MiR-6838-5p过表达下调DDR1基因表达抑制乳腺癌MCF-7细胞的增殖

doi:10.12122/j.issn.1673-4254.2024.09.07

摘要/Abstract

摘要：

目的探索miR-6838-5p调控DDR1基因表达水平对乳腺癌MCF-7细胞增殖的影响。方法采用脂质体转染方法干预乳腺癌MCF-7细胞中miR-6838-5p 及DDR1基因表达，设置组别如下： Control mimic；miR-6838-5p mimic；Control inhibitor；miR-6838-5p inhibitor；Control siRNA、DDR1 siRNA、Control vector、DDR1 vector及miR-6838-5p 过表达+DDR1过表达共转染组。qRT-PCR实验检测miR-6838-5p在正常乳腺上皮细胞及乳腺癌细胞中的表达；通过TargetscanV 8.0等软件预测miR-6838-5p可能作用的靶基因；双荧光素酶报告基因实验验证miR-6838-5p与DDR1存在靶向结合位点；CCK-8实验及EdU实验检测细胞增殖情况；Western blotting实验检测DDR1表达情况；通过裸鼠荷瘤实验验证miR-6838-5p通过调控DDR1表达抑制了乳腺癌MCF-7细胞增殖。结果 miR-6838-5p在乳腺癌细胞中表达量低于乳腺正常上皮细胞（P<0.05）；对比对照组，miR-6838-5p能抑制乳腺癌细胞增殖（P<0.05）；双荧光素酶报告基因实验证明miR-6838-5p可以靶向结合DDR1（P<0.01），Western blotting实验证明miR-6838-5p能调控DDR1基因表达；DDR1促进乳腺癌细胞的增殖（P<0.05）；miR-6838-5p 过表达+DDR1过表达共转染后能回复miR-6838-5p对乳腺癌细胞增殖的抑制作用（P<0.05）；裸鼠荷瘤实验结果表明过表达miR-6838-5p后肿瘤体积明显减小，Western blotting结果表明肿瘤内miR-6838-5p过表达后抑制了DDR1的表达（P<0.05）。结论 miR-6838-5p通过调控DDR1基因表达抑制了乳腺癌细胞的增殖。

关键词: miR-6838-5p, 乳腺癌, DDR1, 细胞增殖

Abstract:

Objective To explore the regulatory effect of miR-6838-5p on DDR1 gene expression and proliferation of breast cancer cells. Methods The expression levels of miR-6838-5p in normal breast epithelial cells and breast cancer cells were detected using qRT-PCR, and the potential target genes of miR-6838-5p was predicted using TargetscanV 8.0. Double luciferase reporter gene experiment was performed to verify the binding between miR-6838-5p and DDR1. Breast cancer MCF-7 cells were transfected via liposome, miR-6838-5p mimic, miR-6838-5p inhibitor, DDR1 siRNA, DDR1-overexpresisng vector, or both miR-6838-5p mimic and DDR1-overexpressing vector, and the changes in cell proliferation were examined with CCK-8 and EdU assays; Western blotting was used to detect the expression of DDR1. The mediating role of DDR1 in miR-6838-5p overexpression-induced inhibition of MCF-7 cell proliferation was verified in a nude mouse model bearing MCF-7 cell xenografts. Results The expression of miR-6838-5p was significantly lower in breast cancer cells than in normal breast epithelial cells. In MCF-7 cells, miR-6838-5p overexpression induced significant inhibition of cell proliferation. Dual luciferase reporter gene experiment demonstrated a binding relationship between miR-6838-5p and DDR1 (P<0.01). Western blotting showed that miR-6838-5p overexpression significantly lowered DDR1 expression in MCF-7 cells, and DDR1 overexpression promoted proliferation of the cells; co-transfection of the cells with DDR1-overexpressing vector significantly attenuated the inhibitory effect of miR-6838-5p mimic on cell proliferation. In the tumor-bearing nude mice, the xenografts overexpressing miR-6838-5p showed a significantly smaller volum with obviously the expression of DDR1. Conclusion Overexpression of miR-6838-5p inhibits breast cancer cell proliferation by regulating DDR1 expression.

Key words: miR-6838-5p, brest cancer, DDR1, cell proliferation

薛良军, 谈秋瑜, 许静文, 冯璐, 李文锦, 颜亮, 李玉磊. MiR-6838-5p过表达下调DDR1基因表达抑制乳腺癌MCF-7细胞的增殖[J]. 南方医科大学学报, 2024, 44(9): 1677-1684.

Liangjun XUE, Qiuyu TAN, Jingwen XU, Lu FENG, Wenjin LI, Liang YAN, Yulei LI. MiR-6838-5p overexpression inhibits proliferation of breast cancer MCF-7 cells by downregulating DDR1 expression[J]. Journal of Southern Medical University, 2024, 44(9): 1677-1684.

图/表 6

表1 引物序列

Tab.1 Sequences of primers for RT-qPCR

Primer	Forward	Reverse
DDR1	5'-GAACACGGACGAGGACATG-3'	5'-CGGCAACGAGGGAGAAAGG-3'
GAPDH	5'-CGAGCCACATCGCTCAGACA-3'	5'-GTGGTGAAGACGCCAGTGGA-3'

图1 miR-6838-5p在乳腺癌细胞及正常细胞表达情况及其对乳腺癌细胞增殖的影响

Fig.1 Expression of miR-6838-5p in breast cancer cells and normal cells and its effect on proliferation of breast cancer cells. A: Expression of miR-6838-5p in breast cancer cells and normal breast epithelial cells. B, C: Results of CCK-8 assay for assessing the effect of miR-6838-5p interference and overexpression on MCF-7 cell proliferation. D, E: EdU assay for assessing the effect of miR-6838-5p interference and overexpression on proliferation of MCF-7 cells (Original magnification: ×100). **P<0.01, ***P<0.0001.

图2 miR-6838-5p调控DDR1基因的表达

Fig.2 MiR-6838-5p regulates DDR1 expression in MCF-7 cells. A: Bioinformatics analysis of miR-6838-5p binding to DDR1. B: Luciferase activity assay. C-G: Western blotting and qRT-PCR for DDR1 expression level. H: Analysis of transfection efficiency of miR-6838-5p using qRT-PCR. *P<0.05, **P<0.01, ***P<0.0001 vs Control.

图3 DDR1促进了乳腺癌细胞的增殖

Fig. 3 DDR1 overexpression promotes proliferation of breast cancer cells. A, B: Western blotting for DDR1 protein level. C, D: CCK-8 assay for assessing the effect of DDR1 interference and overexpression on proliferation of breast cancer cells. E-H: EdU assay for assessing the effect of DDR1 interference and overexpression on proliferation of breast cancer cells (×100). *P<0.05, **P<0.01, ***P<0.0001.

图4 miR-6838-5p调控DDR1基因的表达抑制了乳腺癌细胞的增殖

Fig. 4 MiR-6838-5p inhibits breast cancer cell proliferation by regulating DDR1 expression. A: CCK-8 assay for assessing the effect of co-transfection with miR-6838-5p mimics and DDR1 vector on proliferation of breast cancer cells. B, C: EdU assay for assessing the effect of co-transfection with miR-6838-5p mimics and DDR1 vector on proliferation of breast cancer cells (×100). *P<0.05, **P<0.01.

图5 体内实验证明miR-6838-5p通过靶向DDR1抑制乳腺癌细胞的增长

Fig. 5 MiR-6838-5p overexpression inhibits the growth of breast cancer cells in nude mice by targeting DDR1. A: Representative images of xenografts derived from MCF-7 cells infected with miR-6838-5p or empty lentiviruses. B, C: Growth curve of the xenografts. D: Analysis of miR-6838-5p expression in the xenografts detected by qRT-PCR. E: DDR1 protein levels in the xenografts detected by Western blotting. F: Immunohistochemistry for examining Ki67 and DDR1 expressions in the xenografts (×100). *P<0.05.

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