Evaluation of plasma circ_0006282 as a novel diagnostic biomarker in colorectal cancer

Abstract Background Nowadays, non‐invasive and rapid detection of cancers through molecular biomarkers has received much attention. Therefore, this study investigated the non‐invasive and rapid diagnosis of colorectal cancer through one of the newest biomarkers (circular RNA). Methods For this purpose, we collected tumoral, adjacent normal tissue, and plasma samples from 100 colorectal cancer (CRC) patients, 25 postoperative CRC patients, 28 colitis patients, and 108 healthy donors. First Illumina high‐throughput (Hi Seq 2000) sequencing was performed to identify known and novel differentially expressed circRNAs in the cancerous and adjacent normal tissues (n = 3). We used quantitative real‐time fluorescent polymerase chain reaction (qRT‐PCR) to detect the expression level of hsa_circ_0006282 among the different samples. Moreover, inter‐ and intra‐assays were performed to evaluate the potential of hsa_circ_0006282 as being a biomarker. The receiver operating characteristic curve (ROC) was drawn to appraise its diagnostic efficacy, and the sensitivity of this circ RNA was evaluated. Results Based on RNA‐sequencing results circ_0006282, cirs7, circ‐0001313, circ_0055625, circ_000984, circ_0055625, circ_0001178, circ_0071589, circ‐001569 were upregulated, and circ‐ITGA7, circ‐CDYL, circITCH, circ_0026344, circ_0000038, circ_0002220, circ_0067480, circIGHV3‐20‐1, circ_104916, circ_0009361 were downregulated circRNA. The hsa_circ_0006282 was the highest upregulated differentially expressed circRNA. Expression evaluation of this circRNA on different samples showed upregulation in CRC tissues (p < 0.0001) and plasma samples of CRC patients in comparison to healthy controls (p < 0.0001), while the area under the curve (AUC) was 0.831 (95% CI: 0.779–0.883). Expression of hsa_circ_0006282 in CRC patients decreased to normal after surgery (p < 0.0001). Our results showed high specificity and sensitivity of CRC detection when hsa_circ_0006282, carcinoembryonic antigen (CEA), and carbohydrate antigen199 (CA199) are combined. Conclusion Plasma hsa_circ_0006282 can be used as a novel diagnostic and dynamic monitoring biomarker in CRC patients.


| INTRODUC TI ON
Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is defined as any cancer that affects the colon and rectum. Treatments used for colorectal cancer may include a combination of surgery, radiation therapy, chemotherapy, and targeted therapy. The use of molecular biomarkers in diagnosis of cancers, including colorectal cancer, has great importance. 1,2 Nowadays, liquid biopsy is used as a non-invasive diagnostic method in hospital settings. 3 Currently, carcinoembryonic antigen (CEA) and carbohydrate antigen199 (CA199) are the most used antigens in the diagnosis of gastrointestinal tumors, 4 but their sensitivity and specificity are low. 5 Circular RNAs (circRNAs) are a novel class of endogenous noncoding RNAs characterized by their stable closed-loop structure.
They are considered potential disease biomarkers due to their tissue-and development stage-specific expression pattern. Two methods have been proposed to produce these RNAs: (A) back-splicing and (B) covalent binding. 6,7 Recently, many studies have been conducted on the function and regulatory mechanisms of circRNAs. In general, the critical role of these RNAs is the gene expression regulation of micro RNAs (miRNAs). Since circRNAs participate in different cell signaling pathways, they could be used as a diagnostic and prognostic aid in various cancers, including human CRC. [6][7][8] One of the unique features of these RNAs is their covalently closed cyclic structure which makes them resistant to digestion by exonucleases.
For this reason, their expression in cells is stable, and their halflife is high, particularly in cell-free samples (such as blood, urine, and saliva). 9 Human hsa_circ_0006282 is one of the newly introduced circRNAs with 198bp nt in spliced sequence length. Its gene is located at chr8:74868145-74872053, and its gene symbol is TCEB1. 10 Our study aimed to evaluate the diagnostic efficacy and sensitivity of tissue and plasma hsa_circ_0006282 for early and rapid diagnosis of CRC. After taking the tissue samples, they were stored at −80°C immediately to prevent RNA degradation. The plasma samples included 100 CRC patients, 25 postoperative CRC patients, 28 cases of colitis patients, and 108 cases of age-matched healthy controls. In our research, all CRC patients were diagnosed as primary by clinicians and did not receive chemotherapy or radiotherapy before the surgery.

| Collection of plasma and tissue samples
Patients' documents have been carefully reviewed, and their pathological information was also collected. and supplemented with 10% fetal bovine serum. Cells were cultured under standard conditions and incubated at 37°C and 5% CO 2 .

| Circular-RNA sequencing and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
For total RNA extraction, 700 mg of the frozen tissue, 1 × 10 6 of the cultured cells, and 350 µl of plasma were prepared, then 1 ml TRIZOL reagent (Invitrogen) was added to the centrifuged cells following the manufacturer's instruction. To evaluate the quantity and quality of extracted RNA, we used NanoDrop 2000c (Thermo). In this case, RNA samples with A260/A280 ratios of >2 were selected for sequencing and quantitative analysis. RNase R was used for the treatment of total RNA and to improve the purity of circRNAs. The reactions were incubated in a 72-well optical strip at 95°C for 15 min (enzyme activation), followed by 95°C for 20 s and 60°C for 60 s (40 cycles). All reactions were run in triplicate. After these reactions, the mean Ct was determined from the triplicate PCRs.
We used Ct values to evaluate the expression levels of the hsa_ circ_0006282. The expression value of the mentioned CircRNA relative to internal control was determined using the 2 −△Ct method. 12

| Statistical analysis
The results were analyzed by GraphPad software (GraphPad PRISM V 8.4 analytical software). We used Student's t test and Pearson's χ 2 test, respectively, to compare data between pairs of groups and to evaluate the clinicopathological variables.  To evaluate the repeatability and stability of this circRNA, a mix of plasma was provided and was placed at room temperature for 0, 2, 6, 10, 14, and 24 h. Then, mixed plasma was frozen and thawed for 0, 1, 2, 3, and 5 times, the relative expression rate of hsa_circ_0006282 was detected. Based on our results, there was no significant difference in the expression rate of hsa_circ_0006282 in plasma between different examined periods (p > 0.05) ( Figure 3A,B), which indicated that the detection of hsa_circ_0006282 had good repeatability and stability. Finally, we detected an amplification product of qRT-PCR on the agarose gel (198bp) ( Figure 3C).

| Diagnostic utility of plasma Circ_0006282 in CRC
The receiver operating characteristic curve (ROC) and area under curve (AUC) were drawn for hsa_circ_0006282 plasma levels of 100 patients and 108 age-matched control to evaluate hsa_circ_0006282 as a biomarker for CRC diagnosis. Our results showed that plasma hsa_circ_0006282 could distinguish the primary CRC patients from the healthy samples, and the AUC was 0.812 (95% CI: 0.625-0.801, p < 0.0001) (Figure 4). Based on Table 3

| DISCUSS ION
Colorectal cancer is one of the most common cancers with a high mortality rate, and most cases of CRC patients are found at an advanced stage. 13 Although there are different biomarkers for detecting colorectal cancer, these biomarkers are not very sensitive and specific. Today, the use of molecular biomarkers in the early detection of cancers is very crucial. CircRNAs are among these biomarkers that have been received too much attention in the last three or four years. [14][15][16] In this regard, Chiu  Regarding the role of hsa_circ_0006282 in cancer incidence, only one study was conducted in 2020, which showed an increase in the expression of hsa_circ_0006282 in patients with gastric cancer. 11 They also suggested that the oncogenic function of circ_0006282 is partly attributed to its regulation on miR-155/FBXO22 axis by sponging miR-155 to upregulate the expression of FBXO22. Since one of the essential roles of the circRNAs is the sponge of miRNAs, in the future study, we will investigate the circRNA-miRNA-mRNA axis in CRC patients.
In conclusion, plasma hsa_circ_0006282 can be used as a novel biomarker in the progression of CRC. Plasma hsa_circ_0006282 has the potential to be used as an early screening indicator in the detection of primary CRC. Given that, after surgery or recurrence, the expression level of plasma hsa_circ_0006282 has dynamically changed, suggesting that this circRNAs may have a real-time monitoring function. Furthermore, the differential expression of hsa_circ_0006282 in plasma samples of CRC and colitis patients showed that this biomarker could differentiate between CRC and colitis patients.

ACK N OWLED G M ENTS
We thank all participants for their participation in the current study. We also thank the Cellular and Molecular Research Center of the Qazvin University of Medical Science for providing laboratory facilities.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author [SM]. The data are not publicly available because they contain information that could compromise the privacy of research participants. Note: The bold value means statistical significance.