lncRNA-MIAT对肿瘤相关巨噬细胞M2型极化的作用及其机制

doi:10.13481/j.1671-587X.20220612

摘要/Abstract

摘要：

目的探讨lncRNA-MIAT对肿瘤相关巨噬细胞M2型极化的作用，并阐明其可能的作用机制。方法体外培养THP-1细胞，将过表达（LV-MIAT）与下调lncRNA-MIAT表达（LV-shMIAT）的慢病毒颗粒及空载体（LV-Vector）转染入THP-1细胞中，将THP-1细胞诱导活化为巨噬细胞，并采用Transwell共培养体系将活化后的巨噬细胞与骨肉瘤（OS）MG63细胞共培养，将上述共培养体系分为MG63+LV-Vector组（阳性对照组）、MG63+LV-shMIAT组、MG63+LV-MIAT组和IL-4+LV-Vector组。检测各组THP-1细胞中lncRNA-MIAT表达水平，流式细胞术检测各组细胞中M2型巨噬细胞百分率，ELISA法检测各组THP-1细胞上清液中血管内皮生长因子（VEGF）、白细胞介素4（IL-10）和转化生长因子β1（TGF-β1）水平，检测各组HUVEC中血管内皮细胞生长因子受体2（VEGFR2）、Notch1和δ样蛋白4（DLL4）表达水平。取各培养体系中的巨噬细胞与人脐静脉内皮血管细胞（HUVEC）共培养，EDU染色法检测各组HUVEC增殖活力，成管实验检测HUVEC血管形成数。Western blotting法检测各组THP1中Janus激酶1（JAK1）、信号传导及转录激活蛋白6（STAT6）和磷酸化STAT6（p-STAT6）蛋白表达水平，并计算p-STAT6/STAT6比值。构建OS荷瘤小鼠模型，并将36只荷瘤小鼠分为LV-Vector组、LV-shMIAT组和LV-MIAT组（n=12）。检测干扰lncRNA-MIAT表达后各组小鼠肿瘤体积和质量及肿瘤组织中CD163和CD31阳性表达率。结果采用慢病毒感染成功建立稳定过表达或下调lncRNA-MIAT的THP-1细胞。与LV-Vector组比较，LV-shMIAT组细胞中lncRNA-MIAT表达水平明显降低（P<0.05），LV-MIAT组细胞中lncRNA-MIAT表达水平明显升高（P<0.05）。与MG63+LV-Vector组比较，MG63+LV-shMIAT组THP-1细胞中VEGF、IL-10和TGF-β1水平明显降低（P<0.05），HUVEC中VEGFR2、Notch1和DLL4蛋白表达水平明显降低（P<0.05），MG63+LV-MIAT组和IL-4+LV-Vector组THP-1细胞中VEGF、IL-10和TGF-β1水平明显升高（P<0.05或P<0.01）， HUVEC中VEGFR2、Notch1和DLL4蛋白表达水平明显升高（P<0.05），M2型巨噬细胞百分率、THP-1细胞中p-STAT6/STAT6比值和JAK1蛋白表达水平升高（P<0.05）；与MG63+sh-MIAT组比较，MG63+LV-MIAT组和IL-4+LV-Vector组THP-1细胞中VEGF水平明显降低（P<0.05），IL-10和TGF-β1水平明显升高（P<0.05），HUVEC中VEGFR2、Notch1和DLL4蛋白表达水平均明显升高（P<0.05）；与MG63+LV-Vector组比较，MG63+LV-shMIAT组HUVEC的增殖活力与血管形成数均明显降低（P<0.05），MG63+LV-MIAT组和IL-4+LV-Vector组HUVEC的增殖活力与血管形成数均明显升高（P<0.05）。裸鼠体内成瘤实验，与LV-Vector组比较，LV-MIAT组小鼠移植瘤体积和质量明显升高（P<0.05），肿瘤组织中CD163和CD31阳性表达率明显升高（P<0.05）；LV-shMIAT组小鼠移植瘤体积和质量、肿瘤组织中CD163和CD31阳性表达率均明显降低（P<0.05）。与LV-shMIAT组比较，LV-MIAT组小鼠移植瘤体积和质量明显升高（P<0.05），肿瘤组织中CD163与CD31阳性表达率明显升高（P<0.05）。结论 lncRNA MIAT可能通过促进巨噬细胞的M2极化和上调肿瘤组织血管生成，参与调控OS进展。

关键词: 骨肉瘤, lncRNA-MIAT, M2型巨噬细胞, 肿瘤血管, 人脐静脉内皮血管细胞

Abstract:

Objective To investigate the effect of lncRNA-MIAT on M2-type polarization of tumor-associated macrophages， and to elucidate its possible mechanism. Methods The THP-1 cells were cultured in vitro. The lentivirus particles， which over expressed （LV-MIAT） and down regulated expression of lncRNA-MIAT（LV-shMIAT），and empty vector（LV-Vector） were transfected into the THP-1 cells. The THP-1 cells were activated into the macrophages， and the activated macrophages were co-cultured with the osteosarcoma （OS） MG63 cells by using Transwell co-culture system.The above co-culture systems were divided into MG63+LV-Vector group （positive control group），MG63+LV-shMIAT group， MG63+LV-MIAT group，and IL-4+LV-Vector group. The expression levels of lncRNA-MIAT in the THP-1 cells in various groups were detected；the percentages of M2-type macrophages in various groups were detected by flow cytometry； the levels of vascular endothelial growth factor （VEGF）， interleukin-10（IL-10），and transforming growth factor-β1（TGF-β1） in supernatant of the THP-1 cells in various groups were detected by ELISA method；the macrophages in each culture system were co-cultured with the human umbilical vein endothelial cell （HUVEC）； EDU staining was used to detect the proliferation activities of the HUVEC in various groups；the numbers of angiogenesis in the HUVEC in various groups were detected by tube formation assay；the expression levels of Janus kinase 1 （JAK1），signal transducer and activator of transcription 6 （STAT6），and phosphorylated STAT6 proteins in the THP1 cells and the expression levels of vascular endothelial growth factor receptor 2（VEGFR2），Notch1，and delta like protein 4（DLL4）in the HUVEC in various groups were detected by Western blotting method；the p-STAT6/STAT6 ratio was calculated.The OS tumor-bearing mouse model was constructed， and 36 nude mice were divided into LV-Vector group， LV-shMIAT group，and LV-MIAT group（n=12）.The volumes and weights of the tumor and the positive expression rates of CD163 and CD31 in tumor tissue of the mice in various groups were detected after interfering lncRNA-MIAT expression. Results The THP-1 cells with stable over-expression or down-regulation of lncRNA-MIAT were successfully established by lentivirus infection. Compared with LV-Vector group， the expression level of lncRNA MIAT in the cells in LV-shMIAT group was significantly decreased （P<0.05）， and the expression level of lncRNA MIAT in the cells in LV-MIAT group was significantly increased （P<0.05）. Compared with MG63+LV-Vector group， the levels of VEGF， IL-10，and TGF- β1 in the THP-1 cells in MG63+LV-shMIAT group were significantly decreased （P<0.05），and the expression levels of VEGFR2， Notch1， and DLL4 proteins in the HUVEC were significantly decreased （P<0.05），the levels of VEGF，IL-10，and TNF-β1 in the THP-1 cells in MG63+LV-MIAT and IL-4+LV-Vector groups were increased significantly （P<0.05 or P<0.01）；the expression levels of VEGFR2， Notch1，and DLL4 proteins in HUVEC were increased（P<0.05）， the percentage of M2-type macrophages，and p-STAT6/STAT6 ratio and expression level of JAK1 protein in the THP-1 cells were increased（P<0.05）；compared with MG63+sh-MIAT group， the VEGF level in the THP-1 cells in MG63+LV MIAT group was decreased significantly （P<0.05）， the levels of IL-10 and TGF-β were decreased significantly （P<0.05），the expression levels of VEGFR2， Notch1，and DLL4 proteins in the HUVEC were increased significantly （P<0.05）；compared with MG63+LV-Vector group， the proliferation activity and number of angiogenesis in the HUVEC in MG63+LV shMIAT group were significantly decreased（P<0.05），while the proliferation activities and number of angiogenesis in the HUVEC in MG63+LV-MIAT group and IL-4+LV Vector group were significantly increased （P<0.05）.The results of in vivo tumorigenesis experiment of the nude mice showed that compared with LV-Vector group， the volume and weight of transplanted tumor of the mice in LV-MIAT group were significantly increased （P<0.05），and the positive expression rates of CD163 and CD31 in tumor tissue were significantly increased （P<0.05）； the volume and mass of transplanted tumor and the positive expression rates of CD163 and CD31 in tumor tissue of the mice in LV-shMIAT group were significantly decreased （P<0.05）. Compared with LV-shMIAT group， the volume and weight of transplanted tumor of the mice in LV-MIAT group were significantly increased （P<0.05）， and the positive expression rates of CD163 and CD31 in tumor tissue were significantly increased （P<0.05）. Conclusion LncRNA MIAT may regulate the progression of OS by promoting the M2 polarization of macrophages and up-regulating the angiogenesis in tumor tissue.

Key words: Oteosarcoma, LncRNA-MIAT, M2-type macrophage, Tumor vessel, Human umbilical vein endothelial cell

中图分类号:

许静,郭健,蒲兴魏,李大星. lncRNA-MIAT对肿瘤相关巨噬细胞M2型极化的作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(6): 1462-1473.

Jing XU,Jian GUO,Xingwei PU,Daxing LI. Effect of lncRNA-MIAT on M2-type polarization of tumor-associated macrophages and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2022, 48(6): 1462-1473.

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参考文献 22

1	TAHIR I， ANDREI V， POLLOCK R， et al. Malignant giant cell tumour of bone： a review of clinical， pathological and imaging features［J］. Skeletal Radiol， 2022， 51（5）： 957-970.
2	LIN Z L， WU Z Y， LUO W. Chimeric antigen receptor T-cell therapy： the light of day for osteosarcoma［J］. Cancers， 2021， 13（17）： 4469.
3	LOCATI M， CURTALE G， MANTOVANI A， Diversity，mechanisms， and significance of macrophage plasticity ［J］. Annu Rev Pathol， 2020， 15： 123-147.
4	WU K Y， LIN K J， LI X Y， et al. Redefining tumor-associated macrophage subpopulations and functions in the tumor microenvironment［J］. Front Immunol， 2020， 11： 1731.
5	MOHAPATRA S， PIOPPINI C， OZPOLAT B， et al. Non-coding RNAs regulation of macrophage polarization in cancer［J］. Mol Cancer， 2021， 20（1）： 24.
6	WINKLER L， DIMITROVA N. A mechanistic view of long noncoding RNAs in cancer［J］. Wiley Interdiscip Rev RNA， 2022， 13（3）： e1699.
7	ZHANG Y Y， MAO Q J， XIA Q M， et al. Noncoding RNAs link metabolic reprogramming to immune microenvironment in cancers［J］. J Hematol Oncol， 2021， 14（1）： 169.
8	WANG Y F， FU L Y， LU T T， et al. Clinicopathological and prognostic significance of long non-coding RNA MIAT in human cancers： a review and meta-analysis［J］. Front Genet， 2021， 12： 729768.
9	ZHANG C Y， XIE L S， LIANG H L， et al. LncRNA MIAT facilitates osteosarcoma progression by regulating mir-128-3p/VEGFC axis［J］.IUBMB Life，2019，71（7）： 845-853.
10	JI Q， ZHU J， FANG C L， et al. Down-regulation of MIAT suppresses osteosarcoma progression by acting as a ceRNA for miR-141-3p to regulate SIX1-mediated PI3K/AKT pathway［J］. Eur Rev Med Pharmacol Sci， 2020， 24（5）： 2218-2228.
11	WANG Z Y， YANG K， ZHAO L， et al. LncRNA MIAT downregulates IL-1β， TNF-α to suppress macrophage inflammation but is suppressed by ATP-induced NLRP3 inflammasome activation［J］. Cell Cycle， 2021， 20（2）： 194-203.
12	LI P F， HAO Z F， WU J Y， et al. Comparative proteomic analysis of polarized human THP-1 and mouse RAW264.7 macrophages［J］. Front Immunol， 2021， 12： 700009.
13	HUANG Y F， LU L， SHEN H L， et al. LncRNA SNHG4 promotes osteosarcoma proliferation and migration by sponging miR-377-3p［J］. Mol Genet Genomic Med， 2020， 8（8）： e1349.
14	ZHOU S Y， XU A L， SONG T F， et al. lncRNA MIAT regulates cell growth， migration， and invasion through sponging miR-150-5p in ovarian cancer［J］. Cancer Biother Radiopharm， 2020， 35（9）： 650-660.
15	SONG F C， YANG Y， LIU J X. Long non-coding RNA MIAT promotes the proliferation and invasion of laryngeal squamous cell carcinoma cells by sponging microRNA-613［J］. Exp Ther Med， 2021， 21（3）： 232.
16	WANG L X， ZHANG S X， WU H J， et al. M2b macrophage polarization and its roles in diseases［J］. J Leukoc Biol， 2019， 106（2）： 345-358.
17	ATRI C， GUERFALI F Z， LAOUINI D. Role of human macrophage polarization in inflammation during infectious diseases［J］. Int J Mol Sci， 2018， 19（6）： 1801.
18	RHEE I. Diverse macrophages polarization in tumor microenvironment［J］. Arch Pharm Res， 2016， 39（11）： 1588-1596.
19	HAO J， HU Y X， LI Y M， et al. Involvement of JNK signaling in IL4-induced M2 macrophage polarization［J］. Exp Cell Res， 2017， 357（2）： 155-162.
20	CUI L Y， YANG G D， YE J N， et al. Dioscin elicits anti-tumour immunity by inhibiting macrophage M2 polarization via JNK and STAT3 pathways in lung cancer［J］. J Cell Mol Med， 2020， 24（16）： 9217-9230.
21	HE Y， GAO Y， ZHANG Q， et al. IL-4 switches microglia/macrophage M1/M2 polarization and alleviates neurological damage by modulating the JAK1/STAT6 pathway following ICH［J］. Neuroscience， 2020， 437： 161-171.
22	PARK Y G， CHOI J， JUNG H K， et al. Baicalein inhibits tumor progression by inhibiting tumor cell growth and tumor angiogenesis［J］. Oncol Rep， 2017， 38（5）： 3011-3018.

Group	VEGF	IL-10	TGF-β1
MG63+LV-Vector	3.15±0.93	2.07±0.48	2.53±0.61
MG63+LV-shMIAT	1.91±0.16^*	0.82±0.15^*	1.46±0.28^*
MG63+LV-MIAT	6.44±1.05^*△	4.82±0.71^*△	5.17±0.92^*△
IL-4+LV-Vector	8.21±1.19^*△	9.74±0.83^*△	11.05±1.66^*△

[1]	胡萍,姜楠,李英华,赵红光. 尾骨原发小细胞型骨肉瘤患者 ¹⁸F-FDG PET/CT 显像特点分析1例报告及文献复习[J]. 吉林大学学报(医学版), 2022, 48(1): 216-221.
[2]	李苇航,丁子毅,王栋,潘益凯,刘玉辉,张世磊,李靖,闫铭. 基于骨肉瘤核心驱动基因筛选的生物信息学分析和患者生存期预测基因模型的构建[J]. 吉林大学学报(医学版), 2021, 47(6): 1570-1580.
[3]	陈炳鹏, 任明, 李容杭, 韩青, 王金成. 基因芯片技术检测NOB1对骨肉瘤细胞相关基因的调控作用[J]. 吉林大学学报(医学版), 2018, 44(05): 929-934.
[4]	金凯, 赵刚, 王育波, 张帅, 王东东. 原发性颅内软骨肉瘤2例报告及文献复习[J]. 吉林大学学报(医学版), 2017, 43(06): 1256-1259.
[5]	那键, 戴维享, 马超, 张晓东, 邵长青, 刘勇, 王秀力, 刘颖. 氟尿嘧啶联合顺铂对骨肉瘤MG-63细胞的抑制作用及其对TRPV5和TRPV6蛋白表达的影响[J]. 吉林大学学报(医学版), 2015, 41(06): 1201-1206.
[6]	拉宗，王建霞，崔倪，李明月，邢树刚，任博，张丽红，李伟，李玉林. 乳腺浸润性导管癌微环境中CD4和CD8阳性T细胞表达与 血管新生的关联性分析[J]. 吉林大学学报(医学版), 2014, 40(05): 1064-1073.
[7]	蔡文涛，夏虹，沈宁江，林明侠. Ⅱ型谷氨酰胺转氨酶对骨肉瘤MG-63细胞凋亡的抑制作用及其机制[J]. 吉林大学学报(医学版), 2014, 40(04): 782-789.
[8]	张治宇\|胡硕\|蔡郑东. 光敏剂BCPD-17和PSD007对小鼠骨肉瘤的光动力作用[J]. J4, 2012, 38(4): 644-648.
[9]	万法青,王井,单玉兴\|张海玉. 端粒酶抑制剂对骨肉瘤细胞株增殖的抑制作用[J]. J4, 2012, 38(2): 254-258.
[10]	钱明\|刘敏\|段蒙娜\|刘畅\|贺玺\|周延民. 缩宫素对人骨肉瘤细胞系MG-63增殖及成骨活性的影响[J]. J4, 2012, 38(2): 262-265.
[11]	王井, 刘易军, 赵丁, 单玉兴. MMP-9和PCNA在端粒酶途径和端粒替代途径骨肉瘤细胞中的表达及其临床意义[J]. J4, 2011, 37(3): 495-498.
[12]	葛岩, 王医术, 高婷, 张丽红, 廖翔宇, 郝艳艳, 李玉林. 槲寄生碱对低分化骨肉瘤U2OS的抑制作用[J]. J4, 2009, 35(5): 841-844.
[13]	雷南伟, 潘秀杰, 齐亚灵, 方艳秋. P16和PCNA在骨肉瘤中的表达及临床意义[J]. J4, 2009, 35(3): 519-521.
[14]	吕佳音，吴丹凯，徐春华，张舵舵，吴丹华，高忠礼，赵燕颖. siRNA对骨肉瘤细胞MDM2的表达及细胞增殖的抑制作用[J]. J4, 2008, 34(3): 442-445.
[15]	杨小玉，顾锐，高忠礼，隋福革，孙伟，殷昭阳，张善永. 人骨肉瘤蛋白质组双向凝胶电泳图像分析方法的建立与应用[J]. J4, 2007, 33(6): 1038-1042.