MACE-Seq-based coding RNA and TrueQuant-based small RNA profile in breast cancer: tumor-suppressive miRNA-1275 identified as a novel marker

Abstract

Introduction Disruption of cellular processes in the breast by abnormally expressed miRNA is characterized to develop cancer. We aimed to identify the differential expression of small RNAs (sRNAs) and mRNAs in formalin-fixed paraffin-embedded (FFPE) tissue of the breast cancer (BC) and normal adjacent tissue (NAT). Another aim is to determine the differential expression of miR-1275 as a novel biomarker for BC and also identify its target genes. Methods TrueQuant method for analysis of sRNA expression and MACE-sequencing method for analysis of gene expression were used analyzing. The RT-qPCR technique was used to confirm miR-1275 down expression. Target genes of miR-1275 were computationally identified using target prediction sites and also the expression level of them was experimentally determined among the expressed genes. Results TrueQuant findings showed that 1400 sRNAs were differentially expressed in the FFPE tissue of two Kurdish cases with BC, as compared to NAT. Among the sRNAs, 29 small RNAs were shown to be significantly downregulated in BC cells. The RT-qPCR results confirmed that miR-1275 was significantly down-expressed in 20 Kurdish cases with BC compared to NAT. However, Overall survival (OS) analysis revealed that the correlation between the expression level of miR-1275 and clinical significance was highly corrected in cases with BC (OS rate: P = 0.0401). The MACE-seq results revealed that 26,843 genes were differentially expressed in the BC tissue compared to NAT, but 7041 genes were displayed in a scatter plot. Furthermore, putative target genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were computationally identified as direct targets of miR-1275 in several target predicted sites. The MACE-seq results revealed that the expression level of these targets was increased in BC tissue compared to NAT. The level of these targets was negatively associated with miR-1275 expression. Finally, the role of down-regulated miR-1275 on its targets in biological mechanisms of BC cells was identified; including cell growth, proliferation, movement, invasion, metastasis, and apoptosis. Conclusion Down-expressed miR-1275, a tumor suppressor, is a novel biomarker for early detection of BC. DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA are newly identified to be targeted by miR-1275.

RNA Sequencing-Based Total RNA Profiling; The Oncogenic MiR-191 Identification as a Novel Biomarker for Breast Cancer

“…To understand the differentially expressed mRNA (DEmRNA) transcripts, RNA sequencing was carried out from the seven paired samples that were used to identify DEncRNA molecules by applying Massively Analysis of cDNA End sequencing (MACE-Seq). The materials and methods for extraction of total RNA were previously described and also found in (3). Biotinylation of the cDNA cap structures was done precisely.…”

“…The seven paired samples that were used to identify DEncRNA molecules, were also used to identify differential expression of the identified target genes by applying Massively Analysis of cDNA End sequencing (MACE-Seq). The procedure of these techniques and bioinformatic analysis were described in (3). Then, the result of the identified target genes was statistically analyzed by GraphPad Prism (V 8.0.1).…”

“…Breast cancer (BRCA) is a genomic illness wreaking havoc on women's health worldwide (1)(2)(3). According to the Global Cancer Project 2018, The most frequent cancer diagnosed in women is BRCA (11.7 percent of cases all), which is followed by lung (11.4 percent) colorectal (10.0 percent) cancers (4).…”

“…Specific BRCA clinicopathologic characteristics, such as expression level of estrogen and progesterone receptor, vascular invasion, and tumor stage, are related to miRNA expression. Furthermore, BRCA has been found to have an abnormal expression of miRNAs between normal and cancerous tissues, as well as between breast cancer molecular subtypes, such as Her2+, luminal A, luminal B, and basal-like (3,12). A study revealed that miRNAs in BRCA have significantly contributed to the onset and development.…”

The Regulatory Network and Potential Role of LINC00973-miRNA-mRNA ceRNA in the Progression of Non-Small-Cell Lung Cancer

“…miRNA could trigger degradation of downstream target genes by recognizing the MRE in the 3’non-transcribed region ( 11 , 12 ). Therefore, when lncRNA and mRNA had the same MRE, both of them could competitively bind to miRNA, forming a competitive endogenous RNA (ceRNA) regulatory network ( 13 , 14 ). When miRNA bound to the target mRNA, the stability of mRNA decreased and its translation was hindered, thereby affecting gene expression.…”