徐纪中; 王贵宪; 袁维堂; 周全博; 胡晟云

doi:10.3760/cma.j.issn.1001-9030.2020.01.018

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• 实验研究 •

微小RNA-182靶向调控特定富含AT碱基DNA序列结合蛋白2基因对结肠癌细胞增殖和迁移的影响

中华实验外科杂志, 2020,37(1) : 63-66. DOI: 10.3760/cma.j.issn.1001-9030.2020.01.018

摘要

目的

观察微小RNA(miRNA，miR)-182通过靶向特定富含AT碱基DNA序列结合蛋白2(SATB2)基因对结直肠癌细胞增殖迁移的作用，探讨其对SATB2/烟酰胺腺嘌呤二核苷酸磷酸氧化酶4(nox4)通路的调节机制。

方法

对数期生长人结肠癌细胞(HT-29)细胞，采用脂质体转染法将si-miR-182、Control-si转至HT-29细胞，分别设为Si-miR-182组、N-miR-182组，另取不做任何处理细胞为对照组。测定3组细胞中miR-182基因表达、增殖和迁移、SATB2、nox4、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)mRNA和蛋白表达。重复计量资料比较采用重复测量方差分析，两两样本比较采用LSD-t检验。

结果

Si-miR-182组miR-182基因相对表达量(0.35±0.05)低于对照组(1.24±0.26)和N-miR-182组(1.20±0.25)，差异均有统计学意义(t＝7.517、7.455，P<0.05)；Si-miR-182组培养24、48、72 h MTT试验吸光度(A)值均低于对照组和N-miR-182组，差异均有统计学意义(24 h：t＝2.667、2.664；48 h：t＝4.559、4.524；72 h：t＝7.257、6.981；P<0.05)；与培养24 h比较，3组培养48、72 h MTT试验A值均升高(48 h：t＝5.507、5.092、3.741；72 h：t＝11.330、10.637、9.229；P<0.05)，且72 h MTT试验A值高于48 h，差异均有统计学意义(t＝7.411、6.941、5.214，P<0.05)。Si-miR-182组细胞迁移率[(53.90±3.19)%]低于对照组[(81.66±5.92)%]和N-miR-182组[(80.35±5.40)%]，差异均有统计学意义(t＝9.231、9.430，P<0.05)；Si-miR-182组细胞SATB2、E-cadherin mRNA和蛋白相对表达量高于对照组和N-miR-182组(SATB2：t＝10.930、11.158；E-cadherin：t＝9.288、9.369；P<0.05)，nox4、Vimentin mRNA和蛋白相对表达量低于对照组和N-miR-182组，差异均有统计学意义(nox4：t＝8.955、7.590；Vimentin：t＝6.543、6.644；P<0.05)。

结论

沉默miR-182基因可显著抑制结直肠癌细胞增殖及迁移能力，可能通过激活SATB2、E-cadherin的表达、抑制nox4、Vimentin的表达、参与SATB2/nox4通路共同调控。

引用本文: 徐纪中, 王贵宪, 袁维堂, 等. 微小RNA-182靶向调控特定富含AT碱基DNA序列结合蛋白2基因对结肠癌细胞增殖和迁移的影响 [J] . 中华实验外科杂志, 2020, 37(1) : 63-66. DOI: 10.3760/cma.j.issn.1001-9030.2020.01.018.

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近年来研究显示，微小RNA(miRNA，miR)在结直肠癌发生、发展中起重要作用^[1]。有研究证实，miR-182在结直肠癌、食管癌组织中呈异常高表达状态，与肿瘤转移关系密切，但关于其对结直肠癌细胞增殖、迁移的影响及具体调控机制仍需要探讨^[2]。本研究通过小干扰RNA法抑制结直肠癌HT-29细胞中miR-182基因表达，观察miR-182基因对其增殖及迁移能力的影响，并探讨其相关靶基因及调控通路。

贡献者信息

徐纪中

郑州大学第一附属医院结直肠外科　450052

王贵宪

郑州大学第一附属医院结直肠外科　450052

袁维堂

郑州大学第一附属医院结直肠外科　450052

周全博

郑州大学第一附属医院结直肠外科　450052

胡晟云

郑州大学第一附属医院结直肠外科　450052

通信作者

王贵宪

郑州大学第一附属医院结直肠外科　450052

Email：xuzhong601@163.com

关键词

微小RNA-182; 特定富含AT碱基DNA序列结合蛋白2; 结直肠癌; 增殖; 迁移;

利益冲突

所有作者均声明不存在利益冲突

历史

出版日期：2020-01-08

收稿日期：2019-06-29

Experimental Study

MicroRNA-182 regulates colon cancer cell proliferation and migration by targeting special AT-rich sequence-binding protein 2 gene

Xu Jizhong, Wang Guixian, Yuan Weitang, Zhou Quanbo, Hu Shengyun

Published 2020-01-08

Cite as Chin J Exp Surg, 2020, 37(1): 63-66. DOI: 10.3760/cma.j.issn.1001-9030.2020.01.018

Abstract

Objective

To observe the effect of microRNA (miRNA, miR)-182 on the proliferation and migration of colorectal cancer cells by targeting special AT-rich sequence-binding protein 2 (SATB2) gene, and to explore its effect on SATB2/icotinamide adenine dinucleotide phosphate oxidase 4 (nox4) pathway.

Methods

HT-29 cells in logarithmic phase were transfected with Si-miR-182 and Control-si resectively, by liposome transfection, serving as Si-miR-182 group and N-miR-182 group, respectively. The expression of miR-182 gene, the cell proliferation and migration, the mRNA and protein expression of SATB2, nox4, E-cadherin and vimentin were examined.

Results

The relative expression of miR-182 gene in Si-miR-182 group (0.35±0.05) was lower than that in control group (1.24±0.26) and N-miR-182 group (1.20±0.25) (t=7.517, 7.455, P<0.05). TheA values of methyl thiazol tetrazolium (MTT) test in Si-miR-182 group were lower than those in control group and N-miR-182 group after 24, 48 and 72 h of culture (24 h: t=2.667, 2.664; 48 h: t=4.559, 4.524; 72 h: t=7.257, 6.981; P<0.05). Compared with 24-h culture, theA value of MTT test in 48 and 72 hours in three groups were increased (48 h: t=5.507, 5.092, 3.741; 72 h: t=11.330, 10.637, 9.229; P<0.05), and theA value of MTT test in 72 hours were higher than those in 48 hours(t=7.411, 6.941, 5.214, P<0.05). The cell migration rate of Si-miR-182 group [(53.90±3.19)%] was lower than that of the control group [(81.66±5.92)%] and N-miR-182 group [(80.35±5.40)%] (t=9.231, 9.430, P<0.05). The relative expressions of SATB2, E-cadherin mRNA and protein in Si-miR-182 group were higher than those in control group and N-miR-182 group (SATB2:t=10.930, 11.158; E-Cadherin: t=9.288, 9.369; P<0.05), while the relative expressions of nox4, vimentin mRNA and protein in Si-miR-182 group were lower than those in control group and N-miR-182 group (nox4:t=8.955, 7.590; Vimentin: t=6.543, 6.644; P<0.05).

Conclusion

Silencing of miR-182 gene can significantly inhibit the proliferation and migration of colorectal cancer cells, possibly by activating SATB2, E-Cadherin expression, inhibiting the expression of nox4, Vimentin, and participating in the SATB2/nox4 pathway.

Key words:

MicroRNA-182; Special AT-rich sequence-binding protein 2; Colorectal cancer; Proliferation; Migration

Contributor Information

Xu Jizhong