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中华结直肠疾病电子杂志 ›› 2021, Vol. 10 ›› Issue (03) : 260 -265. doi: 10.3877/cma.j.issn.2095-3224.2021.03.007

论著

多重剪接RNA结合蛋白RBPMS在结肠炎相关结肠癌中的表达及作用
赵新华1, 马怡茗1, 贺龙梅1, 汪红英1,()   
  1. 1. 100021 北京,国家癌症中心/国家肿瘤临床医学研究中心/中国医学科学院北京协和医学院肿瘤医院,分子肿瘤学国家重点实验室
  • 收稿日期:2021-02-23 出版日期:2021-06-25
  • 通信作者: 汪红英
  • 基金资助:
    国家自然科学基金项目(81672891); 中国医学科学院医学与健康科技创新工程(2017-I2M-1-006,2019-I2M-1-003)

Effect of RNA-binding protein with multiple splicing on colitis associated colon cancer

Xinhua Zhao1, Yiming Ma1, Longmei He1, Hongying Wang1,()   

  1. 1. State Key Laboratory of Molecular Oncology, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
  • Received:2021-02-23 Published:2021-06-25
  • Corresponding author: Hongying Wang
引用本文:

赵新华, 马怡茗, 贺龙梅, 汪红英. 多重剪接RNA结合蛋白RBPMS在结肠炎相关结肠癌中的表达及作用[J/OL]. 中华结直肠疾病电子杂志, 2021, 10(03): 260-265.

Xinhua Zhao, Yiming Ma, Longmei He, Hongying Wang. Effect of RNA-binding protein with multiple splicing on colitis associated colon cancer[J/OL]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2021, 10(03): 260-265.

目的

研究多重剪接RNA结合蛋白RBPMS在结肠炎相关结肠癌中的表达及对结肠癌细胞增殖的影响。

方法

联合应用氧化偶氮甲烷(AOM)和葡聚糖硫酸钠(DSS)诱导C57BL/6小鼠发生结肠炎相关的结肠癌,得到结肠癌发生发展的4个不同阶段的小鼠模型(分别为炎症恢复期、轻度不典型增生、腺瘤、腺癌),同时设立无任何处理的对照组。对不同阶段的结肠组织进行RNA测序分析。采用Western blot法检测多种人类结肠癌细胞系中RBPMS的表达情况。使用LipofectamineTM 2 000将特异性小干扰RNA转染HCT116细胞,72 h后提取细胞总蛋白,并采用Western blot法检测敲降效率。采用四甲基偶氮唑蓝法(MTT)检测敲降RBPMS对细胞增殖的影响。采用克隆形成实验检测敲降RBPMS对肿瘤细胞克隆形成能力的影响。采用流式细胞术检测敲降RBPMS对细胞周期的影响。

结果

AOM/DSS结肠炎相关结肠癌小鼠模型的结肠RNA测序结果显示,RBPMS mRNA的表达水平随着结肠癌的发生与发展逐步升高。进一步免疫组化染色结果显示,RBPMS在小鼠结肠炎相关结肠癌组织中高表达。体外实验证实敲降RBPMS显著抑制肿瘤细胞的增殖速度和克隆形成能力,造成细胞G0/G1阻滞,S期细胞比例显著降低。

结论

RBPMS在结肠炎相关结肠癌癌组织中高表达,敲降RBPMS能够抑制HCT116细胞的增殖。

Objective

To investigate the expression of RNA alternative splicing protein RBPMS in colitis associated colon cancer and its effect on the proliferation of colon cancer cells.

Methods

Colitis-associated colon cancer was induced by azoxymethane (AOM) and dextran sulfate sodium (DSS) in C57BL/6 mice. Mice of four different stages during the development of colon cancer were obtained (inflammatory recovery stage, mild dysplasia, Adenoma, Adenocarcinoma), and a control group was established without any treatment. RNA sequencing was performed in different stages of colon tissue. HCT116 cells were transfected with specific small interfering RNA (siRNA) using LipofectamineTM 2 000. The total protein was extracted 72 hours later, and the knock-down efficiency was measured by Western blot. The effect of RBPMS knockdown on cell proliferation and clone formation ability was examined by MTT and clone-forming assay. Cell cycle were measured by Flow cytometry.

Results

The RNA sequencing results showed that the expression level of RBPMS mRNA increased with the development of colitis associated colon cancer. The results showed that knocking down RBPMS significantly inhibited the proliferation and clone forming ability of tumor cells by inducing G0/G1 arrest.

Conclusion

RBPMS is overexpressed in colitis associated colon cancer tissue, and RBPMS knock down can inhibit the proliferation of HCT116 cells.

图1 AOM/DSS小鼠结肠炎相关结肠癌模型中RBPMS表达水平的芯片检测结果。AD1:急性炎症恢复期;AD2:轻度不典型增生;AD3:腺瘤;AD4:腺癌。**P<0.01。
图2 免疫组织化学染色检测AOM/DSS小鼠结肠炎相关结肠癌模型中RBPMS表达。(标尺:100 μm)
图3 Western blot检测RBPMS特异性小干扰RNA的敲降效率
图4 MTT法检测敲降RBPMS对细胞增殖的影响。*P< 0.05,***P<0.001
图5 敲降RBPMS对细胞克隆形成能力的影响。5A:克隆形成实验结晶紫染色代表性结果图片;5B:各组克隆形成数量统计结果。*P<0.05,**P<0.01,***P<0.001
图6 流式细胞术检测敲降RBPMS对细胞周期的影响。6A:流式细胞术检测周期结果图;6B:各组G1期、S期和G2期统计结果图。*P<0.05
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