR-99a对单核巨噬细胞极化的影响及其对子宫内膜癌(endometrial cancer,EC)细胞生长、侵袭的影响。方法:检测EC组织中巨噬唾液酸蛋白(macrosialin)CD68表达并分析其与临床病理学特征之间的关系;运用人EC细胞系HEC-1B、RL95-2培养上清液诱导人单核细胞U937向TAM(M2型巨噬细胞)分化;将人工合成的miR-99a模拟物片段转染至诱导后的巨噬细胞,转染后运用实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR)及流式细胞术检测巨噬细胞相关因子CD68、CD163以及巨噬细胞甘露糖受体(mcrophage mannose receptor)CD206表达量变化,并运用酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测巨噬细胞分泌相关细胞因子IL-12、IL-4和IL-10分泌量变化;将转染miR-99a的诱导后巨噬细胞与EC细胞共培养,运用细胞计数试剂盒(cell counting kit-8,CCK-8)和transwell法检测其对EC细胞增殖和侵袭能力的影响,并初步分析其可能的作用机制。结果:EC组织CD68高表达并与肿瘤肌层浸润及血管生成呈正相关;肿瘤细胞培养上清液成功诱导单核细胞向M2型TAM极化。转染miR-99a后单核细胞组CD68及CD163表达较对照组下降(P<0.01),而CD206表达差异无统计学意义(P>0.05),流式细胞术进一步证实上述表达变化;ELISA结果发现,转染miR-99a诱导后巨噬细胞中IL-12分泌增多(P<0.01),而IL-4、IL-10分泌减少(P<0.01),提示巨噬细胞向M2型极化受抑制。将诱导后巨噬细胞与EC细胞共培养,共培养后EC细胞增殖侵袭能力较对照组增加,而转染miR-99a模拟片段至诱导后巨噬细胞能够抑制其对增殖(P<0.01)及侵袭能力的促进作用(P<0.05)。诱导后巨噬细胞中过表达miR-99a后细胞中mTOR及其通路受到抑制。结论:EC间质巨噬细胞浸润与肿瘤肌层浸润及血管新生相关,miR-99a能够逆转单核细胞向M2表型极化,并抑制EC细胞介导TAM的促EC细胞生长和侵袭作用,其作用可能通过调控mTOR通路产生。

Background and purpose:The infiltration of tumor-associated macrophage(TAM)is closely related to tumor progression,but the mechanism of its action is not yet clear.Therefore,we explored the effects of miR-99 a on macrophage polarization and its putative effects on the cell proliferation and invasion of endometrial cancer cells.Methods:We detected the expression of CD68 in endometrial cancer tissues and analyzed its relationship with the clinicopathological parameters of patients.The supernatants of human endometrial cancer cells HEC-1 B and RL95-2 were used to induce human monocyte U937 to differentiate into M2-type macrophages,namely TAMs.The synthetic miR-99 a mimic fragment was transfected into induced TAM.After transfection,real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and flow cytometry were used to explore the expression of macrophage-associated factors CD68,CD163 and CD206,and enzyme-linked immunosorbent assay(ELISA)were used to measure the secretions of IL-12,IL-4 and IL-10 upon induction.TAMs overexpressing miR-99 a were co-cultured with endometrial cancer cells.Cell counting kit-8(CCK-8)and Matrigel invasion assay were used to detect its effects on the cell proliferation and invasion of endometrial cancer and putative mechanisms.Results:The high expression of CD68 indicating the TAM infiltration were positively related with tumor myometrial invasion and new angiogenesis.The supernatant of cancer cells successfully induced monocytes U937 differentiating into M2-type TAM.Overexpression of miR-99 a in TAM decreased the expression of M2-type macrophage markers,CD68 and CD163,compared with the control group(P<0.01),while the expression of CD206 showed no significant difference(P>0.05).The secretion of IL-12 increased(P<0.01),while the secretions of IL-4 and IL-10 decreased(P<0.01),suggesting that macrophages were polarized towards M1 type.Moreover,transfection with miR-99 a in TAMs attenuated proliferation(P<0.01)and invasion(P<0.05)of endometrial cancer cells.Lastly,the expressions of mTOR and its downstream genes were down-regulated.Conclusion:The high expression of CD68 indicating the TAM infiltration is positively related with tumor myometrial invasion and new angiogenesis of endometrial cancer.Overexpression of miR-99 a can reverse the polarization of monocytes to M2 phenotype,and thus inhibits the cell growth and invasion of endometrial cancer cells,probably through suppressing the signal pathway of mTOR.