Volume 30, Issue 3 (5-2023)                   RJMS 2023, 30(3): 204-218 | Back to browse issues page

Research code: 1645
Ethics code: IR.PII.REC.1398.023
Clinical trials code: کار آزمایی بالینی ندارد

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Yousefi A, Sotoodehnejadnematalahi F, Nafissi N, Zeinali S, Azizi M. Investigating the Inhibitory Effect of miR-143-3P on the Level of MUC1 Gene Expression in Breast Cancer Cell Lines Compared to Normal Breast Cell Lines. RJMS 2023; 30 (3) :204-218
URL: http://rjms.iums.ac.ir/article-1-7617-en.html
Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran , mazizi528@gmail.com
Abstract:   (387 Views)
Background & Aims: In today's societies, breast cancer has significantly threatened women's health (1). Successful cancer treatment can also destroy tumor germ cells (2, 3). Treatment methods based on immune checkpoint molecules are considered (4). PD-L1 overexpression suppresses the immune response to cancer cells and causes epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) phenotypes, metastasis and chemical resistance (5). Overexpression of PD-L1 and MUC1 is observed in approximately 90 TNBCs. In this present study, we investigated the inhibitory effect of miR-143-3p on MUC1 expression as an oncogene that has the most impact on PDL-1 overexpression in BC cell lines MCF-7 (less aggressive BC), MDAMB231, and BT-549 (metastatic BC model). The suppressive effect of miR-143-3p on the expression of this oncogene as a direct effect and PDL1 level as an indirect result in BC cell lines was investigated. Besides, the effect of miR-143-3p overexpression on BC cell lines proliferation through downregulation of candidate oncogene was investigated.
Methods: First, the targeting of miR-143-3P on MUC1 3 'UTR was confirmed by using bioinformatics software and then by dual luciferase assay. The expression level of miR-143-3P and MUC1 were measured in breast cancer lines compared to the normal line. After transfection of miR-143-3P into breast cancer cell lines, MUC1 and PD-L1 expression in mRNA levels were evaluated by using qRT-PCR. The miR-143-3p effects on MUC1 and PD-L1 expression and its impact on cell proliferation was investigated by using MTT assay, (mean ± SD).
Results: The result of the dual luciferase assay was obtained from cloning the 3'UTR of the MUC1 gene in the psi-check2 vector and sequencing. According to the prediction analysis, the 3′-UTR of MUC1 gene has been considered as a putative miR-143-3p binding site (Target scan database). Transfecting miR-143-3p into MDA-MB-231, BT-549, MCF-7 cells harboring MUC1 3′-UTR decreased the luciferase activity to to 55% ± 1/73, 57% ± 1 /15 , 59 % ± 3/51% respectively, in comparison with the control (P < 0.01) (mean ± SD). he expression of miR-143-3p was significantly downregulated in BC cell lines compared to their control, and its downregulation was negatively correlated with MUC1 overexpression (r= -0.6419, p = 0.0244). Increased expression of miR-143-3P, decreased the mRNA level of MUC1 in MDA-MB231 to 1/3 ± 0/7, BT549 to 1/1± 0/45 and in MCF7 to 0/4/0 ±21, compared whit untransfected cells.
MiR-143-3p introduction to BC cells downregulates PDL-1 indirectly. Overexpression of miR-143-3P reduces the MUC1expression, and the reduction of MUC1 expression reduces PD-L1 expression mRNA level in MDA-MB231 to 3/4±0.45, BT549 to 3/1±0.56, MCF7 to 0/53±0.48, compared whit un transfected cells (fig4A). miR-143-3p inhibits BC cellular proliferation. An MTT assay revealed that miR-143-3p overexpression significantly suppressed the proliferation in MCF-7 to 52% ± 1.55, BT549 to 48% ± 2.45 MDA-MB231 to 43% ± 3.36, compared whit non-transfected cells 72 h after transfection.
Conclusion: Breast cancer treatment has made significant progress in recent years, and a new treatment called immunotherapy, which uses the body's immune system to treat cancer, has become popular (6). One of the symptoms of breast cancer tumors is the overexpression of MUC1. A high expression level of MUC1 is associated with poor prognosis in cancer patients (7, 8). In an animal model of breast cancer, intravenous injection of MUC1 suppresses the immune system and accelerates the death of tumor-bearing mice. The serum level of MUCl expression is also associated with suppression of the immune system in patients with metastatic adenocarcinoma treated with active specific immunotherapy (8, 9). As we mentioned earlier, MUC1 can increase the expression of PD-L1 by recruiting MYC and NF -kB p65 to the PD-L1 promoter in some solid tumors, such as triple negative breast cancer (10). the mechanisms underlying PD-L1 expression in MUC1-positive tumor tissue were investigated, and the potential of targeting PD-L1 to inhibit the progression of MUC1-overexpressing colon cancer in mice, Therefore, the role of MUC1 in tumor immune evasion has been confirmed and can be a basis for using PD-L1 inhibitors in MUC1-positive colon cancer (8). microRNAs can regulate the expression of immune checkpoint receptors and their ligands (11). Changes in the expression of miR-143 often play a role in tumorigenesis (12). In 2020, Y Tokumaru and his colleagues showed by studying breast cancer samples with estrogen receptors that the high expression of microRNA-143 is associated with a favorable tumor immune microenvironment and better survival in breast cancer. They reported that miR-143 is a tumor suppressor microRNA that exhibits anti-tumor function by targeting KRAS signalling pathways in various malignancies. The high expression of miR-143 in cancer cells with a favorable tumor immune microenvironment regulates the positivity of anti-cancer immune cells. The suppression of pre-cancerous immune cells is related and leads to better survival of breast cancer patients (13).
In this study, we demonstrated MUC1 was potential as a novel target of miR-143-3p, therefore, the dual-luciferase reporter assay verified that miR-143-3p directly targeted MUC1 and inhibited this transcription. We found miR-143-3p downregulated in breast cancer cell lines compared to the normal line via qRT-PCR. In the current study, the qRT-PCR results illustrated an inverse relation between MUC1 mRNA expression and miR-143-3p expression in breast cancer cell lines. We investigated indirect targeting of PD-L1 via MUC1 targeting by miR-143-3p., be able to be prevent the cell proliferation in breast cancer cell line. Therefore we recognized, targeting MUC1 may be a novel strategy for BC therapy and prevent the progression of breast cancer and miR-143-3p as a promising candidate for miR restoration therapy in BC patients.

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Type of Study: Research | Subject: Biology

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