中国癌症杂志 ›› 2020, Vol. 30 ›› Issue (8): 561-569.doi: 10.19401/j.cnki.1007-3639.2020.08.001

• 论著 • 上一篇    下一篇

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

文 静 1 ,黄 洁 2 ,李云云 3 ,张中卒 4 ,周 勤 1   

  1. 1. 重庆医科大学附属第一医院妇产科,重庆 400016 ;
    2. 陆军军医大学附属大坪医院(陆军特色医学中心)内科,重庆 400042 ;
    3. 重庆医科大学附属永川医院妇产科,重庆 402160 ;
    4. 重庆医科大学附属永川医院骨科,重庆 402160
  • 出版日期:2020-08-30 发布日期:2020-09-03
  • 通信作者: 周 勤 E-mail: 1733638008@qq.com
  • 基金资助:
    国家自然科学基金(81902645);国家自然科学基金预研资助项目(NSFYY201716);
    重庆市教委科学技术研究项目(KJ1600228)。

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

WEN Jing 1 , HUANG Jie 2 , LI Yunyun 3 , ZHANG Zhongzu 4 , ZHOU Qin 1   

  1. 1. Department of Gynecology and Obstetrics, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; 2. Department of Internal Medicine, Daping Hospital of Army Medical University, Chongqing 400042, China; 3. Department of Gynecology and Obstetrics, The Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, China; 4. Department of Orthopedics, The Yongchuan Hospital of Chongqing Medical University, Chongqing 402160, China
  • Online:2020-08-30 Published:2020-09-03
  • Contact: ZHOU Qin E-mail: 1733638008@qq.com

摘要: 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。

关键词: 肿瘤相关巨噬细胞, miR-99a, 子宫内膜癌, 细胞增殖, 细胞侵袭

Abstract: 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-99a 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-1B and RL95-2 were used to induce human monocyte U937 to differentiate into M2-type macrophages, namely TAMs. The synthetic miR-99a 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-99a 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-99a 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-99a 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-99a 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.

Key words: Tumor-associated macrophages, miR-99a, Endometrial cancer, Cell proliferation, Cell invasion