Determination of Expression Profiles of miRNAs Regulating Immune Checkpoint Genes in Breast Cancer Cases

Project no. 22979 · Status: Kapanmış · 2021

Summary

Breast cancer (BC) cells are highly heterogeneous and the general approach to treatment is chemotherapy, radiotherapy, targeted therapies and immunotherapy after tumor resection. In immunotherapy, immune checkpoint inhibitors (ICi) target molecules that cause immunosuppressive environment in the tumor microenvironment and aim to eliminate tumor cells by increasing the activity of the immune microenvironment on cancer cells. The first immunotherapy in BC was approved by the FDA for the treatment of triple negative breast cancer (TNBC), which has a more immunogenic microenvironment compared to other types of BC. Although it is an important treatment option especially in patients with PD-L1 expression and has increased survival in some clinical trials, it has been reported to show insufficient activity in the treatment of TNBC. One of the most important factors determining the efficacy of immunotherapy is the expression profiles of IC molecules, which vary according to the cancer immune microenvironment. Therefore, it is important to increase the sensitivity of ICs and to develop targeted treatment methods by determining the expression profiles of genes involved in ICs and elucidating the molecular pathogenesis. miRNAs are short non-coding RNAs that are involved in oncogenic and many cellular processes such as cell proliferation, apoptosis, invasion and metastasis as regulators of gene expression at the post-transcriptional level. Apart from being a therapeutic target, they can also be used as diagnostic and prognostic markers. In addition, miRNA profiling is thought to contribute to the elucidation of the underlying molecular pathogenesis of diseases and the development of new target therapy models. Little is known about the correlation between microRNAs and the prognostic roles of co-inhibitory molecules involved in ICs or the therapeutic effects of antibodies targeting these molecules.For this purpose, in our study, we performed mRNA expression profiling of PD-1, PDL-1,CTLA-4,LAG-3 genes, which are involved in ICs, and expression profiling of 21 miRNAs, which were determined by in silico analysis to regulate these genes, in patients with BC and healthy control subjects. Thus, it was aimed to contribute to the development of new target treatment models by elucidating the molecular mechanisms underlying the expression of genes involved in ICs.Total RNA and miRNA were isolated from peripheral blood samples obtained from patients with CC (n=50) and healthy control subjects (n=46) and cDNA was synthesized by reverse transcription. Expression of 21 miRNAs was determined by q-RT-PCR method. Expression levels of mRNA of target genes and miRNAs thought to regulate these genes were evaluated by taking the significance of p<0.05 as a reference.There was no significant difference in PD-1, PD-L1, CTLA4 and LAG3 mRNA levels of the patients compared to the healthy control group. However, 21 miRNAs were analyzed in the patients and 6 of them were found to be upregulated or downregulated at different levels compared to the control group. miR-195-5p expression was found to be -3.09-fold lower compared to the control group (p<0.0001), while miR-6838-5p expression was -6.93-fold lower compared to the control group (p<0.0001). Compared to the control group, miR-520-5p expression, miR-302d-3p expression was 2.46-fold (p<0.0001), miR-302d-3p expression was 2.83-fold (p<0.0001), miR-15b-5p expression was 3.08-fold (p<0.0001) and miR-373-3p expression was 2.40-fold (p<0.0001) higher in the case group (Table 9, Graph 5, Graph 6 and Graph 7).The reason why IC molecules were not different in peripheral blood cells constituting our study sample compared to healthy controls may be that inhibitory signals do not occur due to the lack of chronic immunogenic stimuli generated from cancer cells in this region. In addition, upregulation of miRNAs known to play a role in cell proliferation and invasion may contribute to their use as a diagnostic and prognostic marker in CC. Expression analysis in tumor tissue for further studies will be important approaches that will contribute to the development of immune-based therapies.

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