Identication of Tumor-Suppressive miRNA-1275 as a Novel Marker for Breast Cancer (BC) by MACE-Sequencing and RT-qPCR Techniques

Disruption of cellular processes in the breast by abnormally expressed miRNA is characterized to develop cancer. We aimed to determine the differential expression of coding and non-coding RNAs in formalin xed paran embedded (FFPE) blocks of breast cancer (BC) tissue and normal adjacent tissue (NAT). Another aim is to determine differential expression of has-miR-1275 as novel biomarker for BC and identify its target genes using prediction sites and experimentally expression level of them via the MACE-sequencing technique. MACE-sequencing technique was utilized to analyze differential expression of coding RNAs and small RNAs (sRNAs). Among small RNAs, miRNA-1275 expression was focused and conrmed using RT-qPCR technique in 20 Kurdish cases with BC . Moreover, clinical signicance of miR- 1275 and its target genes was studied in a large number of patients with BC using the data obtained from The Cancer Genome Atlas database. miRNA-1275 down-expressed for early detection of breast cancer. DVL3, PPP 2R2D, THSD4, CREB1, SYT7, and PRKACA are novely identied to be targeted by miR-1275 in BC cells. putative targets and their pathways were aimed to explain in the cells of BC. Six genes (DVL3, PPP2R2D, TSHD4, CREB1, SYT7, and PRKACA) were experimentally observed to be overexpressed in the cells of BC. Based on the databases of miRNA target prediction, they were selected and closely correlated with poor prognosis. Among these candidate genes, four genes (PPP2R2D, DVL3, and CREB1) were shown to be strongly targeted by the miR-1275 in the BC cells. Studies showed that these regulators were found to reduce cell survival and migration in cancer cells (40, 41). DVL3 is observed to be implicated in the breast cancer pathways (34) and negatively regulated by miR-1275. The up-regulation of this gene can increase the cancer cell proliferation, migration and invasion in BC cells. The overexpression of which increase cancer cell proliferation ability (42). Another target gene, CREB1 and PRKACA show also negative correlation with miR-1275 level. Whereas CREB1 was found to reduce apoptosis process and increase cell proliferation in breast cancer (43), PRKACA plays a key role in tumorigenesis and development of BC (44). However, the role of THSD4 and SYT7, currently unidentied, may enhance tumor growth in a variety of cancers, especially breast cancer (45, 46).


Introduction
Breast malignant cell is a prominent type of cancers mostly diagnosed in females, and the second most frequent malignancy-associated deaths worldwide, especially in the US and Asian countries (1,2). Approximately two million females are annually diagnosed and more than 620,000 deaths are newly recorded every year (3,4). Frequently, BC is developed as a result of a genomic mutation. However, about 10% of BCs is inheritably come down from parents to their generations; whereas, more than 85% of BCs is developed in their lifetime (4,5). Inherited abnormalities in TP53 and PTEN genes were studied to result in the high risk of the breast malignant cell progression (6,7).
Gene expression pro ling has recently played a critical role in medicinal selection for BC subtypes. The analysis of BC gene expression can be used for molecular category of BC subtypes (8)(9)(10). Two studies reported that this classi cation facilitates the determination of the cure doses. The molecular subtypes of BC can be categorized into luminal-A, luminal-B (including HER2+ /-), HER2+, and triple negative (TN) (11,12). These subtypes are pivotal for cure choice and are correlated to the biological characteristics of BC.
MicroRNA (miRNA), which is a type of untranslated sRNAs, is synthesized from eukaryotic genomes, consisting of a single stranded RNA of about 19-22nt in length (13,14). These short non-coding miRNAs are described as regulators of coding and non-coding (nc) RNAs in eukaryotic cells because they are involved in silencing RNA transcripts and in regulating the stability of their targeted mRNAs (14,15). MiRNAs play also several regulatory roles in several cellular processes; cell development, proliferation, migration, invasion and death (16). Because more than 50% of RNA molecules has been detected to be controlled by miRNA, these mRNAs were damaged because of the effect of aberrant miRNA in malignant cells.
MiRNAs in human malignancies were found to act as oncogenes or ant-oncogenes (tumor suppressors). Oncogenic miRNAs in tumor progress play a negative role in stimulating genes which regulate cell development and apoptosis process. Tumor suppressive miRNAs in human tumor have a key role in silencing genes which modulate cell development and apoptosis (17,18). When normal cells do not undergo normal growth and apoptosis process, they normally cause tumor creation. Numerous recent experiments show that numerous miRNAs are directly implicated in modulating cell growth, proliferation as well as apoptosis (19,20). They play major roles in the pathogenesis of a number of human malignancies; such as breast, colorectal cancer, lung, leukemia liver, and brain (16,18). The miRNA expression level may be either down-or upregulated in these cancers. Several molecular techniques, such as RNA sequencing, miRNA microarray, RT-PCR and northern blot are applied to determine the expression level of them.
Numerous miRNAs have been recognized to be implicated in the pathogenesis of human breast cancer. It was found that the expression patterns of miR-145, -125b, -155, and -21were signi cantly downregulated. In breast malignant cells, these miRNAs were observed to be associated with pathologic properties; cell proliferation, expression of progesterone and estrogen receptors (21). A recent study revealed that tumor suppressive miRNA-204-5p plays a key role in targeting several oncogenic genes which are closely connected to BC pathogenesis (20). Complete information on miR-1275 expression level and its targets in BC have not been available; whereas, the expression pro le of which has be analyzed in some human cancer. A study reported that down-expressed miR-1275 leads to overexpression claudin11 in cancer stem cells or tumor-initiating cells (CSCs/TICs) (22). According to a study carried out on young women with BC, 6 miRNAs; including miR-1275, miR-1228*, miR-139, miR-92b, miR-1207, and miR-3196, were involved in the processes of cell movement, proliferation and invasion (23). It was also found that this miRNA was found to be downregulated in gastric cancer (24). Another study found that miRNA-1275 expression level was signi cantly abnormally deregulated in Ewing's Sarcoma (ES) (25). The signi cance of a large number of miRNAs have been reported to become an appropriate biomarker for human cancer diagnostics. However, the signi cance of miRNA-1275 in BC is not reported. The objective of this study was to determine the expression level of miRNA-1275 as a biomarker for BC diagnostics. Another objective is to identify the potential targeted genes of this miRNA.

Collection of FFPE-blocks of BC samples
Formalin Fixed Para n Embedded (FFPE)-Block of 21 Kurdish cases with BC were collected at clinicopathological laboratories, called as Al Mufti and Luay. For each patient, two para n blocks (one adjacent normal tissue (NAT) and one breast cancer tissue) were collected. The normal tissues were histologically taken nearly 2 cm away from the tumor area. Clinical features of 21 cases were obtained using a questionnaire. The features were displayed in table 1. Permission was accomplished from all cases by signing the con rmed consent. any preoperative chemotherapy or radiotherapy was taken by none of these cases 2.2 Generation of the mRNA and sRNA expression pro les for BC by MACE-sequencing To determine differential expression of protein-coding RNAs (mRNA transcripts) and non-coding RNAs (ncRNAs), analyzing two para n blocks (cancerous and normal) of a BC patient, Massive Analysis of cDNA Ends (MACE)-seq technique was performed (GenXpro GmbH, Frankfurt, Germany). cDNA synthesis, NGS-library and sequencing were subsequently prepared after isolation of sRNAs from mRNA transcripts. The raw sequencing data was bioinformatically analyzed and solved. The raw sequencing data is demultiplexed based on the different barcodes (GenXpro GmbH, Frankfurt, Germany). For removing adapter, the organized reads were cut out for high-quality sequences. Bowtie 2 tool was later used for aligning the sorted reads to the nominated reference sequences and annotating with corresponding properties. The numbers of aligned reads were subsequently normalized to account for distinct sequencing depths. Finally, the normalized and original read numbers were considered during statistical analysis.
2.3 Total RNA puri cation and cDNA synthesis miR-1275 was selected as a candidate from the MACE result and con rmed from 20 cases using Real Time-quantitative polymerase chain reaction (RT-qPCR) technique at Salahuddin University Research Center (SURC). Differential expression of miRNA-1275 was measured in 40 block specimens (20 adjacent normal tissues (NATs) and 20 breast cancer tissues). The total RNA molecules including miRNA were extracted using FFPE RNA/DNA Puri cation Plus kit (Cat. No. 54300, NORGEN BIOTEK CORP, Canada). Complementary DNA (cDNA) was synthesized using miRNA All-In-One cDNA Synthesis Kit (Cat. No. G898, abmgood company, US).
2.4 Total RNA puri cation and cDNA synthesis miR-1275 was selected as a candidate from the MACE result and con rmed from 20 cases using Real Time-quantitative polymerase chain reaction (RT-qPCR) technique at Salahuddin University Research Center (SURC). Differential expression of miRNA-1275 was measured in 40 block specimens (20 adjacent normal tissues (NATs) and 20 breast cancer tissues). The total RNA molecules including miRNA were extracted using FFPE RNA/DNA Puri cation Plus kit (Cat. No. 54300, NORGEN BIOTEK CORP, Canada). Complementary DNA (cDNA) was synthesized using miRNA All-In-One cDNA Synthesis Kit (Cat. No. G898, abmgood company, US).

Most common putative targeted genes regulated by miR-1275
Eleven databases were searched for nding the most common putative targets of miR-1275 (Table 3). Six putative targets (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were determined to possess binding sequence to miR-1275 (Table 4)., Graphpad prsim, version 8.0.1 was used to show the differential expression of these selected target genes were performed by MACE-seq.

Analysis of clinicopathological data associated with BC
Association between miR-1275 and its target genes was computationally analyzed to determine the clinical signi cance using databases of cBioPortal (http://www.cbioportal.org/) and OncoLnc (http://www.oncolnc.org/). Clinical data and expression levels of the miR-1275 and its target genes gained from these sites and then were downloaded on 10 September 2020. SRNA sequencing was carried out to construct the differentia expression of non-coding RNAs in BC compared to NAT. Two small libraries were sequenced for two para n blocks (NAT and BC tissue). Histopathological properties of this specimen were shown in table 1 (Case1). By comparing non-coding RNA expression pro les of BC and NAT, 1400 sRNAs (p<0.05) were ltered out by a SAM software. The raw data were then standardized and log2-transformed to show on a scatter plot (Fig. 1A). Among 1400 sRNAs, 723 non-coding RNAs were downregulated, but 678 sRNAs were upregulated. Each dot on the scatter plot represents the sRNA. Among 1400 sRNAs, 520 microRNAs were differentially expressed. 185 microRNAs were down-expressed, but 335 were overexpressed. The x-axis denotes the data of the NAT and y-axis denotes the data of the BC. Correlation plot was constructed to show expression levels of non-coding RNAs between BC and NAT (Fig. 1B). Blue color denoted the correlation of sRNAs between the BC and NAT. Heat map was designed to show 29 miRNAs which were markedly downregulated in BC compared to NAT (Fig. 1C). Table 2 shows the information on these 29 miRNAs which are notably downregulated. In this study, has-miR-1275, which underlined with red color in heat map, was focused to identify sequence and expression level.

Con rmation of miR-1275 expression level by RT-qPCR
The miR-1275 expression level in RNA-sequencing was observed to be signi cantly downregulated in BC tissue, as compared to NAT. The p.value of this miRNA was 0.614 ( Fig. 2A). Then, this miRNA was selected to con rm. RT-qPCR machine was used to con rm the differential expression of miR-1275 in the laboratory. 40 FFPE blocks for 20 cases was used in this experiment, including 20 BC tissues and 20 normal tissues next to tumoral tissues (2cm away from tumoral tissues). The clinical properties of these cases were brie y explained in table 1. The miR-1275 expression level was detected to be markedly decreased in BC cells compared to adjacent normal cells and the P-value of this was 0.001** (Fig.2B). The mature sequence of which in the BC and NAT was made up of 17 nucleotides and also identical (Fig.2C). Then, Kaplan-Meier overall survival curve was designed to show the effect of miR-1275 expression on the prognosis of cases with BC. Data was used from The Cancer Genome Atlas (TCGA) database and analyzed. Kaplan-Meier overall survival curve displayed that cases were separated into two classes according to its expression. The decreased miR-1275 (P-value=0.0401) was related to overall survival in cases with BC (Fig.2D).
3.3 Differentially expressed genes for adjacent normal and BC tissue by MACE-seq.
Differentially expressed genes in BC and NATs was displayed (Fig. 3A). 26843 differentially expressed genes (P≤0.05) were ltered out by a SAM software. In order to show genes that were more signi cantly different in their expressions, 7041 genes were standardized and log2transformed to show on a scatter plot. 3624 genes were signi cantly overexpressed and 3417 genes were signi cantly down-expressed in tumoral cells compared to adjacent normal cells. The P value for that was ranged from smaller (Blue) to greater (Red). Each point on the scatter plot represents the gene. the x-axis denotes the data of the NAT and y-axis denotes the data of the cancerous tissue. Table 3 showed that eleven computational prediction programs were applied for discovering the strongest candidate genes possessed miR-1275 binding sites in the 3' -UTR. Six predicted genes genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were selected to have binding site to miRNA-1275. The information on these six predicted genes were summarized in table 4. Eleven databases showed that DVL3 and PPP2R2D possess the binding site to miRNA-1275; whereas, THSD4, and CREB1 were con rmed in ten prediction programs to be targeted by miRNA-1275, but SYT7 and PRKACA were con rmed by six tools to be predicted targets. These putative target genes are important for biological analysis of the BC tissues because the over-or down-expression of which can play a damaging role in several cellular processes and contribute to the cancer progression and tumorigenesis.

Determination of expression level of candidate target genes by MACE-seq approach
Among 3624 upregulated genes, the six predicted genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were pointed and named in the BC cells as compared to NAT (Fig.3B). Then the differential expression of which and their binding sites to has-miR-1275 were shown in gure 4. SYT7 gene was more overexpressed in BC, as compared to PRKACA gene. Overexpression level of THSD54 gene was higher than the upregulation of PPP2R2D and DVL3 genes. CREB1 was upregulated but more over-expressed than ST73 gene. Table 5 demonstrates the information on P.value, False Discovering Rate (FDR), and Fold Change (FC) of these six predicted genes possessed miR-1275 binding sites in the 3' -UTR. They were identi ed as potentially modulated by miR-1275 using computational prediction databases and TCGA algorithm.

The role of miR-1275 by targeting selected putative genes in BC
MiRNAs are implicated in silencing mRNA transcripts through matching or mismatching with target mRNAs. As hypothesis of microRNA biogenesis, major strands of miR-1275 come from miRNA duplex are joined into the RISC protein and modulate the mRNA transcripts, but minor strands are broken down and cannot modulate gene expression. miR-1275 can play an essential role in regulating several biological mechanisms; including cell growth, migration, differentiation, proliferation and apoptosis. In this study, the down-expression of which regulate a set of genes and regulators related with tumor development. Six genes (DVL3, PPP2R2D, THSD4, CREB1, SYT7, and PRKACA) were detected to be over-expressed in BC cells. Fig.6 shows the relationship between miR-1275 and these target genes. miR-1275 in breast cancer promotes cancer cell proliferation, cell differentiation, tumor growth, invasion and migration and also inhibits apoptosis through several gene targets. PPP2R2D acts as a tumor suppressor in signaling pathway in BC and is negatively regulated by miR-1275. The overexpression of which decreases AKT and RACK1 abilities. Then these regulators decrease cell survival and migration. DVL3 is implicated in the breast cancer pathways and negatively controlled by miR-1275. The up-regulation of this gene increases the cancer cell proliferation, migration and invasion. The cancer cell proliferation ability is increased when miR-1275 becomes overexpressed. another target gene. CREB1 and PRKACA show also negative correlation with miR-1275 level. Whereas CREB1 was found to reduce apoptosis process and increase cell proliferation in breast cancer, PRKACA plays a key role in tumorigenesis and development of BC. However, the function of THSD4 and SYT7, currently unidenti ed, may boost tumor growth in breast cancer.

Discussion
There is evidence that a single microRNA modulates multiple protein-coding and non-coding genes in different ordinary cells and cancerous cells. In human cancer cells, new RNA groups can be detected using the speci c nature of microRNA from relevant microRNA analysis. Multiple high-throughput approaches, such as DNA microarrays, MACE-sequencing, PCR-based arrays, and RNA-sequencing, are now available and have made microRNA expression pro les of BC, showing the irregular expression of numerous miRNAs (26)(27)(28)(29). One approach to detect the most essential miRNA from numerous miRNAs is to detect differential expression of miRNAs which have been shown in numerous experiments.
Some recent studies revealed that this downregulated miRNA was detected to have an essential effect on cancer cell proliferation, migration, invasion, metastasis, and angiogenesis through targeting multiple oncogenic genes HOXB5, WNT7B and LncRNA HAND2-AS1 (34,36,39). One previous study showed that miR-1275 regulates IGF1, NFIX, Claudin11 in very young women with BC (23). Whereas, down-expression of miR-1275 in all subtypes of para n BC tissues was not fully investigated. In this study, down-expression of miRNA was observed in all subtypes of para n embedded BC tissues of 21 cases with different ages.
After that, the miR-1275-modulated putative targets and their pathways were aimed to explain in the cells of BC. Six genes (DVL3, PPP2R2D, TSHD4, CREB1, SYT7, and PRKACA) were experimentally observed to be overexpressed in the cells of BC. Based on the databases of miRNA target prediction, they were selected and closely correlated with poor prognosis. Among these candidate genes, four genes (PPP2R2D, DVL3, and CREB1) were shown to be strongly targeted by the miR-1275 in the BC cells. Studies showed that these regulators were found to reduce cell survival and migration in cancer cells (40,41). DVL3 is observed to be implicated in the breast cancer pathways (34) and negatively regulated by miR-1275. The up-regulation of this gene can increase the cancer cell proliferation, migration and invasion in BC cells. The overexpression of which increase cancer cell proliferation ability (42). Another target gene, CREB1 and PRKACA show also negative correlation with miR-1275 level. Whereas CREB1 was found to reduce apoptosis process and increase cell proliferation in breast cancer (43), PRKACA plays a key role in tumorigenesis and development of BC (44). However, the role of THSD4 and SYT7, currently unidenti ed, may enhance tumor growth in a variety of cancers, especially breast cancer (45,46).

Conclusion
In the present study, differential expression pro les of total mRNA transcripts and sRNAs were identi ed in BC para n tissue (NAT and tumoral tissue) by MACE-sequencing. Decreased miRNA-1275 expression develops breast cancer by increasing the activity of biological processes; such as growth, migration, invasion and metastasis. Upregulated miRNA-1275 prevented BC development by modulating direct expression of DVL3, PPP2R2D, TSHD4, CREB1, SYT7, and PRKACA. This is the rst study revealing that miR-1275 function as a tumor-suppressive miRNA in BC cells, regulating numerous targets which were closely related with BC pathogenesis and oncogenesis. Sevan also discussed and interpreted the data. He also did the manuscript mapping and submission, but Suhad supervised the project.

Funding
This study was supported by Sevan Majed who is a Ph.D. student.

Availability of data and materials
Although row data of MACE-sequencing and sRNA may be available in the database of GenXpro, at https://genxpro.net/, These data will be further studied for another research in the future. The ndings described in this manuscript were provided by the Co-author.
Ethics approval and consent to participate This study was followed and approved by Human Research Ethics Committee at Science College in Salahuddin University-Erbil (Approval no.4c/132). Informed consent was received from all patients in accordance with the requirements of the Human Research Ethics Committee.

Consent for publication
Not applicable.

Competing interest
The author announces no con ict of interest. Mr. Sevan is a teacher of Salahaddin University-Erbil, a subsidiary of ministry of higher education in Kurdistan region government (KRG).

Figure 1
Differential expression analysis of non-coding RNAs by MACE-sequencing. A The expression pro le of 1400 sRNAs in BC compared to NAT is plotted. Red dots represent the sample sRNAs. A light green dot represents miRNA-1275 expression level in BC tissue compared to NAT. B Heatmap based clustering of several downregulated miRNAs in BC compared to NAT. Has-miRNA-1275 is underlined. C correlation between BC and NAT in differential expression of sRNAs.

Figure 3
A Scatter plot analysis of gene expression pro le displays up-or down-regulation of genes in BC tissue, compared to NAT. Each point denotes the average value of one transcript in the experiment. The expression difference is taken account of signi cance for a Pvalue (0.05). B Outlined points and names denote the selected target genes for has-miR-1275.

Figure 3
A Scatter plot analysis of gene expression pro le displays up-or down-regulation of genes in BC tissue, compared to NAT. Each point denotes the average value of one transcript in the experiment. The expression difference is taken account of signi cance for a Pvalue (0.05). B Outlined points and names denote the selected target genes for has-miR-1275.

Figure 4
Target genes and has-miR-1275 are combined in the seed region, including 6 to 8 nucleotides in the 5′ end, showing in red color. Differential expression level of target genes in BC compared to NAT.

Figure 4
Target genes and has-miR-1275 are combined in the seed region, including 6 to 8 nucleotides in the 5′ end, showing in red color. Differential expression level of target genes in BC compared to NAT. An association between the expression levels of six genes (DVL3, PPP2R2D, TSHD4, CREB1, SYT7, and PRKACA) and histopathological signi cance was shown using data from TCGA database. The Kaplan-Meier overall survival curves show that patients with BC were separated into 2 classes according to their expression levels.

Figure 5
An association between the expression levels of six genes (DVL3, PPP2R2D, TSHD4, CREB1, SYT7, and PRKACA) and histopathological signi cance was shown using data from TCGA database. The Kaplan-Meier overall survival curves show that patients with BC were separated into 2 classes according to their expression levels.