Circulating RP11-445H22.4 and TINCR are Potential Biomarkers for Invasive Ductal Carcinoma of the Breast

Background: Recently introduced molecules namely long non-coding RNAs (lncRNAs) are associated with various diseases, including breast cancer, and they may have the potential to act as biomarkers. Invasive ductal carcinoma (IDC) is a frequently observed type of breast cancer that diagnosis through immunohistochemical methods. However, no reliable non-invasive markers such as circulating markers have been introduced for it, so far. Methods: The relative quantitation (RQ) of two lncRNAs, RP11-445H22.4 and TINCR, was measured in tumoral tissues and paired adjacent non-tumoral tissues (PANTs) as well as in plasma samples using real-time polymerase chain reaction (RT-PCR). Furthermore, promoter methylation of the two lncRNAs genes was assessed using methylation-specic PCR (MSP). In order to compare the diagnostic values, plasma levels of CA 15-3 were measured as a conventional circulating biomarker using ELISA. The receiver operating characteristic (ROC) curve was used to illustrate the sensitivity and speci�city of the potential biomarkers. Results: The RP11-445H22.4 and TINCR genes were upregulated in tumor tissues compared to PANTs (RQRP11-445H22.4 = 2.7, RQTINCR = 4.4). Furthermore, the presence of RP11-445H22.4 and TINCR in the plasma of patients was signi�cantly more than healthy controls (RQc.RP11-445H22.4 = 4.12, RQc.TINCR = 4.16, P<0.001). The promoters of RP11-445H22.4 and TINCR genes were hypomethylated in the tumoral tissues with a negative correlation with their gene expression. Conclusion: Based on the �ndings, it can be speculated that the expression levels of these two lncRNAs, as well as their promoter methylation, can be considered as a therapeutic target and potential biomarker for breast carcinoma.


Introduction
Breast cancer is one of the primary health concerns worldwide [1].According to the report published by the Non-Communicable Diseases Research Center (NCDRC) of Iran, breast cancer is the most prevalent type of cancer among women in Kermanshah in western Iran (33.02 new cases per 100000 women in 2016) [2].Nowadays, it has been well demonstrated that the earlier diagnosis of breast cancer is associated with the better the survival rate will be.Conventional serum biomarkers for the detection of breast cancer are not highly convincing for early detection.In recent years, new types of potential biomarkers have been introduced for the prognosis, diagnosis, and treatment of cancer, involving microRNAs and long noncoding RNAs (lncRNAs) [3,4].LncRNAs are classi ed as transcripts with more than 200 nucleotides, with important functions in biological processes.[5].Abnormal promoter methylation takes part in tumorigenesis, including in human breast carcinoma [6].Therefore, lncRNAassociated methylation received increased attention, for example, the investigation of the role of lncRNA MEG3 in breast cancer [7,8].Hence, the identi cation of new genes and mechanisms for their regulation and functions may result in therapeutic approaches or novel biomarkers for breast carcinoma.
KCNK15 and WISP2 antisense RNA 1 (KCNK15-AS1) a.k.a.RP11-445H22.4, is a newly identi ed antisense lncRNA with a length of 862 nucleotides, which has been found in the reverse strand of human chromosome 20q13.12[9].Tissue differentiation-inducing long non-coding RNA (TINCR) is a lncRNA which is necessary for regular epidermal differentiation [10].The TINCR gene is located on human chromosome 19 and produces a 3.7-kb RNA transcript [11].
The main objective of the current study was to assess tissue and plasma expression of the RP11-445H22.4and TINCR in a sample of female Kurdish patients with invasive ductal carcinoma (IDC) of the breast.Moreover, to our knowledge, this is the rst study to evaluate the promoter methylation patterns of these two lncRNAs.

Participants and sample collection
Patients were recruited at the time of the diagnosis at the Bistoon Hospital, between 2016 and 2017.The ethical committee of Kermanshah University of Medical Sciences approved this study, and informed consent was signed by all subjects.Only new con rmed patients with invasive ductal carcinoma of breast were entered in the study without previously diagnosed any malignancy and without manifestation of acute diseases or injury at the time of sampling.The status of hormone receptors and other clinicopathological data were extracted from the patient's medical history.The samples consisted of 20 paired frozen tumoral tissues and adjacent non-tumoral tissues (PANTs) from patients with IDC of the breast.Moreover, 40 patients who were sampled only for taken peripheral blood at the time of diagnosis were studied.In addition, the blood sample was taken from 40 healthy volunteers.The results of histopathological examination con rmed by an expert pathologist in all patient samples to be invasive ductal carcinoma and paired adjacent margin tissues were con rmed as free of cancer cells.

Stock of plasma sample
The whole peripheral blood samples were collected in 5 ml EDTA containing tubes.To separate cellular fraction from plasma, samples centrifuged with 3500 g for 20 min around one hour after blood collection.The collected plasma was stored at − 80°C.

Quantitative real-time PCR (qRT-PCR)
In order to extract RNA, fresh frozen tissues were powdered in liquid nitrogen, afterward, total RNA was isolated by Qiazol reagent (QIAGEN, US) according to the manufacturer's procedure.In addition, total RNA was isolated from 200 microliters of obtained plasma by the miRNeasy Plasma/ Serum extraction Kit (QIAGEN, US) and was eluted into 14µl of elution buffer.The concentration and purity of the extracted RNA were assessed by the Nanodrop spectrophotometer (Thermo Scienti c, Waltham, MA).Complementary DNA (cDNA) was synthesized by applying a QuantiTect Reverse Transcription Kit (QIAGEN, US) from the isolated total RNA.The qRT-PCR assay was performed by speci c primers for RP11-445H22.4,TINCR, and U6 snRNA, as an internal control, using by the Rotor-gene (Corbett Research, Germany) with SYBR Premix Ex Taq II (TaKaRa, China).The PCR reaction conditions were an initial denaturation at 95°C for 30sec, followed by 45 PCR cycles at 95°C for 10sec and 60°C for 60sec.We used the 2 −△△Ct method to determine the relative quantitation (RQ) in the cancerous samples relative to the control samples.

Methylation Speci c PCR (MSP)
DNA was extracted from frozen tissue using previously described modi ed salting-out DNA extraction method [12].In order to Bisul te treatment of extracted DNA was performed as follow, 30 µL of melted 2% low melt agarose was added to about 200 ng DNA and was incubated at 65°C for a few minutes.Then for denaturation of DNA strands, it was incubated with NaOH at 80°C for 15 minutes and resolidify the agarose bead by chilling on ice.Treatment was done by adding 10 mM hydroquinone and 40.5% Sodium bisul te at 50°C for about 4 hours.Finally, in order to stop bisul te reaction and desulfonation, the beads were washed with TE buffer, 0.2 M NaOH, 1M hydrochloric acid, and pure water, respectively.The washed beads were diluted in 30 ul deionized water, heated at 80°C and aliquoted for PCR reaction.An MSP method was used in the determination of the methylation status of the two lncRNAs gene promoters.For this reason, the promoters of these genes and CpG islands were has predicted by the UCSC Genome Browser (https://genome.ucsc.edu/) on Human (GRCH38/hg38) assembly and con rmed by the Genomatix software (https://www.genomatix.de/).The MSP primers for RP11-445H22.4and TINCR genes were designed using the MethPrimer (https://www.urogene.org/methprimer/).The MSP-PCR was performed using two pairs of speci c primers for each gene (Table 1); one pair speci c for methylated and the other for the unmethylated template.
Table 1 The sequence of primers and PCR product size for qRT-PCR and MSP.

Stability analysis of c.RP11-445H22.4 and c.TINCR in plasma
In order to the assessment of c.RP11-445H22.4 and c.TINCR stability in plasma, three plasma samples were examined; each sample divided into four equal parts and incubated at room temperature for 0-, 6-, 12-, and 24-hours.

Measurement of CA 15 − 3 in plasma
The CA15-3 test was done in accordance with the instructions of the manufacturer.The plasma level of CA15-3 was measured in 40 patients with IDC and 40 healthy controls using the ELISA kit (Monobind, USA).The company recommended the threshold value equal to 35 UI/mL.The samples were considered to be positive when its level exceeded a threshold value; if not, they were considered to be negative.

Statistical Analysis
Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were applied to calculate the feasibility of circulating lncRNAs application as a diagnostic tool.Comparisons between two and three groups were analyzed using Student's t-test and One-Way ANOVA, respectively.A paired ttest was used for comparison of lncRNAs expression between tumor tissue and paired adjacent nontumor tissues.Statistical analyses were done by SPSS (v.16.0) software.A P-value of less than 0.05 was considered signi cant.

Results
All participants were women with Kurdish ethnic background from Kermanshah Province of Iran.The mean age of patients was about 49 years (range of 30-68), while the healthy controls had a similar age distribution with a mean of 48 years (range of 26-64, p = 0.14).A number of important clinicopathological characteristics, as well as the results of the immunohistochemical (IHC) analyses of the patients, are presented in Table 2.   Pearson correlation: r = -0.84,95% CI (-0/91 to -0/72), P < 0/0001 The expression of the RP11-445H22.4

and TINCR tumoral tissues
Using the relative expression of the two lncRNAs were measured in 20 pairs of breast tumor tissues and PANTs samples.The expression levels of RP11-445H22.4 and TINCR were signi cantly higher in tumor tissues compared to PANT tissues (RQ RP11−445H22.4= 2.7, RQ TINCR = 4.4, Fig. 1A).Given that the lncRNAs in circulation were most probably originated from the tumor [13], we only evaluated the presence of RP11-445H22.4 and TINCR in the plasma since they had been overexpressed in tumor tissues.

Signi cant elevation of plasma levels of c.RP11-445H22.4 and c.TINCR in patients
The plasma levels of c.RP11-445H22.4 and c.TINCR were signi cantly higher in IDC patients compared to healthy donors (the mean RQ c.RP11−445H22.4= 4.12 and RQ c.TINCR = 4.16).In fact, the level of both circulating lncRNAs in IDC of breast increased more than four-fold in breast cancer patients (P < 0.0001, Fig. 1b).The analysis showed no signi cant correlation between RP11-445H22.4 and TINCR expression and the status of Hormone-Responsiveness Markers (i.e., ER, PR, or HER2) or tumor stage.

Stability analysis of and c.TINCR in plasma
In order to assess the stability of c.RP11-445H22.4 and c.TINCR in plasma, three plasma samples were examined; each sample was divided into four equal parts and incubated at room temperature for 0, 6, 12, and 24 hours, respectively.Since no considerable variation was observed in plasma levels of c.RP11-445H22.4 and c.TINCR (P < 0.001, Fig. 4), the results indicated that they were stable in plasma.

Discussion
According to the de nition provided by World Health Organization (WHO), "a biomarker is any substance, structure, or process that can be measured in the body or its products and can in uence or predict the incidence of outcome or disease".A cancer biomarker evaluates the risk of cancer development, progression, and recurrence [14,15].CA15-3, CEA, CA125, and CA19-9 are conventional blood-based biomarkers for cancer [16].Since these factors are not speci c and have low sensitivity, attempts to discover new biomarkers seem necessary.
It has been demonstrated that long non-coding RNAs (lncRNAs) are dysregulated in tumors, and they can show a high degree of tissue and disease-speci city [17].Since lncRNAs have been detected within cells and body uids of patients, such as serum, plasma, and urine, it has been speculated that they could be used as potential diagnostic and prognostic biomarkers for different types of cancer [17].A known example of lncRNAs, with approval from the US Food and Drug Administration (FDA), is Prostate Cancer Antigen 3 (PCA3, a prostate-speci c lncRNA) [18,19].Nowadays, the urinary PCA3 is used broadly, and it is even preferable to digital rectal examination and the serum prostate-speci c antigen (PSA) test [17].
In this study, the increased expression of RP11-445H22.4 was detected in breast tumor tissues, as well as in the plasma of patients.Comparing the sensitivity of circulating RP11-445H22.4with CA15-3 in the plasma of IDC patients revealed the potential diagnostic value of this antisense lncRNA.A review of the literature shows that there are a limited number of studies regarding RP11-445H22.4.Two separate studies on the Chinese population reported high expression of circulating RP11-445H22.4 in breast cancer patients [9,20].However, two other studies reported the downregulation of RP11-445H22.4 in the tissue of pancreatic and gastric cancer patients [21,22].
Deacetylation of histones and methylation of DNA are two epigenetic mechanisms that regulate gene expression [22].One of the most important outcomes of hypomethylation is the overexpression of protooncogenes, which ultimately promotes cancer [23].Most recently, it was reported that the promoter of RP11-445H22.4 is methylated in gastric tumors, especially in stages III and IV [22].However, no study has been reported on promoter methylation of this gene in breast tumors.For the rst time, we reported a lowfrequency rate of methylation in the promoter of the RP11-445H22.4gene in breast cancer tissues.
TINCR is a lncRNA with an oncogenic role in breast carcinoma [10,24].It has been uncovered that TINCR is regulated by SP1, miR-137/miR-133a, and CREB-binding protein (CBP)-mediated H3K27 acetylation [10,25,26].Acetylation of H3K27 by CBP in the promoter region of TINCR is involved in the upregulation of this gene [26].SP1, which is a transcription factor with the binding site in the promoters of multiple genes, can induce the upregulation of TINCR [10], while miR-137/miR-133a directly targets TINCR and suppresses it [25].In addition, it has been shown that the upregulation of TINCR is mainly caused by gene ampli cation in the genome [24].A number of studies have reported high expression of TINCR in breast carcinoma, especially the HER2-positive subtype, while has been demonstrated that it is related to relapse-free survival (RFS), size of the tumor, and poor tumor prognosis in breast cancer [10,24,26,27].Moreover, TINCR negatively regulates apoptosis and triggers cell cycle progression [24].2018) suggested that the TINCR-miR7-KLF4 axis played a role in breast carcinoma.Indeed, TINCR, is a sponge for miR-7 released KLF4, which is a transcription factor with various physiological and pathophysiological functions [10].
Here, we detected the overexpression and hypomethylation of the TINCR gene in breast tumor tissues.Furthermore, we reported circulating level of TINCR was signi cantly higher in the plasma of patients, compared to healthy controls.The comparison of the c.TINCR sensitivity with CA15-3 revealed the potential diagnostic value of this lncRNA in plasma of patients with invasive ductal carcinoma of the breast.For the rst time, we reported a low-frequency rate of methylation in the promoter of the TINCR gene.

Conclusions
In addition to the overexpression of RP11-445H22.4 and TINCR genes, this is the rst report to investigate the methylation of these two lncRNAs in breast tumors.Overall, it can be concluded that the expression levels and promoter methylation of RP11-445H22.4 and TINCR can be considered as potential biomarkers and therapeutic targets for breast carcinoma.Undeniably, more investigations on a larger population are required to validate the results of this pilot study.The stability assessment of c.RP11-445H22.4 and c.TINCR of three samples right after sampling (0h), and also after 6-, 12-, and 24-hour incubation of plasma samples at room temperature.

Table 3
Correlation between promoter methylation and gene expression in the three lncRNAs.