• Investigation of miR-185 downregulation and its regulatory correlation with competing endogenous long non-coding RNA MIR155HG in breast carcinoma patients
  • Reyhane Hosseini,1,* Fateme Ruhollah,2 Majid Sadeghizadeh,3 Sadegh Babashah,4
    1. Master of Cellular and Molecular Sciences Islamic Azad University of Medical Sciences, Tehran, Iran
    2. Professor, Department of Cellular and Molecular Biology, Islamic Azad University of Medical Sciences, Tehran, Tehran, Iran
    3. Professor, Department of Cellular and Molecular Biology, Islamic Azad University of Medical Sciences, Tehran, Tehran, Iran
    4. Professor, Department of Cellular and Molecular Biology, Islamic Azad University of Medical Sciences, Tehran, Tehran, Iran


  • Introduction: Abstract The aim of this study was to evaluate the down-regulation of miR-185 and its regulatory relationship with non-coding competitive endogenous RNA MIR155HG in breast carcinoma patients and to determine its relationship with pathological indicators such as stage, grade, metastatic involvement of lymph vessels and HER activity status. -2. So far, few studies have been performed to evaluate the down-regulation of miR-185, especially its regulatory association with non-coding competitive endogenous RNA MIR155HG in breast carcinoma patients. In the present study, the expression of an lncRNA called MIR155HG in patients with breast cancer was compared with healthy individuals and the results showed a significant increase in its expression in patients. In the present study, miR-185, which was studied, had a tumor suppressant role and as it was observed, its expression in exosomes showed a significant decrease. The results indicate a regulatory relationship between these two non-coding RNAs. Introduction Breast cancer is one of the most common cancers in women. More than 5.1 million women (25% of all women with cancer) are diagnosed with breast cancer each year [1]. The genetic factors involved in causing cancer have been significantly studied. However, evidence has shown that a significant proportion of cancer predisposing factors can not be attributed to changes in protein coding sequences [2]. The identification of a large number of long non-coding RNAs (lncRNAs) with a length of more than 200 bp in humans has helped to unveil the position of these molecules in cancer pathology and their role as important components in thrombosis. [3]. Recently, biological prophylaxis, mainly known as monoclonal antibodies to breast cancer, has been developed to improve the quality of life in patients with breast cancer [4]. One of the main targets of these monoclonal antibodies is HER2 [5]. Early detection of the disease can lead to a good prognosis and a high survival rate. Breast cancer is the most common malignancy in women, causing high mortality. miRNAs are secreted by exosomes and are highly diverse microRNAs act as oncogenes or tumor inhibitors by inhibiting the expression of cancer-related target genes [6]. Early detection of breast tumors is one of the most important challenges in cancer management. Due to the importance of non-coding RNAs in the pathogenesis of breast cancer, this study determined the expression levels of the long non-coding RNA MIR155HG, which functions as a ceRNA, and its associated microRNA, miR-185.
  • Methods: Methods Serum samples were selected from patients with breast carcinoma and serum samples from healthy individuals without any disease. The serum is a fluid part of the blood that lacks the coagulation proteins provided by the project [7]. Exosomes were isolated from serum using Exoquick kit. Then complete RNA extraction steps, quantitative evaluation of the extracted RNA, cDNA synthesis, real-time quantitative polymerase chain reaction, data analysis using threshold cycle comparison method. Normalization of changes in miRNA expression levels was performed in comparison with endogenous U6 snRNA expression levels [8]. [(Control) mCT reference gene - (control) mCT gene] = ΔCT: control [(Test) mCT reference gene - (Test) mCT gene] = ΔCT: Test (Test) ΔCT- (control) ΔCT = ΔΔCT Data on average ± Standard deviation (SD) was presented and analyzed using Graph Pad software. Due to the normality of data distribution, the comparison between the treatment group and the control group was analyzed using T-test and p-value was calculated [9].
  • Results: Results Determining the specificity of real-time PCR reaction using melting curve In this study, SYBR Green dye and its ability to bind to double-stranded DNA formed in PCR reaction were used. This dye is attached to it by replacing it in a small DNA groove. One of the advantages of this method is that it is cheap, convenient and sensitive. One of its major disadvantages is that it connects to double strands such as Primer dimer and other non-specific bands, the results are estimated to be higher than the original concentration and therefore optimization should be done in such a way that the dimer primer and non-specific product is minimized. , Each strand of DNA in the product is single-stranded based on the length and content of GC bases at its own specific melting temperature of 3, and this state change is displayed by the system as a peak. By examining the resulting peaks, it can be seen that the peaks formed at low temperatures are directly related to the amount of nonspecific products formed at the end of the PCR process to confirm the results of the analysis. Melt Curve Analysis is used. Figure 1- As shown in the figure, each gene studied has a unique melting temperature, indicating that the product of amplification was real-time PCR. Evaluation of MIR155HG regulatory RNA expression and miR-185 secretory microRNA in serum extracellular vesicles of breast carcinoma patients Figure 2 In the first step to investigate the possibility of biomarkers of non-coding RNAs selected in this study, their expression levels were examined in exosomal samples of patients and healthy individuals. As shown in the figure, the expression of MIR155HG lncRNA in the samples of breast cancer patients was significantly higher than normal samples (** P <0.01) and also the expression of miR-185 in the samples of breast cancer patients was significantly higher than the sample. Normals were lower. (** P <0.01) The results suggest an inverse regulatory relationship between these two non-coding RNAs. These results are consistent with the function of MIR155HG ceRNA in regulating miR-185 expression. The figure below shows the gene amplification curve in the Real-time PCR reaction. Evaluation of long non-coding regulatory RNA expression of MIR155HG and regulatory secretory microRNA miR-185 in patients in different stages of the disease Figure 3 Since one of the objectives of this study was to determine the relationship between the expression levels of selected noncoding RNAs and the clinical parameters of breast carcinoma, we found that the expression of miR-185 and MIR155HG lncRNA among patients in different groups based on disease stage Were checked. As can be seen in the figure, the expression level of MIR155HG in Stage III showed increased expression compared to the group of patients in Stages I and II (** P <0.01). In the case of miR-185, the results showed that its expression level in advanced stage III of the disease showed reduced expression compared to the lower stages I, II (** P <0.01). Evaluation of expression levels of long non-coding regulatory RNA of MIR155HG and regulated secretory microRNA of miR-185 in breast carcinoma patients with different Her-2 activity status Because the expression status of Her2 in breast carcinoma patients is directly related to metastasis, the expression levels of long non-coding regulatory RNA MIR155HG and regulated secretory microRNA miR-185 in serum extracellular vesicles of breast carcinoma patients with Her- activity status We paid 2 differently. Figure 4 As can be seen in the figure, MIR155HG lncRNA expression levels showed higher expression in patients with Her-2 (Her-2 positive) expression than in patients with Her-2 (Her-2 negative) expression (* P <0 . 05). In contrast, the microRNA regulated by this ceRNA, miR-185, showed lower expression levels in patients with Her-2 (Her-2 positive) activity than in patients with Her-2 (Her-2 Negative) inactivity (* P < 0. 05). These results are consistent with the function of MIR155HG ceRNA in regulating the expression and function of miR-185 as well as the oncogenic function of MIR155HG lncRNA and the role of miR-185 tumor-expression. Assessing the biomarker potential of non-transfecting RNAs MIR155HG lncRNA and miR-185 in the diagnosis of breast carcinoma Due to the fact that the expression of miR-185 and MIR155HG lncRNA in the two groups of patients and healthy showed a significant difference and inversion with each other and the relationship between their expression with some clinical pathological features of the disease such as disease stage and activity status of Her-2 It was identified, then by examining the ROC (Receiver Operating Characteristics) curve to determine the biomarker capability (biomarker) of these two non-coding RNAs. In this study, miR-185 had decreased expression and MIR155HG lncRNA had increased expression in serum extracellular vesicles compared to the healthy sample. Area under curve (AUC) was 0.81 for miR-185 and 0.87 for MIR155HG lncRNA Calculated. These numbers suggest biomarker potential for these two ncRNAs. Figure 5 Heatmap analysis of MIR155HG lncRNA and miR-185 expression in breast tumor serum and healthy controls Heat map analysis was used to visualize the expression changes of non-coding RNAs between tumor samples compared to healthy individuals. As can be seen in the figure, green indicates higher expression levels and red indicates lower expression levels. Figure 6 In the present study, the expression of an lncRNA called MIR155HG in patients with breast cancer was compared with healthy individuals and the results showed a significant increase in its expression in patients. In the present study, miR-185, which was studied, had a tumor suppressant role and as it was observed, its expression in exosomes showed a significant decrease. The results indicate a regulatory relationship between these two non-coding RNAs.
  • Conclusion: Conclusion Therefore, according to the results of this study, the following can be mentioned: The results showed that the expression ratio of MIR155HG in the serum exosome of breast carcinoma was significantly higher than the normal serum exosome. MIR155HG expression levels at higher stages of expression disease are somewhat higher than at lower stages. MIR155HG expression levels in patients who were HER2 positive showed higher expression than in patients who did not express HER2. The results showed that the normalized expression ratio of miR-185 in the serum of patients was significantly lower than that of healthy individuals. MiR-185 expression levels in the higher stages of the disease show somewhat lower expression than in the lower stages. MiR-185 expression levels in patients who were HER2 positive showed less expression than in patients who did not express HER2.
  • Keywords: Key words Decreasing adjustment -miR-185-Non-coding RNA-MIR155HG