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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 1  |  Page : 124-128

Role of circulating micro-RNA-21 as a noninvasive biomarker for detection of colorectal cancer


1 Department of Internal Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Oncology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
4 Department of Hepatology and Gastrology, Nasser Institute, Cairo, Egypt

Date of Submission14-Jul-2019
Date of Decision27-Aug-2019
Date of Acceptance06-Sep-2019
Date of Web Publication27-Mar-2021

Correspondence Address:
Mohamed H Gohar
El-Bagour, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_207_19

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  Abstract 


Objective
To assess serum micro-RNA-21 (miR-21) expression level as a stable blood-based biomarker for colorectal cancer (CRC).
Background
CRC represents the third most common tumor worldwide with male predominance, with significant morbidity and mortality despite the recent therapeutic improvements. Therefore, a noninvasive diagnostic marker with good sensitivity and specificity is very important, which leads to better clinical outcomes. MiR-21 is an oncogenic miRNA that modulates the expression of multiple cancer-related target genes. MiR-21 expression was upregulated in the serum and in the malignant tissues in relation to the normal tissues. Its elevated level is claimed to be associated with poor survival and poor response to treatment.
Patients and methods
This cross-sectional study was carried on 50 patients and measured their serum miR-21 (CYBR Green). The cases group comprised 20 consecutive patients with CRC proved by colonoscopy and histopathological biopsy examination, the second group comprised 20 patients with colorectal polyps with no malignant transformation proved by histopathological examination, and the third group included 10 patients with normal colonoscopy finding as the control group. The studied cases were attending the Endoscopy Unit Menoufia University Hospital and Nasser Institute. The three groups agreed to written consent to participate in this study after the approval of the local ethical committee.
Results
Serum miR-21 expression was significantly upregulated in the cases group of patients with CRC compared with the other groups, with P value less than 0.001, showing sensitivity of 85% and specificity of 83.3% at a cutoff point of −2.31.
Conclusion
Mi-RNA-21 shows upregulation in the serum in CRC cases, with statistically significant difference compared with the control group and the polyp group, showing promising results as a noninvasive biomarker of CRC.

Keywords: benign colorectal polyps, colorectal cancer, grading and staging of colorectal cancer, micro-RNA-21, tumor markers in colorectal cancer


How to cite this article:
Abdel-Atti EA, El-Shaib ESI, Shehata MA, Sonbl AA, Badr MH, Gohar MH. Role of circulating micro-RNA-21 as a noninvasive biomarker for detection of colorectal cancer. Menoufia Med J 2021;34:124-8

How to cite this URL:
Abdel-Atti EA, El-Shaib ESI, Shehata MA, Sonbl AA, Badr MH, Gohar MH. Role of circulating micro-RNA-21 as a noninvasive biomarker for detection of colorectal cancer. Menoufia Med J [serial online] 2021 [cited 2021 May 8];34:124-8. Available from: http://www.mmj.eg.net/text.asp?2021/34/1/124/312015




  Introduction Top


Colorectal cancer (CRC) is the third most prevalent cancer worldwide and is a leading cause of cancer-related mortality for both men and women [1]. CRC incidence rates are increasing owing to the effect of many risk factors, including smoking, physical inactivity, obesity, red and processed meat consumption, and excessive alcohol consumption [2]. In Egypt, CRC is one of the most common malignant tumors. Its incidence ranges between 2 and 6% of the total number of cancer cases reported annually, and it ranks the sixth most common cancer in both males and females [3]. The CRC mortality rates can be decreased by early diagnosis through screening. However, the present CRC screening techniques [colonoscopy, fecal occult blood test, and carcinoembryonic antigen (CEA) test] are limited by their difficulties and costs, besides uncertain or delayed results [4]. Although colonoscopic detection of CRC is the most reliable diagnostic tool, its availability, patient tolerability, and costs have hampered its wide application. On the contrary, many blood-based biomarkers do exist such as carbohydrate antigen 19-9 (CA19-9) and CEA. Unfortunately, they have low sensitivity, particularly for early-stage CRC [5]. Hence, there is a pressing need for new noninvasive biomarkers to improve the early detection of CRC [6]. MicroRNAs (miRNA) are a family of small noncoding RNAs (19-22 nucleotides) that posttranscriptionally regulate gene expression. In general, miRNAs are transcribed as a group called the pri-miRNA complex, which is cleaved in the nucleus to form the pre-miRNA, which is then translocated to the cytoplasm where they undergo final maturation into a functional miRNA [7]. Studies have shown that profiles of miRNA expression differ between normal tissue and tumor tissues and vary among different tumor types [8]. Aberrant miRNA expression profiles have been identified and emerged as potential screening biomarkers for CRC [9]. Micro-RNA-21 (miR-21) is an oncogenic miRNA that modulates the expression of multiple cancer-related target genes such as PTEN, TPM1, and PDCD and has been shown to be overexpressed in various human tumors [10]. MiR-21 expression was found to be upregulated in CRC tissues. It is elevated during tumor progression and was claimed to be associated with poor survival and poor response to chemotherapy [11].


  Aim Top


The aim of the present study was to assess serum miR-21 expression level as a stable blood-based biomarker for detection of CRC.


  Patients and methods Top


The study was approved by the ethical committee of the faculty of medicine Menoufia University and patients gave an informed consent. This cross-sectional case-control observational study was conducted on 50 patients selected from the Gastroenterology and Oncology Departments at Menoufia University Hospitals and Nasser Institute hospital during the period from January 2015 till April 2016. They were divided into three groups: group 1 included 20 consecutive patients with endoscopic and histological evidence of CRC, group 2 included 20 patients with histological evident benign colonic polyps, and group 3 included 10 apparently healthy individuals without remarkable colonoscopic findings, who served as the control group (the indications for colonoscopy in such healthy participants were either long history of abdominal pain, flatulence, and/or altered bowel habits). Patients who received chemotherapy or radiotherapy underwent previous surgical colectomy, or had any other malignancies were excluded from the present study. After obtaining an informed written medical consent, all studied patients were subjected to full history taking, thorough clinical examination, and laboratory investigations, including baseline investigation (complete blood count, liver and kidney function tests), tumor markers including CEA and CA19-9, and detection of miR-21 expression level using real-time PCR technique. Extraction of total RNA including miR-21 from plasma samples using miRNA extraction kit was done according to Wu et al. [12]. Relative quantitation of miR-21 level using real-time quantitative PCR was done according to Huang et al. [13] (CYBR Green). Computed tomography scan of the abdomen and pelvis with oral and intravenous contrast was done. Complete colonoscopy was done by an expert gastroenterologist.

Statistical analysis

The collected data were tabulated and analyzed using SPSS, version 17.0, on IBM compatible computer (SPSS Inc., Chicago, Illinois, USA). Categorical data were presented as number and percentages, whereas quantitative data were expressed as mean ± SD, median, and range. χ2 or Fisher's exact test was used to analyze categorical variables. Quantitative data were tested for normality using Kolmogorov–Smirnov test, using Student t if normally distributed, or Mann–Whitney U test, Kruskal–Wallis test, and Spearman's correlation coefficient (rho) if not normally distributed. ROC curve was used to determine cutoff values of miR-21 with optimum sensitivity and specificity in prediction of patients with CRCs and in detecting certain T stage and size and modified Astler-Coller (MAC) stage. The accepted level of significance in this work was stated at 0.05 (P < 0.05 was considered significant) [14],[15].


  Results Top


In group 1 (CRC), 60% of the patients were males and 40% were females; their mean age was 50.8 ± 14.42 years, with a range between 22 and 77 years. However, in group 2 (polyp group), 75% of patients were males and 25% of them were females, with mean age of 44.35 ± 9.98 years, with a range between 23 and 60 years. We have selected 10 age-matched and sex-matched individuals as a different studied group (group 3 or control group). In this group, males represent 60% of them and females were 40%, with the mean age of 41.4 ± 9.16 years, with a range between 27 and 55 years. There was also a trend toward association between female sex and abnormal pathology, although this did not meet statistical significance (P = 0.052) [Table 1]. Regarding the site of the lesion in group 1 (CRC), six (30%) patients had right-side lesion, five (25%) patients had left-side lesion, and nine (45%) patients had lesion in the rectum. However, in group 2 (polyp group), one (5%) patients had right-side lesion, eight (45%) patients had left-side lesion, eight (40%) patients had lesion in the rectum, and three (15%) patients had multiple polyps all over the colon [Table 2]. Regarding tumor characteristics in group 1, all examined patients had adenocarcinoma with different grades. Grade II adenocarcinoma was present in 13 (65%) patients and grade III adenocarcinoma was present in seven (35%) patients. However, in group II, the histopathological examination of the benign polyps revealed tubulous polyp in six (30%) patients, villous polyp in two (10%) patients, tubulovillous polyp in seven (35%) patients, inflammatory polyp in four (20%) patients, and only one (5%) patient had hamartomatous polyps. In group 1, small-sized lesions (<5 cm) were present in six (30%) patients, whereas large sized lesions (<5 cm) were present in 14 (70%) patients. Lymph node affection was present in 12 (60%) patients of group 1, and it was absent in eight (40%) patients. However, distant metastasis was present only in two (10%) patients of the CRC group [Table 3]. Serum level of CEA showed area under the curve (AUC) of 0.93, sensitivity of 65%, and specificity of 75% of CEA in patients with CRC [Table 4].
Table 1: Age and sex distribution among the studied groups

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Table 2: Site of lesion among the patient groups

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Table 3: Tumor characteristics among colorectal cancer group

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Table 4: Validity of carcinoembryonic antigen and carbohydrate antigen 19-9 for diagnosis of colorectal carcinoma

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The level of miR-21 is upregulated in the CRC group compared with the other studied groups, with significant P value less than 0.001, and serum miR-21 showed sensitivity of 85% and specificity of 83.3% at cutoff point of −2.31, with AUC of 0.88 [Table 5].
Table 5: Comparison of micro-RNA-21 among the studied groups

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  Discussion Top


The results of the current study showed significant results regarding miR-21 as a noninvasive marker for early detection for CRC, which may improve clinical outcomes. Statistical analysis revealed that the serum expression of the studied marker (miR-21) was elevated in the CRC cases (group 1), with a mean range of 6.47 ± 3.37, and it is relatively elevated in the benign polyp cases (group 2), with a mean range of 1.13 ± 3.10, with relative normal expression in the control group (group 3), with a mean range of − 0.33 ± 4.74. Moreover, it revealed significant statistical difference in the correlation between group 1 and group 3 and between group 1 and group 2 but insignificant difference in the correlation between group 2 and group 3. These results agreed with Liu et al. [16] who found that the serum expression of miR-21 is elevated in both the CRC group and the advanced adenoma group than the control group. Moreover, Toiyama et al. [17] also reported a significant elevation of serum miR-21 in the CRC group and in the adenoma group in relation to the control group, with significant difference in comparing CRC group with the control. Moreover, there was a significant difference in comparing adenoma group with control group. In the current study, tumor markers (CEA and CA19-9) were studied in the different selected groups. Sensitivity, specificity, and accuracy of these tumor markers were assessed individually and in relation to the marker miR-21. The results showed insignificant difference in the level of CA19-9, with a mean of 15.2 ± 12.55 in CRC group (group 1), a mean of 5.22 ± 6.06 in the benign polyp group (group 2), and a mean of 5.65 ± 6.4 in the control group (group 3). The results show a significant statistical difference in the level of CEA between the cases in CRC group (group 1) and the other groups in comparing group 1 with group 2 and group 1 with group 3, with sensitivity of 65% and specificity of 70%. These results come in general agreement with what was reported by Duffy [18], who showed a sensitivity of 80% and specificity of 70% of CEA in patients with CRC. In addition, Kanaan et al. [19] showed AUC of 0.808, sensitivity of 36%, and specificity of 87% of CEA in patients with CRC. In the current study, the correlation of the results of the CEA with the marker miR-21 in the CRC cases (group 1) was statistically significant, which comes in agreement with Toiyama et al. [17] who showed a positive correlation between miR-21 and CEA, and it also comes in agreement with Menéndez et al. [20] who showed a positive correlation. However, this comes in disagreement with Liu et al. [16] who showed no significant correlation between them. In this study, we have searched for relations between the expression of our marker miR-21 and the different tumor characteristics (grading, differentiation, tumor size, lymph node involvement, and the presence of distant metastasis). Comparing the present results with Toiyama et al. [17] there were no significant statistical difference in the level of miR-21 in different characteristic groups in CRC studied cases as regards to the tumor size and the differentiation of the tumor in both studies. On the other hand (LN and distant metastasis) were different between both studies which attributed to limited sample size. Moreover, Bastaminejad et al. [21] studied the relation between the level of miR-21 and the size of the tumor, and the findings showed no significant difference in this variant, which also comes in agreement with our results. In the present study, we found that serum upregulation of miR-21 is a good diagnostic marker for detection of the CRC with sensitivity of 85%, specificity of 83.3%, and AUC of 0.88. This goes hand in hand with other studies such as Bastaminejad et al. [21] who found that sensitivity of 86.05%, specificity of 72.97%, and an AUC of 0.783 in the serum miR-21, and also Kanaan et al. [19], who described miR-21 as a potentially noninvasive biomarker for CRC with near-optimal results for discriminating CRC with sensitivity of 90%, specificity of 90%, and an AUC of 0.910. Honghe and Peiwei Li Pesta [22] reported that circulating miR-21 level has potential value for CRC early detection, whereas high tissue miR-21 level is associated with adverse outcomes in CRC and its sensitivity of 76%, specificity of 81%, and an AUC of 0.810. Toiyama et al. [17] demonstrated the potential role of serum miR21 in the early detection of CRC. This is supported by the markedly high AUC values of 0.919 derived from comparisons between patients with CRC and healthy controls, with sensitivity of 91.9% and specificity of 81.1%. Yu et al. [23] indicated that miR-21 demonstrates good accuracy for CRC diagnosis. with sensitivity of 72%, specificity of 85%, and AUC of 0.87. Liu et al. [16] stated that miR-21 serum levels have potential value for early detection of CRC with sensitivity of 65%, specificity of 85%, and AUC of 0.802.


  Conclusion Top


MiR-21 shows upregulation in the serum in the CRC cases with statically difference compared with the control group and the polyp group of benign nature, and it shows promising results as a noninvasive serum biomarker for detection of CRC. Further studies should be conducted on a larger number of patients with correlation of tissue levels of the miR-21 and also correlations with other miRNA markers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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