MiR-6838-5p overexpression inhibits proliferation of breast cancer MCF-7 cells by downregulating DDR1 expression

doi:10.12122/j.issn.1673-4254.2024.09.07

Abstract

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

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.

Figures/Tables 6

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'

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.

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.

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.

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.

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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