[1]李 阳,包 刚.LncRNA UCA1 介导Raf/MEK/ERK 信号通路调控乳腺癌细胞生物学作用的机制研究[J].现代检验医学杂志,2022,37(06):40-45.[doi:10.3969/j.issn.1671-7414.2022.06.008]
　LI Yang,BAO Gang.Mechanism of LncRNA UCA1 Mediating Raf/MEK/ERK Signaling Pathway to Regulate the Biological Effects of Breast Cancer Cells[J].Journal of Modern Laboratory Medicine,2022,37(06):40-45.[doi:10.3969/j.issn.1671-7414.2022.06.008]
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LncRNA UCA1 介导Raf/MEK/ERK 信号通路调控乳腺癌细胞生物学作用的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年06期
页码:
40-45
栏目:
论著
出版日期:
2022-11-15

文章信息/Info

Title:
Mechanism of LncRNA UCA1 Mediating Raf/MEK/ERK Signaling Pathway to Regulate the Biological Effects of Breast Cancer Cells
文章编号:
1671-7414(2022)06-040-06
作者:
李 阳; 包 刚
(贵州医科大学附属医院乳腺外科，贵阳 550004 )
Author(s):
LI Yang; BAO Gang
(Department of Breast Surgery，the Affiliated Hospital of Guizhou Medical University，Guiyang 550004, China)
关键词:
乳腺癌细胞; 长链非编码RNA 尿路上皮癌胚抗原1; 生物学作用; Raf/MEK/ERK 通路
分类号:
R737.9；R730.43
DOI:
10.3969/j.issn.1671-7414.2022.06.008
文献标志码:
A
摘要:
目的 探讨长链非编码RNA(long non-coding RNA, LncRNA)尿路上皮癌胚抗原1(urothelial carcinoma antigen1,UCA1) 在乳腺癌细胞中的表达及其对乳腺癌细胞生物学作用的机制研究。方法 实时定量聚合酶链式反应(quantitativereal-time PCR , qRT-PCR) 法检测LncRNA UCA1 在正常人乳腺上皮细胞MCF-10A 与乳腺癌细胞BT-474，MCF-7，SKBR-3 和MDA-MB453 中的表达； 选择BT-474 和MCF-7 细胞随机分为三组， 分别转染UCA1-siR，NC-siR 及Control，再通过qRT-PCR 法验证转染后BT-474 和MCF-7 细胞中LncRNA UCA1 表达；通过CCK-8 法、Transwell 实验和流式细胞仪检测BT-474 和MCF-7 细胞增殖、侵袭、凋亡能力；利用Western blot 检测两种细胞中RAF/MEK/ERK通路相关蛋白的表达。结果 与MCF-10A 相比，LncRNA UCA1 在BT-474，MCF-7, SKBR-3 和MDA-MB453 细胞中表达水平分别上调了133.8%，169.2%，35.4% 和73.8%，差异有统计学意义(F=34.152，P=0.002)。转染处理后，BT-474 细胞Control 组、NC-siR 组和UCA1-siR 组Lnc RNA UCA1 表达量分别为1.52±0.36，1.49±0.17 和0.63±0.11，差异有统计学意义(F=42.628，P ＜ 0.001) 。MCF-7 细胞Control 组、NC-siR 组和UCA1-siR 组Lnc RNA UCA1 表达量分别为1.75±0.25，1.70±0.22 和0.74±0.08，差异亦有统计学意义(F= 39.372，P ＜ 0.001)。CKK-8 结果表明，与Control 组和NC-siR 组相比，UCA1-siR 组BT-474 和MCF-7 细胞增殖能力均显著下降，差异有统计学意义(F=57.382 ～ 198.251, 均P ＜ 0.001)。Transwell 结果显示，BT-474 细胞Control 组、NC-siR 组和UCA1-siR 组穿膜数分别为205±12 个，192±16 个和114±9 个，差异有统计学意义(F=108.250，P ＜ 0.001)，MCF-7 细胞Control 组、NC-siR 组和UCA1-siR 组穿膜数分别为187±9 个，175±13 个和96±12 个，差异亦有统计学意义(F= 139.062，P＜ 0.001)。流式结果显示，BT-474 细胞Control 组、NC-siR 组和UCA1-siR 组凋亡率分别为4.25%，4.44% 和10.28%，差异有统计学意义(F=49.134，P ＜ 0.001)，MCF-7 细胞Control 组、NC-siR 组和UCA1-siR 组凋亡率分别为2.30%，3.05%和8.98%，差异亦有统计学意义(F=62.750，P ＜ 0.001)。Western blot 结果显示，与Control 组和NC-siR 组相比，UCA1-siR 组BT-474 和MCF-7 细胞中Raf，p-MEK/MEK 及p-ERK1/2/ ERK1/2 蛋白表达均显著下调，差异有统计学意义(F=42.384 ～ 76.092，均P<0.001)。结论 LncRNA UCA1 在乳腺癌细胞系中呈高表达，沉默LncRNA UCA1 基因可以抑制乳腺癌细胞增殖和侵袭，促进凋亡，其作用机制可能与阻断Raf/MEK/ERK 磷酸化通路有关。
Abstract:
Objective To investigate the expression of long non-coding RNA(LncRNA)urothelial carcinoma antigen 1 (UCA1) in breast cancer cells and its biological mechanism on breast cancer cells. Methods Quantitative real-time PCR (qRT-PCR) method was used to detect the expression of LncRNA UCA1 in normal human breast epithelial cells MCF-10A and breast cancer cells BT-474, MCF-7, SKBR-3 and MDA-MB453. BT-474 and MCF-7 cells were selected and randomly divided into 3 groups, respectively transfected with UCA1-siR, NC-siR and control, and then the expression of LncRNA UCA1 in BT-474 and MCF-7 cells after transfection was verified by qRT-PCR method. The proliferation, invasion and apoptosis ability of BT-474 and MCF-7 cells were detected by CCK-8 method, flow cytometry and Transwell assay. Western blot was used to detect the expression of RAF/MEK/ERK pathway-related proteins in the two cells. Results Compared with MCF-10A, the expression levels of LncRNA UCA1 in BT-474, MCF-7, SKBR-3 and MDA-MB453 cells were up-regulated by 133.8%, 169.2%, 35.4% and 73.8%, respectively, and the difference was statistically significant (F=34.152, P=0.002). After transfection, the expression levels of Lnc RNA UCA1 in the control group, NC-siR group and UCA1-siR group of BT-474 cells were 1.52±0.36, 1.49±0.17 and 0.63±0.11, respectively, and the differences were statistically significant(F=42.628, P ＜ 0.001).The expression levels of Lnc RNA UCA1 in MCF-7 cells control group, NC-siR group and UCA1-siR group were 1.75±0.25, 1.70±0.22 and 0.74±0.08, respectively, and the difference was also statistically significant (F = 39.372, P ＜ 0.001). The results of CKK-8 showed that compared with the control group and the NC-siR group, the proliferation ability of BT-474 and MCF-7 cells in the UCA1-siR group was significantly decreased, and the differences were statistically significant (F=57.382 ～ 198.251, all P ＜ 0.001). Transwell results showed that the number of BT-474 cells that penetrated the membrane in the control group, the NCsiR group and the UCA1-siR group were 205±12, 192±16 and 114±9, respectively, and the difference was statistically significant (F=108.250, P ＜ 0.001), the number of MCF-7 cells in the control group, the NC-siR group and the UCA1-siR group were 187±9, 175±13 and 96±12, respectively, and the difference was also statistically significant (F=139.062, P ＜ 0.001). Flow cytometry results showed that the apoptosis rates of BT-474 cells in control group, NC-siR group and UCA1-siR group were 4.25%, 4.44% and 10.28%, respectively, and the difference was statistically significant (F=49.134, P ＜ 0.001). The apoptosis rates of MCF-7 cells in control group, NC-siR group and UCA1-siR group were 2.30%, 3.05% and 8.98%, respectively, and the differences were also statistically significant (F=62.750, P ＜ 0.001). Western blot results showed that compared with the control group and the NC-siR group, the protein expressions of Raf, p-MEK/MEK and p-ERK1/2/ERK1/2 in the BT-474 and MCF-7 cells of the UCA1-siR group were significantly higher down-regulation, the differences were statistically significant (F=42.384 ～ 76.092, P ＜ 0.001). Conclusion LncRNA UCA1 was highly expressed in breast cancer cell lines. Silencing the LncRNA UCA1 gene can inhibit the proliferation and invasion of breast cancer cells and induce apoptosis. The regulatory mechanism may be related to the inhibition of Raf/MEK/ERK phosphorylation pathway.

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备注/Memo

备注/Memo:
作者简介：李阳(1996-)，女, 硕士, 规培医师, 研究方向：乳腺肿瘤基础及临床，E-mail：okoK0643@163.com。
通讯作者：包刚(1970-)，男，硕士，副主任医师，研究方向：乳腺肿瘤基础与临床，E-mail：18984846632@189.com。
更新日期/Last Update: 2022-11-15