miRNA-1236通过靶向FOXO3a调控结直肠癌细胞增殖的作用及机制研究

doi:10.3877/cma.j.issn.2095-3224.2019.04.011

中华结直肠疾病电子杂志 ›› 2019, Vol. 08 ›› Issue (04) : 383 -389. doi: 10.3877/cma.j.issn.2095-3224.2019.04.011

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论著

miRNA-1236通过靶向FOXO3a调控结直肠癌细胞增殖的作用及机制研究

孙宇¹, 朱琳¹, 刘祯璐¹, 杨明¹, 余贞¹, 孙静¹, 王妍¹, 蒋雨含², 李红岩³, 孙辉⁴^,^†(

)

^1. 150081　哈尔滨医科大学药学院
^2. 150081　哈尔滨医科大学基础医学院
^3. 150081　哈尔滨，黑龙江中医药大学附属第一医院
^4. 150081　哈尔滨医科大学药学院；150001　哈尔滨医科大学药学院　药学实验中心

收稿日期:2018-06-17 出版日期:2019-08-25
通信作者: 孙辉
基金资助:
哈尔滨医科大学创新科学研究基金(No.2016JCZX55); 黑龙江省大学生创新创业训练计划项目(No.201810226069)

The effect and mechanism of miRNA-1236 suppresses colorectal cancer cell proliferation by targeting FOXO3a

Yu Sun¹, Lin Zhu¹, Zhenlu Liu¹, Ming Yang¹, Zhen Yu¹, Jing Sun¹, Yan Wang¹, Yuhan Jiang², Hongyan Li³, Hui Sun⁴^,^†()

^1. College of Pharmacy, Harbin Medical University, Harbin 150081, China
^2. College of Basic Medical, Harbin Medical University, Harbin 150081, China
^3. Traditional Chinese Medicine University of Heilongjiang, Harbin 150081, China
^4. College of Pharmacy, Harbin Medical University, Harbin 150081, China; Pharmaceutical Experiment Teaching Center, College of Pharmacy, Harbin Medical University, Harbin 150001, China

Received:2018-06-17 Published:2019-08-25
Corresponding author: Hui Sun
About author:
Corresponding author: Sun Hui, Email: sunhui@hrbmu.edu.cn

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引用本文：

孙宇, 朱琳, 刘祯璐, 杨明, 余贞, 孙静, 王妍, 蒋雨含, 李红岩, 孙辉. miRNA-1236通过靶向FOXO3a调控结直肠癌细胞增殖的作用及机制研究[J]. 中华结直肠疾病电子杂志, 2019, 08(04): 383-389.

Yu Sun, Lin Zhu, Zhenlu Liu, Ming Yang, Zhen Yu, Jing Sun, Yan Wang, Yuhan Jiang, Hongyan Li, Hui Sun. The effect and mechanism of miRNA-1236 suppresses colorectal cancer cell proliferation by targeting FOXO3a[J]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2019, 08(04): 383-389.

目的

探讨miRNA-1236对结直肠癌细胞增殖的作用及其相关机制。

方法

通过实时定量PCR检测转染miR-1236后miRNA-1236的表达，使用CCK-8以及克隆形成方法检测转染miRNA-1236后HCT116细胞的活性，ki67方法检测转染miRNA-1236后HCT116细胞增殖能力的变化，生物信息方法筛选miR-1236的潜在靶基因FOXO3a。Luciferase报告检测miRNA-1236与FOXO3a的结合位点，通过实时定量PCR方法检测转染miRNA-1236后FOXO3a基因的变化；通过免疫组化的方法检测结直肠癌患者组织中FOXO3a的表达，通过western blot检测低表达miRNA-1236后HCT116细胞中FOXO3a、PCNA以及Bax蛋白的变化。通过TCGA数据库检测FOXO3a基因在结直肠癌患者癌-癌旁，不同肿瘤阶段与不同性别中的表达。

结果

本研究发现高表达miRNA-1236后HCT116细胞活性相对于阴性对照组明显增加，而低表达miRNA-1236后活性降低。低表达miRNA-1236后HCT116细胞增殖能力相对于阴性对照组明显降低。生物信息学预测miR-1236与FOXO3a有潜在的结合位点。采用Western blot以及逆转录实时定量PCR验证，过表达miRNA-1236后FOXO3a表达相对于阴性对照组明显降低，而低表达miRNA-1236后，FOXO3a表达明显升高，同时luciferase结果显示，FOXO3a是miRNA-1236的靶基因。并且，FOXO3a在结直肠癌症组织中明显降低。

结论

miR-1236通过靶向FOXO3a的表达调节结直肠癌细胞HCT116增殖，同时为miR-1236成为诊断、预防以及治疗结直肠癌的生物学标记物提供理论基础。

关键词: 结直肠肿瘤, miR-1236, FOXO3a, 增殖

Objective

To investigate the role and mechanism of miRNA-1236 on proliferation in colorectal cancer.

Method

The expression of miRNA-1236 was detect by quantitative real-time PCR. The cell viability of HCT116 cells after transfected with miRNA-1236 was detected by CCK-8. The proliferation of miR-1236 in HCT116 cell was tested by colony formation assay and Ki67 staining. The potential target gene FOXO3a of miR-1236 was screened by bioinformatics. Observing the level of FOXO3a gene with transfection of miRNA-1236 by real-time quantitative PCR detection. Luciferase reports detected the binding site of miRNA-1236 with FOXO3a; The expression of FOXO3a in colorectal cancer was detected by immunohistochemistry. Detected the protein level of FOXO3a, PCNA and Bax in HCT116 cells after transfection with miR-1236 by western blotting. The expression of FOXO3a in colorectal Cancer-Para cancer patients with different cancer stages and genders was detected by TCGA database.

Results

The cell viability of HCT116 cells was significantly increased with the miRNA-1236 mimic compared with negative control group, while it was decreased with the miRNA-1236 inhibitor. The proliferation of HCT116 cells was significantly reduced compared with the negative control group by colony formation assay and Ki67 staining. Bioinformatics predicted potential binding sites between miR-1236 and FOXO3a. Using Western blot and quantitative real-time PCR of reverse transcription to verify that FOXO3a expression was significantly reduced after overexpression of miRNA-1236 than the negative control group, while FOXO3a expression was significantly increased after treat with miRNA-1236 inhibitor. Meanwhile, luciferase results showed that FOXO3a was a direct target gene of miRNA-1236. Simultaneously, FOXO3a was significantly reduced in colorectal cancer tissue.

Conclusion

miRNA-1236 regulate the proliferation of colorectal cancer by targeting FOXO3a, and significantly affects survival of colorectal cancer patients.

Key words: Colorectal neoplasms, miRNA-1236, FOXO3a, Proliferation

图1 miR-1236对于结直肠癌细胞HCT116活性的影响。1A：逆转录实时定量PCR检测转染miR-1236mimic或者miR-1236inhibitor后miR-1236的表达；1B：CCK-8检测转染miR-1236后HCT116细胞的活性变化；1C：克隆形成检测转染miR-1236后HCT116细胞的活性变化；1D：ki-67检测低表达miR-1236对于HCT116细胞增殖能力的影响；1E~1F：Western blot检测低表达miR-1236对于HCT116细胞中PCNA以及Bax蛋白的变化，与对照组相比^*P<0.05

图2 mir-1236通过靶向FOXO3a基因调节HCT116细胞增殖。2A：TargetScan软件预测，FOXO3a基因与mir-1236潜在种子序列；2B：荧光素酶报告分析；2C、2D：分别转染mir-1236-mimic或mir-1236 inhibitor后FOXO3a的蛋白和mRNA表达。^*与对照组相比P<0.05

图3 FOXO3a在结直肠癌中的表达。3A：TCGA数据库检测FOXO3a在癌症-癌旁中的表达；3B：TCGA数据库检测FOXO3a在不同肿瘤阶段中的表达；3C：TCGA数据库检测FOXO3a与不同性别的表达；3D：免疫组化检测FOXO3a在男性患者中结直肠癌组织中的表达

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The effect and mechanism of miRNA-1236 suppresses colorectal cancer cell proliferation by targeting FOXO3a

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The effect and mechanism of miRNA-1236 suppresses colorectal cancer cell proliferation by targeting FOXO3a