biomolecules Article The Influence of rs1859168 Polymorphism on Serum Expression of HOTTIP and Its Target miR-615-3p in Egyptian Patients with Breast Cancer Omayma O. Abdelaleem 1, Olfat G. Shaker 2 , Marwa N. AbdelHafez 3, Noha K. Abdelghaffar 4, Hanaa M. Eid 5,*,†, Mohamed Zaidan 6, Abeer A. Khalefa 7, Naglaa A. Ahmed 7, Nada F. Hemeda 8 , Othman M. Zaki 9, Aeshah Ali A. Awaji 10 and Shereen R. Mohammed 1 1 Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Fayoum University, Fayoum 63511, Egypt; [email protected] (O.O.A.); [email protected] (S.R.M.) 2 Department of Medical Biochemistry and Molecular Biology, Faculty of Medicine, Cairo University, Cairo 11511, Egypt; [email protected] 3 Department of Medical Oncology, National Cancer Institute, Cairo University, Cairo 11511, Egypt; [email protected] 4 Department of Clinical pathology, Faculty of Medicine, Fayoum University, Fayoum 63511, Egypt; [email protected] 5 Department of Microbiology and Immunology, Faculty of Medicine, Fayoum University, Fayoum 63511, Egypt 6 Department of General Surgery, Faculty of Medicine, Fayoum University, Fayoum 63511, Egypt; [email protected] Citation: Abdelaleem, O.O.; Shaker, 7 Department of Physiology, Faculty of Medicine, Zagazig University, Zagazig 44523, Egypt; O.G.; AbdelHafez, M.N.; [email protected] (A.A.K.); [email protected] (N.A.A.) Abdelghaffar, N.K.; Eid, H.M.; 8 Department of Genetics, Faculty of Agriculture, Fayoum University, Fayoum 63511, Egypt; Zaidan, M.; Khalefa, A.A.; Ahmed, [email protected] N.A.; Hemeda, N.F.; Zaki, O.M.; et al. 9 Department of Clinical Pathology, Faculty of Medicine, Damietta University, Damietta 34511, Egypt; The Influence of rs1859168 [email protected] 10 Polymorphism on Serum Expression Department of Biology, Faculty of Science, University College of Taymaa, Tabuk University, of HOTTIP and Its Target miR-615-3p Tabuk 47711, Saudi Arabia; [email protected] in Egyptian Patients with Breast * Correspondence: [email protected]; Tel.: +20-0101-154-2894 † Present address: Medical Microbiology and Immunology, Faculty of Medicine, Fayoum University, Egypt. Cancer. Biomolecules 2021, 11, 733. https://doi.org/10.3390/biom11050733 Abstract: Background: Polymorphisms of long noncoding RNAs are lately documented as hazardous Academic Editor: Cecilia M. factors for the development of numerous tumors. Furthermore, the evaluation of noncoding RNAs P. Rodrigues has emerged as a novel detector of breast cancer patients. We aimed to genotype the HOXA transcript at the distal tip (HOTTIP) rs1859168 and assess its relationship with the levels of the serum HOTTIP Received: 4 April 2021 and its target miR-615-3p in patients with breast cancer (BC). Methods: One hundred and fifty- Accepted: 7 May 2021 one patients with BC, 139 patients with fibroadenoma (FA), and 143 healthy participants were Published: 14 May 2021 incorporated into the current study. The genotyping of rs1859168 and the measurements of the HOTTIP and miR-615-3p levels were assessed using quantitative real-time PCR. Results: We revealed Publisher’s Note: MDPI stays neutral a significant association between each of the CC genotypes, C allele, dominant and recessive models, with regard to jurisdictional claims in and the increased risk of BC (p = 0.013, p < 0.001, p < 0.001, and p < 0.001, respectively) relative published maps and institutional affil- to the healthy controls. Similarly, the CC genotype, C allele, and recessive model were observed iations. to be related to the increased incidence of BC with respect to FA (p < 0.001 for all). A significant upregulation of HOTTIP and a marked decrease of miR-615-3p were verified in patients with BC compared to each of the healthy individuals, patients with FA, and the non-BC group (healthy subjects + FA) (p < 0.001 for all). A significant negative correlation was demonstrated between the Copyright: © 2021 by the authors. expression of HOTTIP and miR-615-3p in the serum of patients with BC. The HOTTIP expression was Licensee MDPI, Basel, Switzerland. upregulated, while that of miR-615-3p was downregulated in patients with BC who carried the CC This article is an open access article distributed under the terms and genotype with respect to those who carried the AA or AC genotypes (p < 0.05 for all). Conclusions: conditions of the Creative Commons The genetic variants of rs1859168 are linked to an increased susceptibility to BC. Moreover, HOTTIP Attribution (CC BY) license (https:// and miR-615-3p may be used as novel indicators and targets for the treatment of patients with BC. creativecommons.org/licenses/by/ 4.0/). Biomolecules 2021, 11, 733. https://doi.org/10.3390/biom11050733 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 733 2 of 15 Keywords: breast cancer; fibroadenoma; rs1859168; HOTTIP; miR-615-3p 1. Introduction Breast cancer (BC) is considered the most frequent malignancy diagnosed among women [1]. Genetic alterations play an essential role in BC pathogenesis [2]. Most breast cancers are diagnosed at a progressive stage due to difficulties in the early detection of the disease. Therefore, detecting new biomarkers is necessary to diagnose BC and predict its behavior, allowing the development of new therapeutic targets [3]. Long noncoding RNAs (lncRNAs) are recognized as RNA molecules with more than 200 nucleotides in length and without a protein-coding capability [4]. Emerging evidence has recently shown that lncRNAs play an essential role in the regulation of some genes concerning the suppression of tumors or oncogenes affecting tumor initiation and progression [5]. The HOXA transcript at the distal tip (HOTTIP) is a lncRNA found at the 50-end of the HOXA cluster [6]. Numerous works have revealed that HOTTIP regulates HOXA gene expression so that it may play a role in cancer pathogenesis. The upregulation of HOTTIP expression has been described in a variety of malignancies [7]. Interestingly, Yang and his colleagues also reported a positive correlation in BC between HOTTIP expression and lymph node metastasis, tumor size, and TNM staging [8]. However, the prevalence and functional significance of HOTTIP in breast cancer still requires more investigation. Many recent researchers have revealed that single-nucleotide polymorphisms (SNPs) present in lncRNA genes affect the function and expression of those lncRNAs, representing an important risk factor of disease susceptibility [9]. Furthermore, numerous studies have explained the associations between lncRNA polymorphisms and the susceptibility to different cancers [10,11]. Recently, Hu et al. identified that the functional variant rs1859168 (A/C) in the HOTTIP regulation region contributed to the risk of pancreatic cancer [12], considering A as the wild allele and C as the mutant one. However, there were no studies that reported the associations between this SNP and BC risk. MicroRNAs (miRNAs) are small noncoding RNAs (19–25 nucleotides in length). Accumulating evidence has linked miR-615-3p to many cancers, it was revealed that miR-615-3p was downregulated in hepatoma cells and breast cancer cells, as well as pancreatic ductal adenocarcinoma [13–15]. Additionally, Bai et al. verified that miR- 615 was significantly repressed in BC tissues and inhibited the expression of AKT2 [14]. However, little is known about its role in BC. Moreover, it was proven that HOTTIP directly targets miR-615 (as it acts as a competing endogenous RNA (ceRNA) for miR-615-3p, resulting in the activation of its endogenous targets), affecting the growth of renal cell carcinoma [16]. In the current study, we aimed to evaluate whether the rs1859168 SNP is associated with breast cancer or the development of fibro adenoma (FA). Additionally, we performed this work to find out whether this polymorphism could affect the serum expression level of HOTTIP and its target microRNA (miR-615-3p) and explore any correlations with the clinicopathological features. Furthermore, we investigated the possibility of using HOTTIP and miR-615-3p as noninvasive biomarkers of BC, analyzing the correlations between their serum expression levels and pathology, as well as the clinical data in BC. 2. Materials and Methods 2.1. Subjects The current study included 151 BC patients who were histologically or cytologically confirmed to have BC. Additionally, 139 female patients with FA were incorporated into this study. Patients were recruited from the outpatients and inpatients of the Surgery Biomolecules 2021, 11, 733 3 of 15 Department, Faculty of Medicine, Fayoum University and from The National Cancer Institute, Cairo University during the period from December 2018 to May 2020. Patients with a history of any other malignancies, current infectious or inflammatory conditions, or those who received any direct or specific treatments for cancer were excluded from the study. A total of 143 healthy age-matched women who underwent a medical examination in the outpatient clinic of the Surgery Department, Faculty of Medicine, Fayoum University volunteered as the control subjects. Neither they nor their families had a history of cancer. All the enrolled participants gave signed informed consent. Ethical approval was obtained from the Faculty of Medicine, Fayoum University Local Ethics Committee, which was in-line with the ethical guidelines of the Declaration of Helsinki. 2.2. Blood Sample Processing Venous blood samples were taken from each participant. Samples were delivered into a plain vacutainer with a gel separator. The blood was allowed to clot for 15 min, and the serum was separated by centrifugation at 4000× g for 10 min. Serum samples were immediately stored at −80 ◦C until use. Another group of whole-blood samples was drawn in EDTA-containing tubes for the DNA extraction and genotyping of rs1859168. 2.3. Total RNA Extraction and Reverse Transcription MiRNeasy extraction kit (Qiagen, Hilden, Germany) was used to get the total RNA (including microRNAs and lncRNAs) extracted from the serum samples after adding the QIAzollysis reagent following the manufacturer’s protocol.