INTRODUCTION
Colorectal cancer (CRC) is characterized as a tumor growth on the inner lining of the rectum or colon (1,2). It is the third most common cancer among both men and women (3). Growing westernized diet have led to an increase in the incidence and mortality of CRC (4). Indeed, CRC is complicated by the difficulty of early diagnosis, due to the lack of reliable cancer-specific diagnostic biomarkers (5). MicroRNAs (miRNAs) are a class of non-coding RNAs of nearly 22 bases in length that play crucial roles in both physiological and pathological processes. In addition, they are involved in cancer pathogenesis from initiation to metastasis, primarily through interaction with the 3' of target mRNA, which leads to post-transcriptional inhibition or mRNA degradation (6). In this respect, growing body of evidence have indicated that certain miRNAs released from tumor cells are chemically stable and can be detected in a broad range of body fluids, including plasma, which makes quick diagnosis feasible (7,8). Therefore, miRNAs have emerged as promising novel biomarkers for CRC diagnosis. So far, hundreds of miRNAs have been reported to be associated with CRC progression and metastasis (9). A large number of studies have been performed to investigate the expression level of circulating miR-21 in blood samples of cancer patients and healthy individuals. However, results of the studies on the diagnostic value of circulating miR-21 have been inconsistent (10,11). Here, we intended to investigate the expression level of mir-21 in Iranian patients with hereditary CRC and healthy individuals.
 
MATERIALS AND METHODS
A total of 39 whole blood samples were collected from CRC patients before and after surgical intervention at Qaem hospital in Mashhad, Iran. Age- and gender-matched healthy individuals (39 controls) with no history of cancer were also enrolled in the study. The study was performed between March 2018 and September 2019 in the Department of Biotechnology at Golestan University of Medical Sciences, Gorgan, Iran. Ethical approval was obtained from the Ethics Committee of Golestan University of Medical Sciences. In addition, an informed written consent form was obtained from all participants. First, sera were separated from the blood samples by centrifugation at 12,000 g for 10 min at 4 ◦C. Then, miRNA extraction was carried out using RNx-PLUS kit (Sinagene Company) according to the manufacturer’s protocol. The purity and quality of the extracted RNAs was checked and samples with absorbance of 1.8-2 at 260/280 nm were used. Moreover, the concentration of the extracted RNAs was assessed using a bio-photometer (Eppendorf, Germany), which produced an average concentration of 110 ng/μl. Finally, real-time PCR was performed using specific primers for the miR-21. Internal control (let-7d and let-7g) was used due to the relativity of the method. For the real-time PCR reaction, 10 μl SYBER Green master mix, 5 μl of the synthetized cDNA and 4 μl specific primers were mixed in a total volume of 20 μl. The reaction was carried out using the ABI 7300 model (Applied Biosystems) at optimal temperatures. Next, miR-21 expression level was calculated based on the ΔΔCT method. Data were analyzed using SPSS statistical software (version 22) and Graph Pad.
 
RESULTS
Based on the results, miR-21 was overexpressed in in postoperative serum samples from CRC patients who underwent curative surgery (P=0.022) (Table 1). The obtained Ct values from the real-time expression analysis indicated thatmiR-21 level increased almost 4.5-fold in sera of patients after tumor removal (Figure 1)
 
 
Table 1. Expression analysis of miR-21 after tumor removal

Result	P(h1)	95% c.i.	Std.error	Expression	Reaction efficiency	Type	Gene
up	0.022	0.096 - 378.272	0.566 - 41.907	4.683	1.0	Target	miR-21
				0.772	1.0	Reference	let7-D
				1.296	1.0	Reference	let7-G

 
 
 
 Figure 1. Ct values of real-time PCR experiment for hereditary CRC patients and healthy controls
 
DISCUSSION
In this study, we aimed to explore the association between miRNA expression patterns and the diagnostic outcome of hereditary CRC.  Since the beginning of the new century, the link between miRNAs and human diseases, particularly cancer, has been established. Although most miRNAs are intracellular, a large number of miRNAs have been detected outside cells in various body fluids. They are stable in cell culture supernatants and in many body fluids such as blood, urine, saliva and milk (12). Results from recent studies indicate that circulating miRNAs could serve as potential diagnostic and prognostic markers in different types of cancers and other diseases. Additionally, early and timely diagnosis of cancer is one of the main objectives of today's cancer research projects worldwide. Thus, there has been a need for non-invasive biomarkers that would improve the early detection of different types of cancer including CRC (13, 14). Furthermore, the expression pattern of miRNAs in the circulation could be relevant to the risk of disease progression in CRC. However, inclusion criteria for evaluating miRNA expression from patients with hereditary cancer have long been considered to use patients without diabetes, metabolic diseases and hereditary cancer. Moreover, cases with history of medications, radiation therapy or even smoking have been excluded from the miRNA expression analysis studies (15).
 
CONCLUSION
Our study verified previous results concerning the exclusion of samples from patients with hereditary cancer when investigating diagnostic potential of miRNAs expression in cancer studies. 
 
ACKNOWLEDGEMENTS
The authors would like to thank Deputy of Research and Technology of Golestan University of Medical Sciences and all those who participated in this study.
 
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.