摘要
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。
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-rel
作者
李燕
周雄坤
刘静
苟亚军
Li Yan;Zhou Xiongkun;Liu Jing;Gou Yajun(Department of Breast Surgery,the First Affiliated Hospital of Military Medical University,Chongqing 400038,China;Department of General Surgery,the Second Affiliated Hospital of Military Medical University,Chongqing 400037,China;Department of Emergency and Critical Care Medicine,Chenjiaqiao Hospital of Shapingba District,Chongqing 401331,China)
出处
《中华细胞与干细胞杂志（电子版）》
2019年第1期23-28,共6页
Chinese Journal of Cell and Stem Cell（Electronic Edition）
