Effects of miR-34a-5p on Triple Negative Breast Cancer Cells and Related Mechanisms

doi:10.13523/j.cb.2206036

China Biotechnology

2023, Vol. 43

Issue (1): 18-26 DOI: 10.13523/j.cb.2206036

Effects of miR-34a-5p on Triple Negative Breast Cancer Cells and Related Mechanisms

DENG Si-yu¹,LIANG Bing^1,²,WEI Wei¹,WANG Meng-na¹,CAO You-de^1,^2,^**(

)

1 College of Basic Medicine, Chongqing Medical University, Chongqing 400016, China
2 The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China

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Abstract

Objective: To investigate the expression of miR-34a-5p in triple negative breast cancer (TNBC), its role in TNBC cell proliferation, apoptosis and migration, its effects on tumor growth in TNBC mice and its impact on the expression of B7-H1 in TNBC. Methods: RT-qPCR and Western blot were used to analyze the expression of miR-34a-5p and B7-H1 in TNBC cells, respectively. Kaplan-Meier was used to analyze the association between the expressions of miR-34a and B7-H1 with the survival rates of TNBC patients. The effects of miR-34a-5p on the proliferation, apoptosis and migration of TNBC cells were detected by CCK-8, flow cytometry and wound healing. The interaction between miR-34a-5p and B7-H1 was verified by Dual-luciferase reporter assays. The expression levels of miR-34a-5p and B7-H1 were detected by RT-qPCR and Western blot after transfected with miR-34a-5p mimics into TNBC cells. RT-qPCR, Western blot and IHC were used to detect the effect of miR-34a-5p agomir on the expression of miR-34a-5p and B7-H1 in MDA-MB-231 tumor bearing mice. Results: The expression level of miR-34a-5p in TNBC cells was lower than that in normal breast cells, and the expression level of B7-H1 was higher than that in normal breast cells, low expression of miR-34a and high expression of B7-H1 were related to the poor prognosis of TNBC patients, and the difference was statistically significant (P<0.01). miR-34a-5p could inhibit cell proliferation and migration, promote cell apoptosis, and target downregulate the expression of B7-H1 in TNBC cells. miR-34a-5p agomir inhibited tumor growth and B7-H1 expression of MDA-MB-231 tumor bearing mice. Conclusion: miR-34a-5p plays an important role in the development and progression of TNBC. Targeting miR-34a-5p/B7-H1 may become a new treatment strategy for TNBC patients.

Key words： MicroRNA-34a-5p Triple negative breast cancer B7 homolog 1

Received: 19 June 2022 Published: 14 February 2023

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	Si-yu DENG
	Bing LIANG
	Wei WEI
	Meng-na WANG
	You-de CAO

Cite this article:

DENG Si-yu, LIANG Bing, WEI Wei, WANG Meng-na, CAO You-de. Effects of miR-34a-5p on Triple Negative Breast Cancer Cells and Related Mechanisms. China Biotechnology, 2023, 43(1): 18-26.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2206036 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I1/18

Table 1 Primer sequences

Fig.1 The difference of miR-34a-5p expression in MCF 10a, MDA-MB-231 and BT-549 cell lines and its correlation with prognosis (a) RT-qPCR analysis showed that the expression of miR-34a-5p in MDA-MB-231 and BT-549 was lower than that in MCF 10A (^** P<0.01, ^*** P<0.001) (b) Kaplan Meier analysis showed that low expression of miR-34a was associated with poor prognosis of TNBC patients (P<0.01)

Fig.2 Effect of miR-34a-5p on proliferation, apoptosis and migration of TNBC cells (a) miR-34a-5p inhibited TNBC cells at 24h, 36h and 48h after transfection (^** P<0.01, ^*** P<0.001) (b) Transfection of miR-34a-5p promoted TNBC cell apoptosis (c) Transfection of miR-34a-5p inhibited TNBC cell migration

Fig.3 Targeting relationship between miR-34a-5p and B7-H1 (a) Dual-luciferase reporter gene assay was performed to detect the relative luciferase activity of TNBC cells (^* P<0.05) (b) Western blot showed that the expression of B7-H1 in MDA-MB-231 and BT-549 was lower than that in MCF 10A (c) Kaplan Meier analysis showed that high expression of B7-H1 was associated with poor prognosis of TNBC patients (P<0.01)

Fig.4 Transfection of miR-34a-5p inhibited the expression of B7-H1 in TNBC cells (a) RT-qPCR demonstrated that overexpression of miR-34a-5p was successfully transfected (^*** P<0.001) (b) Western blot and quantitative analysis showed that B7-H1 expression changed (^** P<0.01, ^*** P<0.001)

Fig.5 miR-34a-5p inhibited tumor growth and downregulated B7-H1 expression in TNBC tumor bearing mice (a) Representative images of tumor bearing mice in different treatment groups after each injection, the statistics of tumor volume and mice weight (b) RT-qPCR was used to analyze the relative expression of miR-34a-5p in tumor of different treatment groups (^*** P<0.001) (c) Western blot and quantitative analysis of B7-H1 relative expression in different treatment groups (^* P<0.05) (d) The expression of B7-H1 was detected by IHC

Fig.6 Lollipop chart of correlation between B7-H1 and immune related cells in breast cancer


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