Potential diagnostic value of novel circular RNA hsa_circ_0060927 in human colorectal cancer REVIEW

Background CYP24A1 plays a role in strictly regulated vitamin D metabolism pathway and has been nominated as a prognostic biomarker for colorectal cancer (CRC). Increasing evidence suggests that circular RNAs (circRNAs) are involved in cancer initiation and progression; however, their diagnostic values in cancers as the new potential biomarkers are still not fully understood. Methods and Results In the present study, by using quantitative real-time polymerase chain reaction (qRT-PCR), we demonstrated that CYP24A1 and hsa_circ_0060927 were only transcribed in 33 and 25 out of 83 CRC tissues, respectively. In these samples, we demonstrated that CYP24A1 was up-regulated in both linear and circular forms ( P < 0.05). Furthermore, we showed that CYP24A1 and hsa_circ_0060927 transcription were induced after vitamin D treatment in HCT-116 and HT-29 colon cancer cell lines. Importantly, analysis of the ROC curve in expressed samples indicated that CYP24A1 and hsa_circ_0060927 could serve as the potential diagnostic markers of CRC, but due to the fact that these genes are not expressed in many samples, the diagnostic role of these genes could be rejected. the however, further studies are required to validate the present results.

Background CYP24A1 plays a role in strictly regulated vitamin D metabolism pathway and has been nominated as a prognostic biomarker for colorectal cancer (CRC). Increasing evidence suggests that circular RNAs (circRNAs) are involved in cancer initiation and progression; however, their diagnostic values in cancers as the new potential biomarkers are still not fully understood.

Methods and Results
In the present study, by using quantitative real-time polymerase chain reaction (qRT-PCR), we demonstrated that CYP24A1 and hsa_circ_0060927 were only transcribed in 33 and 25 out of 83 CRC tissues, respectively. In these samples, we demonstrated that CYP24A1 was up-regulated in both linear and circular forms ( P < 0.05).
Furthermore, we showed that CYP24A1 and hsa_circ_0060927 transcription were induced after vitamin D treatment in HCT-116 and HT-29 colon cancer cell lines. Importantly, analysis of the ROC curve in expressed samples indicated that CYP24A1 and hsa_circ_0060927 could serve as the potential diagnostic markers of CRC, but due to the fact that these genes are not expressed in many samples, the diagnostic role of these genes could be rejected.
Conclusion Taken together, for the first time, the results of our study illustrated the expression levels hsa_circ_0060927 in CRC; however, further studies are required to validate the present results.

Background
According to the global cancer project (GLOBOCAN 2018) estimation, colorectal cancer (CRC) is one of the most common cancers representing third in terms of incidence and second in terms of mortality worldwide. In 2018, GLOBOCAN estimated that over 1.8 million new CRC cases and 881,000 deaths in the world [1]. Although many different altered molecular targets have been reported in CRC, only a few of them are clinically functional, and the exact mechanism underlying the development and progression of CRC remained generally unknown [2][3][4]. One of the most important altered pathways in CRC is the vitamin D metabolism pathway. Vitamin D decreases the epithelial cell proliferation and promotes differentiation in human colon cells [5]. Cytochrome P450 family 24 subfamily A member 1 (CYP24A1) gene encodes a mitochondrial enzyme that plays a role in vitamin D inactivation and is over-expressed in many cancers, including CRC [6]. A recent study emphasized that CYP24A1 serves as a potential biomarker for the progression and prognosis of CRC [7]; however, there is no evidence concerning the role of CYP24A1 related circular RNAs (circRNAs) in the CRC. circRNAs are an abundant newly accepted class of widely expressed non-coding RNAs in the eukaryotic transcriptome. These molecules are highly conserved and feature tissue and cell-type specificity and relative stability [8,9]. Therefore, it is valuable to find stable and ideal circRNAs as biomarkers in colorectal cancer. Recent studies have shown that circRNAs are closely associated with several diseases, including CRC and participate in biological processes such as cell proliferation, differentiation, and apoptosis [10,11]. For instance, Wang J et al. showed that hsa_circ_0000567 expression is down-regulated in CRC tissues and cell lines, therefore, could be served as a diagnostic biomarker. Furthermore, they showed that knockdown of hsa_circ_0000567 promoted CRC cells proliferation and migration in vitro [12]. In the present study, after assessment of CYP24A1 gene expression, we, for the first time, estimated the expression level of hsa_circ_0060927 in CRC tissues. To determine the expression of CYP24A1 and hsa_circ_0060927 is influenced by vitamin D, we assessed the expression level of these genes in HCT-116 and HT-29 colon cancer cell lines after vitamin D treatment. Consequently, the aims of the present study were the assessment the expression levels of CYP24A1 and hsa_circ_0060927 in colorectal cancer tissues compared to control noncancerous tissues in order to determine that these genes are good diagnostic biomarkers or not. Furthermore, we investigate the correlation between expression levels of CYP24A1 and hsa_circ_0060927 and clinicopathological data of CRC

RNA extraction and reverse transcription
Total RNA was directly extracted from the colorectal tissues, normal mucosa, and cell lines using a GeneAll Hybrid-R™ RNA purification kit (Geneall Biotechnology Co. Ltd, Seoul, Korea) as described by the manufacturer's protocol. RNA was visualized on 1% agarose gel to evaluate the RNA integrity. Additionally, the quantity and the quality of the total extracted RNA was estimated using NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, USA). The RNA purity was evaluated according to the A260/A280 ratio.
First-strand cDNA was synthesized from 1 µg of extracted total RNA using a Revert Aid  Table 1). Each reaction consisted of 10 µl 2X RealQ-PCR Master Mix®, 1 µl cDNA (10 ng), 1 µl of each primer (10 pmol) and 7 µl of nuclease-free water to conduct PCR in a 20 µl of reaction mixture. We carried out the reactions in duplicate and β2M gene was used as normalization control. This gene was selected according to previous research for identification of housekeeping control genes in colorectal cancer [16]. Two-step thermal cycling and real-time data acquisition were performed using the following conditions: 95 °C for 15 min × 1 cycle, and 95 °C for 15 s, followed by 60 °C for 1 min × 40 cycles followed by melt curve stage assessment. The melting curve profile and agarose gel electrophoresis were performed to verify the specificity of primers and the authenticity of the PCR products.

Hsa_circ_0060927 existence in colorectal cancer cells and tissues
We analyzed the hsa_circ_0060927 existence in CRC tissues. In addition, the existence of We did not find any CYP24A1 and hsa_circ_0060927 expression in 60% and 70% of the tissue samples, respectively, but in the rest, CYP24A1 and hsa_circ_0060927 expression analysis results revealed up-regulation of both transcripts in CRC tissues compared with adjacent tissue (Fig. 2). Similar to previous researches, the CYP24A1 gene expression is generally over-expressed 6 , but, in our study, it was not expressed in all colon tissues.
Surprisingly, in HT-29 and HCT-116 cell lines, CYP24A1 and hsa_circ_0060927 were not expressed before vitamin D treatment; however, both were transcribed after treatment.
Potential diagnostic values of C Y P 2 4 A 1 a n d h s a _ c i r c _ 0 0 6 0 9 2 7 i n c o l o r e c t a l c a n c e r Since we observed CYP24A1 and hsa_circ_0060927 over-expression in 40% and 30% of tissue samples, respectively, we further analyzed the potential diagnostic value of these molecules in CRC. The potential diagnostic value was evaluated by ROC curve. The larger the area under the ROC curve (AUC), the higher diagnostic value. The area under the ROC curve for CYP24A1 and hsa_circ_0060927 have shown in Fig. 3A and 3B, respectively.
Besides, we analyzed the correlation between clinicopathological date and the expression levels of CYP24A1 and hsa_circ_0060927. Our results showed that the hsa_circ_0060927 expression level was correlated with and gender (P = 0.04). However, no association was found between CYP24A1 and hsa_circ_0060927 levels and other clinicopathological features such as clinical age and clinical stages (Tables 2 and 3). Table 2 The correlation of CYP24A1 expression levels (ΔCt) and clinicopathological features of the CRC patients.  Table 3 The correlation of hsa_circ_0060927 expression levels (ΔCt) and clinicopathological features of the CRC patients.

Factors
No Circular RNAs are formed by non-linear reverse splicing, which had been considered as the result of splicing errors [17]. By the development of bioinformatics and statistical methods, lots of circular RNAs were found [18]. Although the mechanisms underlying the function of circRNAs are not fully understood, it has been suggested that circRNAs could act as microRNA sponges, RNA-binding proteins, protein and/or RNA transport in order to regulate transcription [17,19]. In this study, we investigated the expression level of CYP24A1 and its related cirRNA, hsa_circ_0060927, using 83 fresh CRC samples and adjacent normal tissues by qRT-PCR. The results showed that CYP24A1 and hsa_circ_0060927 expressed in 40% and 30% of the tissue samples, respectively. In these samples, the expression level of CYP24A1 and hsa_circ_0060927 was obviously upregulated. Because these genes were not expressed in many specimens, the role of these genes as biomarkers can not be accepted. Contrary to our results, some studies introduce the CYP24A1 as the biomarker [7,30]. The co-expression of CYP24A1 and hsa_circ_0060927 has given us the idea that these genes are under common regulation.
So, we investigated the existence of hsa_circ_0060927 in human HT-29 and HCT-116 colon cancer cell lines before and after treatment with the vitamin D. The HCT-116 and HT-29 colon cancer cell lines did not show expression of CYP24A1 and hsa_circ_0060927, but after vitamin D treatment the CYP24A1 and hsa_circ_0060927 were transcribed. According to Lemay J et al. the CYP24A1 gene is the most responsive primary vitamin D target gene [28]. Our results also confirmed the role of vitamin D as a key contributor of the transcription for both hsa_circ_0060927 and CYP24A1. The hsa_circ_0060927 mechanism of function has not been studied, but as mentioned, circRNAs act as microRNA (miRNA) sponges, thus could be part of the competing endogenous RNA network. For instance, circ_001569 acts as a miRNA sponge to bind and inhibit miR-145 activity in CRC cells and circHIPK3 sponges the miR-7 and promotes colorectal cancer growth and metastasis [10,20]. A recent study predicted that hsa_circ_0060927 targets the hsa-miR-224-3p, hsa-miR-29b-1-5p, hsa-miR-522-3p, hsa-miR-661 and hsa-miR-1264 [14]. Considering this study, we predicted the miRNAs that target both hsa_circ_0060927 and CYP24A1 by TargetScan to specify the common miRNAs. The results showed mechanically, circ_0060927 acts as a miRNA sponge to directly inhibit hsa-miR-224-3p, hsa-miR-522-3p and hsa-miR-1264 (Fig. 4). These miRNAs have various functions and are dysregulated in some cancers.
Previous reports displayed miR-224-3p up-regulation in colon cancer, cervical cancer, and hepatocellular carcinoma [21,22]. Moreover, it was suggested that this miRNA induces cell proliferation and metastasis in lung cancer cells by inhibition of TNF-α-induced apoptosis [23]. Hsa-miR-1264 targets DNA methyltransferase-1 (DNMT1) and is overexpressed in ovarian cancer cells [24,25]. miR-522 promotes cell proliferation in CRC and is over-expressed in colon cancer, hepatocellular carcinoma, non-small cell lung cancer, and glioblastoma [26,27]. These data provide important clues that up-regulation of hsa_circ_0060927 may sponge and suppress the inhibitory role of these miRNAs and hence result in the up-regulation of the miRNA target genes such as CYP24A1 (Fig. 4). We predicted the hsa-miR-224-3p, hsa-miR-522-3p and hsa-miR-1264 targeted genes according to DIANA-miRPath online tool and then the DAVID functional annotation was performed for target genes analysis. The analysis showed that the nominated genes were significantly correlated with transcription regulation. To our best knowledge, this is the first report of the expression of hsa_circ_0060927 in CRC tissues and HCT-116 and HT-29 colon cancer cell lines. Due to some specific features of circRNAs such as highly conserved sequences and a high degree of stability compared to other non-coding RNAs, such as miRNAs and long non-coding RNAs (lncRNAs), these molecules may serve as the diagnostic markers in some diseases such as CRC [20]. Further studies concerning the relationship between circular RNAs and CRC have suggested hsa_circ_0000567 and hsa_circ_0001649 as promising diagnostic biomarkers in human CRC [12,29]. We didn't find significant correlation between expression of CYP24A1 in colorectal cancer tissue and sex, age, and clinical stages; however, hsa_circ_0060927 up-regulation was correlated with gender (P < 0.05) (Tables 2 and 3).

Conclusion
Taken together, the findings of the present study indicated that hsa_circ_0060927 the same as CYP24A1 common transcript had different expression levels between CRC tissue and adjacent normal tissue but could not be served as diagnostic biomarkers for CRC, but future studies with larger sample size are needed to prove our results as a fact.
Additionally, functional analysis in colorectal cancer cells and tissues are needed to uncover the exact underlying mechanisms of hsa_circ_0060927.

Ethics approval and consent to participate
This study was approved by the Ethics Committee of Baqiyatallah University of Medical Sciences (Code: IR.BMSU.REC.1398.217).

Consent for publication
Written consent was obtained from all patients who were informed that the data would be used for research.

Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare no conflict of interest.

Figure 2
The relative expression levels (-ΔCt) of CYP24A1 and hsa_circ_0060927 in colorectal cancer tissues and control noncancerous tissues. The figures suggest that CYP24A1 and hsa_circ_0060927 were over-expressed in CRC tissues compared with adjacent normal tissues. **P < 0.01, ****P < 0.0001.  Hsa_circ_0060927 is frequently up-regulated in CRC and acts as miRNA sponges to directly bind and inhibit hsa-miR-224-3p, hsa-miR-522-3p and hsa-miR-1264, consequently, this mechanism increases the CYP24A1 gene expression, which is targeted by the same miRNAs.