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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2019, Vol. 09 ›› Issue (01) : 23 -28. doi: 10.3877/cma.j.issn.2095-1221.2019.01.005

所属专题： 文献；

论著

长链非编码RNA Hotair通过调控巨噬细胞表型转换促进胃癌细胞增殖与侵袭
李燕1, 周雄坤2, 刘静1, 苟亚军3,†()   
  1. 1. 400038　重庆，陆军军医大学第一附属医院乳腺外科
    2. 400037　重庆，陆军军医大学第二附属医院普通外科
    3. 401331　重庆市沙坪坝区陈家桥医院急诊重症医学科
  • 收稿日期:2018-12-04 出版日期:2019-02-01
  • 通信作者: 苟亚军

Long non-coding RNA Hotair promotes proliferation and invasion of gastric cancer cells by regulating macrophage phenotypic transformation

Yan Li1, Xiongkun Zhou2, Jing Liu1, Yajun Gou3,†()   

  1. 1. Department of Breast Surgery, the First Affiliated Hospital of Military Medical University, Chongqing 400038, China
    2. Department of General Surgery, the Second Affiliated Hospital of Military Medical University, Chongqing 400037, China
    3. Department of Emergency and Critical Care Medicine, Chenjiaqiao Hospital of Shapingba District, Chongqing 401331, China
  • Received:2018-12-04 Published:2019-02-01
  • Corresponding author: Yajun Gou
  • About author:
    Corresponding author: Yan Yajun, Email:
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引用本文：

李燕, 周雄坤, 刘静, 苟亚军. 长链非编码RNA Hotair通过调控巨噬细胞表型转换促进胃癌细胞增殖与侵袭[J]. 中华细胞与干细胞杂志(电子版), 2019, 09(01): 23-28.

Yan Li, Xiongkun Zhou, Jing Liu, Yajun Gou. Long non-coding RNA Hotair promotes proliferation and invasion of gastric cancer cells by regulating macrophage phenotypic transformation[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(01): 23-28.

目的

探讨胃癌细胞是否通过长链非编码RNA Hotair作用于巨噬细胞，将其转化为癌症支持细胞，进而促进胃癌的增殖与侵袭。

方法

实验分为对照组与AGS细胞共培养组，转染实验分为对照组、Hotair过表达组和RNA干扰组胃癌细胞与巨噬细胞共培养实验检测巨噬细胞表型转换。进行原位杂交实验对M2型巨噬细胞与lncRNA Hotair共定位。通过RT-?PCR实验检测并筛选出促进巨噬细胞表型转换的关键lncRNA。通过RNA干扰技术敲低胃癌细胞lncRNA水平并进行共培养实验。随后通过CCK-8实验与Transwell实验检测转型后的癌症相关巨噬细胞对胃癌细胞增殖与侵袭能力的影响。两组间均数比较采用t检验，多组均数间比较采用单因素方差分析，组间多重比较采用SNK-q检验。

结果

共培养实验结果表明，相比于对照组（5.63±1.97）个，AGS细胞组中含有更多的CD206阳性M2型癌症相关巨噬细胞（32.51±5.44）个，两者比较差异有统计学意义（t =25.742，P = 0.001）；ELISA实验也证明AGS细胞组的巨噬细胞分泌更多的抑炎因子[TGF-β：（163.45±54.91）pg/ml，对照组：（87.32±19.24）?pg/ml；IL-4：（156.83±69.25）pg/ml，对照组：（49.94±17.55）pg/ml；IL-10：（385.65±24.75）pg/ml，对照组：（98.82±46.26）pg/ml]，两组间比较差异有统计学意义（t?= 7.167，8.203，29.991，P均< 0.05）。GEO数据库鉴定并使用RT-PCR筛选出Hotair为关键lncRNA，随后的RNA干扰实验表明，Hotair敲低会抑制巨噬细胞的转变（CD206阳性细胞数量由41.12±6.91变为21.45±2.19），进而降低胃癌细胞的增殖与侵袭能力。

结论

胃癌细胞的lncRNA Hotair会被巨噬细胞摄取，将其转化为癌症相关巨噬细胞，进而促进胃癌细胞的增殖与侵袭能力，这为胃癌的治疗提供了新的可能的靶点。

关键词: lncRNA, 胃癌, 巨噬细胞, 细胞增殖, 细胞侵袭
Objective

To explore whether gastric cancer cell acts on macrophages through lncRNA Hotair to and convert them into cancer supporting cells, and promote the invasion and proliferation of gastric cancer cell.

Methods

The experiment was divided into the control group and AGS cells co-culture group. Co-culture experiment was conducted to detect the phenotypic transformation of macrophages. In situ hybridization was performed to co-localize M2-type macrophages with lncRNA. Key lncRNAs promoting phenotypic transformation of macrophages were detected and screened by RT-PCR. LncRNA levels of gastric cancer cells were knocked down and co-cultured by RNA interference. Subsequently, CCK-8 assay and Transwell assay were used to detect the effect of transformed cancer-related macrophages on the proliferation and invasion ability of gastric cancer cells. Analysis of variance and t test were used for statistical analysis.

Results

The results of co-culture experiment showed that compared with the control group (5.63±1.97) , AGS cell group contained more CD206+ m2-type cancer-related macrophages (32.51±5.44) , and the difference was statistically significant (t = 25.742, P = 0.001) . ELISA also showed that macrophages in AGS cell group secreted more anti-inflammatory factors [TGF-β (163.45±54.91) pg/ml, control group: (87.32±19.24) pg/ml; il-4: (156.83±69.25) pg/ml, control group: (49.94±17.55) pg/?ml; il-10: (385.65±24.75) pg/ml, control group: (98.82±46.26) pg/ml], the difference between the two groups was statistically significant (t = 7.167, 8.203, 29.991, P < 0.05) . Hotair was screened as the key lncRNA through the GEO database and RT-PCR, and subsequent RNA interference experiments showed that the knockdown of Hotair inhibited the transformation of macrophages (the number of CD206 positive cells changed from 41.12±6.91 to 21.45±2.19) , thus reducing the proliferation and invasion ability of gastric cancer cells.

Conclusion

LncRNA Hotair of gastric cancer cells will be absorbed by macrophages and transformed into cancer-related macrophages, thereby promoting the proliferation and invasion ability of gastric cancer cells.

Key words: LncRNA, Macrophage, Gastric cancer, Cell proliferation, Cell invasion
表1 两组巨噬细胞不同表型数量（个， ± s）
分组 样本量 CD86 CD206
对照组 30 46.82±7.45 5.63±1.97
AGS细胞组 30 7.86±2.44 32.51±5.44
t值 ? 27.464 25.742
P值 ? 0.001 0.001
图1 倒置荧光显微镜观察两组胃腺癌细胞对邻近巨噬细胞的影响（免疫荧光染色，×400）
表2 两组炎症相关因子水平检测（pg/ml， ± s）
分组 样本量 IL-6 TNF-α IL-1β TGF-β IL-4 IL-10
对照组 30 986.51±239.13 2986.43±731.21 567.92±143.47 87.32±19.24 49.94±17.56 98.82±46.26
AGS细胞组 30 321.63± 96.32 998.34±314.73 212.41±101.24 163.45±54.91 156.83±69.25 385.65±24.75
t值 ? 14.136 13.680 11.094 7.167 8.203 29.991
P值 ? 0.001 0.001 0.001 0.001 0.001 0.001
表3 巨噬细胞两种lncRNA相对水平检测
分组 样本量 Hotair PICSAR
对照组 30 1.04±0.22 1.06±0.17
AGS细胞组 30 2.10±0.56 1.13±0.23
t值 ? 10.108 2.490
P值 ? 0.001 0.016
图2 荧光共聚焦显微镜观察Hotair与巨噬细胞共定位（×400）
表4 各组流式细胞各表型细胞比例、450 nm吸光度与侵袭细胞数量
分组 样本量 CD86（﹪） CD206（﹪） 450 nm吸光度 侵袭细胞数量（个）
对照组 10 27.81±3.26 41.12±6.91 0.66±0.10 174.81±24.24
Hotair过表达组 10 14.63±2.57a 59.26±5.93a 1.05±0.23b 326.90±34.62a
RNA干扰组 10 47.42±6.78a 21.45±2.19a 0.46±0.09b 126.78±21.85a
F值 ? 133.154 123.514 38.447 145.131
P值 ? 0.001 0.001 0.001 0.001
图3 Hotair将巨噬细胞转变为癌支持细胞
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