MicroRNA -383-5p restrains the proliferation and migration of breast cancer cells and promotes apoptosis via inhibition of PD-L1.

MicroRNAs (miRs) play pivotal roles in breast cancer development. The dysregulation of miRs has been associated with PD-L1-mediated immune suppression. This study aimed to examine the effect of transfected miR-383-5p on breast cancer cells and T-cells and its association with clinicopathological features in affected patients. Initially, miR-383-5p and PD-L1 expression levels were investigated in breast cancer tissues. Then, MDA-MB-231 cells were transfected with miR-383-5p mimics to perform analyses. Cell viability was investigated using the MTT assay, and the annexin V/PI staining assay was performed to examine apoptosis induction. Furthermore, the effect of miR-383-5p on cell migration and cell cycle progression was analyzed using the wound-healing assay and flow cytometry, respectively. Gene and protein expressions were studied using qRT-PCR and western blotting. Finally, the effect of miR-383-5p on T-cells, which were co-cultured with cancer cells, was investigated. Compared to non-malignant tissues, PD-L1 was up-regulated, and miR-383-5p expression was downregulated in breast cancer tissues. Moreover, miR-383-5p reduced breast cancer cell viability via inducing apoptosis and modulating the expression of apoptosis-related genes. Besides, miR-383-5p could inhibit the migration of breast cancer cells via down-regulating metastasis-related genes. Besides, transfected miR-383-5p induced the secretion of pro-inflammatory cytokines from T-cells. Furthermore, the results showed that miR-383-5p might exert its tumor-suppressive effect via inhibiting the PI3K/AKT/mTOR pathway. The inhibitory effect of transfected miR-383-5p on the PI3K/AKT/mTOR pathway might be the underlying mechanism for inhibiting tumoral PD-L1 expression. Overall, miR-383-5p can be a promising therapeutic agent for treating breast cancer.

Azarbarzin S ，Hosseinpour-Feizi MA ，Banan Khojasteh SM ，Baradaran B ，Safaralizadeh R ... -  《-》

Tumor suppressive activity of miR-424-5p in breast cancer cells through targeting PD-L1 and modulating PTEN/PI3K/AKT/mTOR signaling pathway.

MicroRNAs (miRs) are key modulators of cellular processes such as proliferation, apoptosis, as well as anti-cancer immune responses. Here, we evaluated the role of miR-424-5p in breast cancer (BC) and investigated its effects on T cell-related immune response. BC tissues and cell lines were prepared and the expression of miR-424-5p and PD-L1, as well as the underlying molecular pathways, were assessed via qRT-PCR and western blotting. The MTT assay and flow cytometry were used to assess the effect of miR-424-5p on proliferation, apoptosis, autophagy, and cell cycle progression. The co-culture of T cells with MDA-MB-231 was performed for evaluating the role of miR-424-5p in rescuing T cell exhaustion. The results indicated the down-regulation of miR-424-5p and up-regulation of PD-L1 expression in BC tissue specimens. MiR-424-5p transfection into PD-L1 overexpressing MDA-MB-231 cells decreased the expression of PD-L1. Also, miR-424-5p could reduce MDA-MB-231 cell viability through modulating apoptosis and autophagy pathways. Furthermore, miR-424-5p transfection leads to decreased colony formation and increased cell number at the G2/M phase. Western blot analysis illustrated that miR-424-5p could exert its anti-proliferative effect via modulating PTEN/PI3K/AKT/mTOR pathway. Moreover, it was demonstrated that suppression of PD-L1 by miR-424-5p could participate in regulating the expression of effector cytokines in T cells. MiR-424-5p could be considered as a potential tumor-suppressor miR in regulating BC cellular growth, apoptosis, and T cell-related immune response through targeting PD-L1, and its downstream mediators. Therefore, we recognized miR-424-5p as a promising candidate for miR restoration therapy in BC patients.

Dastmalchi N ，Hosseinpourfeizi MA ，Khojasteh SMB ，Baradaran B ，Safaralizadeh R ... -  《-》

MiRNA-138-5p: A strong tumor suppressor targeting PD-L-1 inhibits proliferation and motility of breast cancer cells and induces apoptosis.

MicroRNAs are important regulators in multiple cellular processes and are closely related to a variety of cancers including breast cancer (BC). Immunotherapy using different methods such as modulating immune check points has been known as an advanced and successful procedure in cancer treatment. Here we investigated the effects of miRNA-138-5p restoring on Programmed Death Ligand 1 (PD-L-1) expression, BC biological behaviors and T-cell exhaustion. Breast cancer specimens and cell lines were provided and qRT-PCR and western blotting were used to measure the expression of miRNA-138-5p, PD-L-1 and other underlying genes. MTT and colony formation assays and scratch test were employed to specify proliferation, cloning and migration in miRNA-138-5p-transfected MDA-MB-231 cells respectively. DAPI staining assay and flow-cytometry were used to investigate apoptosis rate and cell cycle development. Finally, isolated T-cells were co-cultured with transfected BC cells to explore the effect of miRNA-138-5p on T-cell exhaustion. qRT-PCR revealed down-regulation ofmiRNA-138-5p conversely, up-regulation of PD-L-1 in BC tissues and cell lines. Transfection of miRNA-138-5p into MDA-MB-231 cells inhibited PD-L-1 expression. Western blotting, MTT and colony formation assays affirmed the anti-proliferative effect ofmiRNA-138-5p through down-regulating PI3K/AKT pathway. Also, miRNA-138-5p induced apoptosis in BC cells via up-regulating Caspase-9 and Caspase-3 and arresting cell cycle at sub-G1 phase. Moreover, scratch test and western blotting indicated that miRNA-138-5p inhibits cell motility via targeting MMP2, MMP9 and vimentin but up-regulating E-cadherin. Finally, miRNA-138-5p restrains T-cell exhaustion via suppressing PD-L-1 expression in BC cells leading to disrupt PD-L-1/PD-1 interaction and modulate effector cytokines in T-cells.

Rasoolnezhad M ，Safaralizadeh R ，Hosseinpourfeizi MA ，Banan-Khojasteh SM ，Baradaran B ... -  《-》

The combined therapy of miR-383-5p restoration and paclitaxel for treating MDA-MB-231 breast cancer.

The deregulation of microRNAs (miRs) has been identified in tumor development. Indeed, the restoration of tumor-suppressive miRs has been associated with inhibited tumor development in various cancers. Herein, we aimed to evaluate the impact of combined miR-383-5p restoration, as a tumor-suppressive miR, with taxol therapy in suppressing MDA-MB-231 breast cancer development. MDA-MB-231 cell line was restored with miR-383-5p and treated with paclitaxel both in combined and separate manners. The MTT experiment was carried out to measure the cytotoxicity of the therapeutic approaches on the tumoral cells. Besides, flow cytometry was conducted to assess apoptosis and cell cycle status following the treatments. Furthermore, the expression levels of critical factors contributed to tumor proliferation, migration, apoptosis were investigated via the qRT-PCR and western blotting techniques. The outcomes pointed out that the miR-383-5p might substantially enhance the chemosensitivity of MDA-MB-231 to taxol. Besides, miR-383-5p restoration and the combined therapy of miR-383-5p restoration with paclitaxel could remarkably increase apoptosis, decrease cell viability, arrest the cell cycle, inhibit clonogenicity, suppress tumor migration, suppress the PI3K/Akt signaling pathway, and down-regulate PD-L1 expression of BC cells. The restoration of miR-383-5p can enhance the chemosensitivity of MDA-MB-231 cells to taxol. Despite the anti-tumoral effects of miR-383-5p restoration on MDA-MB-231 breast cancer development, the combined therapy of miR-383-5p restoration with paclitaxel can be more effective in repressing MDA-MB-231 breast cancer development.

Dastmalchi N ，Azarbarzin S ，Safaralizadeh R ，Khojasteh SMB ，Shadbad MA ，Amini M ，Baghbanzadeh A ，Asl ER ，Baghbani E ，Lotfinejad P ，Baradaran B ... -  《-》

Protective effect of hsa-miR-570-3p targeting CD274 on triple negative breast cancer by blocking PI3K/AKT/mTOR signaling pathway.

To find out the role of hsa-miR-570-3p targeting CD274 in triple negative breast cancer (TNBC) via PI3K/AKT/mTOR signaling pathway. Hsa-miR-570-3p and CD274 expressions in 175 TNBC patients were detected by qRT-PCR and immunohistochemistry respectively. The human TNBC cell lines (MDA-MB-468 and MDA-MB-231) were used to verify the targeting relationship between hsa-miR-570-3p and CD274 via dual-luciferase reporter gene assay. Then, MDA-MB-468 and MDA-MB-231 cells were divided into Blank, miR-NC, miR-570-3p mimics, NC siRNA, CD274 siRNA, and miR-570-3p inhibitors + CD274 siRNA groups. Next, the biological activities of cells were detected by MTT, Cell-Light EdU, Annexin-V-FITC/PI, wound healing and Transwell invasion assays. Western blotting was conducted to detect protein expressions.MiR-570-3p expression was lower in tumor tissues than that in adjacent normal tissues, which was more obvious in CD274-positive TNBC patients, which targeted CD274 in TNBC cell lines. MiR-570-3p inhibited cell proliferation, invasion and migration, but induced cell apoptosis accompanying the upregulation of apoptotic proteins and downregulation of anti-apoptotic protein. CD274 siRNA had the similar results of miR-570-3p mimics, which could be reversed by miR-570-3p inhibitors. Besides, both miR-570-3p mimics and CD274 siRNA blocked PI3K/AKT/mTOR signaling pathway in TNBC cell lines. Hsa-miR-570-3p was downregulated and CD274 was upregulated in TNBC patients. Besides, hsa-miR-570-3p targeted CD274 to inhibit cell proliferation, invasion, migration, and induce cell apoptosis, which may be related to the suppression of PI3K/AKT/mTOR pathway.

Wang LL ，Huang WW ，Huang J ，Huang RF ，Li NN ，Hong Y ，Chen ML ，Wu F ，Liu J ... -  《-》