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ORIGINAL ARTICLE
Ahead of print publication  

Circulating serum miR-1246 and miR-1229 as diagnostic biomarkers in colorectal carcinoma


1 Department of Cellular and Molecular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2 Department of Medical Genetics, Faculty of Medical Sciences, TarbiatModares University, Tehran, Iran
3 Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
4 International Collaborative Center on Growth Factor Research, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China
5 Sarem Cell Research Center, Sarem Hospital, Tehran, Iran

Date of Submission08-Jun-2020
Date of Decision16-Jul-2020
Date of Acceptance30-Sep-2020
Date of Web Publication23-Sep-2021

Correspondence Address:
Fatemeh Rouhollah,
Department of Cellular and Molecular Sciences, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran
Iran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_752_20

 > Abstract 


Background: Colorectal cancer (CRC) is one of the most common cancers worldwide. Although colonoscopy is considered as the “Gold Standard” technique to detect CRC, its application is invasive and cost incurred. Thus, noninvasive or minimally invasive approaches are of utmost importance. The aberrant expression of some microRNAs (miRNAs, miRs) has been suggested in association with CRC pathogenesis. This study aimed to validate if circulating serum miR-1229 and miR-1246 are diagnostic biomarkers for CRC.
Materials and Methods: Serum samples were isolated from 45 CRC patients and also 45 healthy controls (HC). The expression levels of circulating serum-derived miR-1229 and miR-1246 were evaluated by quantitative real-time polymerase chain reaction. Receiver operating characteristic (ROC) curves were constructed to evaluate the CRC diagnostic accuracy of selected miRNAs. Furthermore, the association of candidate miRNAs and clinicopathological characteristics were evaluated. Functional enrichment of the candidate miRNAs was applied using in silico tools.
Results: The expression of miR-1229 and miR-1246 was significantly higher in CRC patients than HC (P < 0.0001) and also was found in association with lymph node metastasis (P < 0.05). We demonstrated a significant up-regulation of serum-derived miR-1246 in advanced tumor–node–metastasis stage III of CRC patients (P < 0.05). Areas under the ROC curve of miR-1229 and miR-1246 were 0.81 and 0.84, respectively (P < 0.0001).
Conclusion: We confirmed the capability of circulating serum miR-1229 and miR-1246 as novel diagnostic biomarkers for CRC.

Keywords: Biomarker, circulating microRNAs, colorectal cancer, diagnosis



How to cite this URL:
Rafiee R, Razmara E, Motavaf M, Mossahebi-Mohammadi M, Khajehsharifi S, Rouhollah F, Babashah S. Circulating serum miR-1246 and miR-1229 as diagnostic biomarkers in colorectal carcinoma. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 6]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=326583




 > Introduction Top


Colorectal cancer (CRC) is a major public health issue, being the third leading cause of cancer-related deaths worldwide. Similar to other types of cancers, both genetic and environmental factors justify the etiology and progression of CRC. The majority of CRC cases are sporadic, though approximately 20%–30% of CRC cases are imputed to inherited mutations. CRC arises through the transformation of normal colonic mucosa to an adenomatous polyp, which may develop into invasive carcinoma.[1],[2]

Since CRC is almost asymptomatic in the early stages, it is often diagnosed in the late stages. Therefore, this can put the patients on the brink of metastasis, i.e., approximately 90% of CRC-related deaths are due to distant organ metastases.[3] In the early stages, CRC detection increases the 5-year survival rate up to 90%, compared to 12% if distant metastasis develops.[4] The currently conventional methods for screening or diagnosis of CRC are colonoscopy, fecal occult blood test, and fecal immunochemical testing. While these approaches effectively reduce CRC mortality, they have limited sensitivity and also are invasive that restrict their applications.[5],[6] Keeping this in mind that early diagnosis of CRC notably improves the survival rate, establishing novel sensitive and noninvasive/minimally invasive diagnostic strategies seems imperative.[7]

microRNAs (miRNAs, miRs) are a highly conserved group of small noncoding RNA molecules that function in multitudinous developmental, physiological, and pathophysiological processes.[8],[9] They are, in fact, an abundant class of endogenous small RNA molecules (20–25 nucleotides in length). Due to their role in cancer development, they classified as oncogenes (oncomiRs) which can promote tumor formation or tumor suppressors that in turn prevent tumor development by negatively inhibiting oncogenes and/or genes that control cell differentiation or apoptosis (reviewed in[10]). Dysregulation of miRNAs has added a new layer of complexity to the understanding of the development and progression of human cancers.[11],[12] For instance, for the first time, Michael et al. reported that the expression levels of miR-143 and miR-145 were significantly lower in both colonic adenocarcinoma and adenomatous polyps tissues.[13] This study opened a new door to the potential application of miRNAs as diagnostic or prognostic biomarkers in CRC.[14],[15]

Accumulating evidence suggests that circulating miRNAs in biofluids (e.g., serum, plasma, and urine) may contribute to different pathogenesis aspects of cancer from initiation to metastatic spread.[16],[17] Recently, differentially expressed circulating miRNAs were found to be potential noninvasive biomarkers for cancer diagnosis.[17],[18],[19] Herein, we investigated the expression of two candidate circulating serum miRNAs (i.e., miR-1229 and miR-1246) in CRC patients to show whether these miRNAs could be used as novel non-invasive biomarkers for CRC diagnosis. Additionally, we assessed the possible correlations between the expression of these miRNAs with some clinicopathological parameters in CRC patients.


 > Materials And Methods Top


Clinical samples

Forty-five patients (38–67 years old) with primary tumors, diagnosed at Shariati and Rasoul Akram hospitals, Tehran, Iran, were recruited as CRC groups. Written informed consent was obtained from each patient, and the study protocol was approved by the ethics committee at the participating centers. After doing a complete physical screening, 55 individuals without any malignant or neoplastic lesions were registered as healthy control (HC). The peripheral blood from each patient and the healthy individual was collected and then centrifuged at 1000 ×g for 10 min at 4°C. The sera samples were then transferred to sterile vials and stored at-20°C until further investigation. The clinicopathological characteristics of the patients enrolled in this study are summarized in [Table 1].
Table 1: The clinicopathological features of subjects enrolled in the study

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RNA isolation and complementary DNA synthesis

Total RNA was isolated from the sera samples using TRIzol (Invitrogen, USA) according to the manufacturer's instruction and purity ratios were determined using Nanodrop™ 2000c (Thermo Fisher Scientific, USA). Poly-(A)-tailing and complementary DNA (cDNA) synthesis were performed using miR-Amp Kit (ParsGenome co., Iran), and the resultant cDNA diluted in RNAs-free water.

Quantitative real-time polymerase chain reaction

The expression of candidate mature miRNAs were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) using SYBR® Premix Taq™II (TAKARA, Japan). QRT-PCR was performed on an ABI StepOne Sequence Detection System (Applied Biosystems, Foster City, USA) tally with the following conditions: 95°C for 5 min, followed by 40 cycles at 95°C for 10 s, and 60°C for 30 s. U48 snRNA was used as an internal control for normalizing qRT-PCR data. The relative expression levels of miR-1229 and miR-1246 were calculated using the comparative threshold cycle (Ct) values. The fold change was calculated using the equation 2−ΔΔCt.[20],[21]

Identification of miRNAs associated with prognosis in colorectal cancer

To further validate, we used the Kaplan-Meier plotter online database[22],[23] and miRpower[24] for the pan-cancer tool to analyze the relationship between miR-1229 and miR-1246 expression and overall survival of 160 CRC tissue samples from the aforementioned database. Hazard ratio, 95% confidence intervals (CIs) and log-rank P values were calculated. P < 0.05 was considered statistically significant.

Functional enrichment analysis of miR-1229 and miR-1246

To gain further insight into the function of the dysregulated miRNAs and also their possible common targets, the DNA Intelligent Analysis (DIANA)-miRPath v3.0 software (DIANA Lab, Thessaly, Greece)[25] was applied to identify the significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.[26] The algorithms of miRTarBase v7.0[27] and DIANA-microT-CDS (v5.0)[28] were used to identify the validated target pathways of miR-1229 and miR-1246. DIANA-microT-CDS v. 5.0 algorithm recognizes potential miRNA binding sites located in both the coding sequence and in the 3′-untranslated region (3′-UTR) of target mRNAs, according to complementary pairing with nucleotides in position 1-9 at the 5′ end of the miRNA. We used the 'Pathways Union' option of the miRPath software. The P value threshold was determined <0.05 and the false discovery rate (P < 0.05) were calculated by Fisher's exact test using the Benjamini and Hochberg method.[27] Using miRTargetLink Human,[29] we retrieved experimentally supported and shared target genes for selected miRNAs and also provided the interaction network.

Statistical analysis

Statistical Analysis were performed using GraphPad Prism Version 7.0 (GraphPad Software Inc, San Diego, California, USA). Data were presented as mean ± standard deviation from three independent tests. T-test was used for statistical analysis of data changes and P < 0.05 was considered statistically significant. Receiver operating characteristic (ROC) curves were generated, and areas under the ROC curves (AUC) were calculated to evaluate the diagnostic performance of the candidate miRNAs.


 > Results Top


Elevated expression levels of miR-1229 and miR-1246 in colorectal cancer patients

According to literature for the candidate miRNAs, the identified targets, and the previous experimental findings aimed to identify the possible contributing miRNAs to CRC pathogenesis, we selected miR-1229 and miR-1246 as candidate miRNAs. To determine the expression status of these miRNAs, qRT-PCR was conducted and the relative expression for each CRC and HC individual was evaluated. The expression of circulating miR-1229 and miR-1246 was significantly higher in CRC than control samples (P < 0.001) [Figure 1]a and [Figure 1]b. Importantly, unsupervised hierarchical cluster analysis showed that the pattern of significantly higher expression of the candidate miRNAs in serum samples could discriminate CRC from healthy samples [Figure 1]c.
Figure 1: The expression levels of circulating miRNAs in the serum of CRC patients and healthy controls. (a and b) Relative expression analysis showed a significant up-regulation of miR-1229 (fold change: 2.07, P < 0.0001) and miR-1246 (fold change: 2.34, P < 0.0001) in the serum of the patients with CRC compared to healthy controls. (c) Unsupervised hierarchical cluster analysis using the differentially expressed serum miRNAs discriminated CRC patients and HC individuals. The heatmap (Euclidian distance, complete linkage) represents miRNAs with high relative expression in green and miRNAs with low relative expression in red. The heatmap was drawn by Heatmapper (http://www. heatmapper. ca). CRC=Colorectal carcinoma patients, HC=Healthy controls

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The amplification efficiency calculated for the candidate miRNAs and also the internal control was approximately equal with high linear correlation, affirming the validity of the assay for relative expression quantification [Supplementary Table 1]. Furthermore, the specificity and uniqueness of each product was confirmed using dissociation curve analysis. In other words, a single and sharp melting curve excluded the possible presence of primer-dimer or nonspecific products that were generated during the amplification reaction [Supplementary Figure 1].



Correlation between miRNA expression and clinicopathological features

We also assessed and compared the expression levels of the candidate circulating miRNAs (i.e., miR-1229 and miR-1246) in CRC patients with different clinicopathological features. Although the expression of circulating serum miR-1229 inclined to increase in the advanced stages of CRC, the experiment did not show any statistical significance [Figure 2]a. As indicated in [Figure 2]b, a higher expression level of miR-1246 (P < 0.05) was detected in CRC patients with tumor–node–metastasis (TNM) stage III. Results also showed that the overexpression of serum-derived miR-1229 and miR-1246 was positively associated with lymph node metastasis (LNM). As depicted in [Figure 2]c and [Figure 2]d, the expression of circulating serum miR-1229 and miR-1246 was significantly higher in the serum of the CRC patients with LNM than those without LNM (P < 0.05).
Figure 2: Possible correlation between circulating serum-derived miRNA levels and some clinical features in CRC patients. (a and b) Relative expression levels of circulating miR-1246 and miR-1229 in CRC serum samples categorized by tumor stages. Results showed that miR-1246 was significantly increased in the late stage of CRC (TNM stage III) compared to the patients within the early stages (TNM stage I-II). No statistically significant differences were found in the expression levels of serum-derived miR-1229 between the two groups. (c and d) Relative expression levels of circulating miR-1246 and miR-1229 in CRC serum samples categorized by LNM status. Elevated expression levels of circulating miR-1229 and miR-1246 were positively associated with LNM status. LNM=Lymph node metastasis, CRC=Colorectal carcinoma patients, TNM=Tumor–node–metastasis

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Diagnostic potential of circulating serum miR-1229 and miR-1246 in colorectal cancer

ROC curves were utilized to assess the diagnostic accuracy of individual serum miR-1229 and miR-1246 for discrimination of CRC patients from HCs. Results (according to the sensitivity and specificity of miRNA expression) showed that the AUC with 95% CIs for miR-1229 and miR-146 were 0.81 (P < 0.0001) and 0.84 (P < 0.0001), respectively, confirming that these miRNAs can be used as reliable CRC biomarkers [Figure 3]a and [Figure 3]b.
Figure 3: The up-regulation of circulating serum miR-1229 and miR-1246 suggests a potential diagnostic marker for CRC. ROC curve analyses of circulating serum-derived miR-1229 (a) and miR-1246 (b) was used to discriminate patients from healthy controls. The AUC of serum-derived miR-1229 and miR-1246 were calculated 0.81 and 0.84, respectively (P < 0.0001). ROC=Receiver-operating characteristic, AUC=Area under the curve

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miR-1229 and miR-1246 associated with poor overall survival in colorectal cancer patients

Using the Kaplan-Meier plotter database, we determined that the overall survival of 160 CRC patients (from the Kaplan–Meier plotter online database[23]) is low in cases with higher expression of miR-1229 and miR-1246. The follow-up time was determined in 120 months. The plots showed that as patients enter the late stages of CRC, their overall survival decreases [Supplementary Figure 2].



Target prediction and correlated pathways for miRNA expression clusters

We also performed a computational target prediction analysis coupled with pathway analysis using DIANA-miRPath v. 3.0 software. Using the algorithms of miRTarBase v. 7.0 and DIANA-microT-CDS (v5.0), we demonstrated the common pathways shared between miR-1229 and miR-1246. The results are reported as heatmaps [Figure 4]a and [Figure 4]b, and the pathways are clustered based on significance levels.
Figure 4: Heatmap of differentially expressed miRNAs versus significantly enriched functional pathways. (a) The attached dendrograms on both axes represent hierarchical clustering results for miRNAs and pathways, respectively. The figure was obtained from the output of Diana miRpath V.2[27] using the algorithm of miRTarBase v7.0. (b) Predicted pathways heat map using DIANA-microT-CDS (v5.0) algorithm. In the heatmap, reddish colors indicate lower P values (more significant), and higher interaction of each miRNA with a specific molecular pathway

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Predicted and reported mRNA targets of the validated miRNAs were mapped using MirTargetLink. We screened overlapped gene targets between the miRNAs. To facilitate the interpretation of a list of gene targets and aid in understanding of the potential function of the candidate miRNAs, we performed pathway enrichment analysis for KEGG pathways. Only one gene, TAO Kinase 1 (TAOK1; OMIM: 610266), remained after selecting the “weak interactions” [Figure 5].
Figure 5: The network of interactions between candidate miRNAs and their validated target genes. (a) The network of interactions between miR-1229 and its validated target genes provided by miRTargetLink Human.[29] (b) The same network depicted for miR-1246. (c) After selecting the “Weak Interactions,” only one target, TAOK1, remained. In this figure, orange describes genes interacting with at least three miRNAs, while blue shows genes targeted by two miRNAs. miR-1229 and miR-1246 did not show any strong interaction in this network

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 > Discussion Top


CRC is one of the leading causes of cancer death worldwide. The main reason for the high mortality rate of CRC is late diagnosis influenced by CRC asymptomatic nature.[1],[2] Current screening or diagnostic methods range from semi-invasive tools (e.g., colonoscopy detects colorectal abnormalities) to novel noninvasive tests that detect lesions indirectly using biofluids. While these methods have clinically improved the detection of CRC, there are some obstacles (e.g., low sensitivity in early CRC stage and being invasive and cost-incurred) limiting their applications.[6],[7] As early detection of CRC has a significant impact on patients' prognosis, the development of novel sensitive and minimally invasive diagnostic methods is of great essential.[7]

Since the discovery, the profound role of miRNAs in molecular pathways contributing to cancer development and progression has been explored. Besides their fundamental roles in cellular functions, their application as biomarkers has been intensely focused.[19] In the present study, we demonstrated that the expression levels of circulating serum miR-1229 and miR-1246 were significantly higher in CRC patients. As an effective approach, integrative computational bioinformatics procedures have been used to detect the potential contributing miRNAs in CRC. Thus, as a rational approach, preliminary detection of candidate miRNAs derived from large-scale expression profiling data and low-throughput experimental verification for the selected miRNAs was used to select the candidate miRNAs. A recent study showed that exosomal miR-1229 and miR-1246 levels in the plasma from CRC patients decreased after surgery.[30],[31] However, there is little information about their contribution and functions as potential biomarkers to CRC.

Further analysis revealed a significant correlation between the elevated levels of miR-1229 and some clinicopathological features, including LNM. The present study is consistent with the findings of Hu et al.[32] who indicated that high expression level of circulating exosomal miR-1229 was significantly associated with clinicopathological characteristics in CRC tissues. Although we could not find the significant difference among TNM stages, this miRNA increased apparently in TNM stage III. We believe that future studies should aim to replicate results in a larger cohort of CRC patients in different populations.

miR-1229 inhibits the protein expression of HIPK2 which, in turn, can activate Vascular endothelial growth factor factor leading to angiogenesis.[32] Overexpressed miR-1229 enhanced breast cancer cell tumorigenesis via activating Wnt/β-catenin signaling pathway through targeting its negative regulators e.g. adenomatous polyposis coli (APC), glycogen synthase kinase-3 β (GSK-3 β), an inhibitor of β-catenin and T cell factor.[33] Wnt/β-catenin signaling takes a center stage in intestinal homeostasis and its excessive activation through APC inactivation aberrantly occurs in most CRCs. As APC is a target of miR-1229,[33] the up-regulation of this miRNA potentially contributes to CRC development via APC attenuation and consequently over-activation of the Wnt/β-catenin pathway. Furthermore, Nishibeppu et al. showed that plasma miR-1229 might be a clinically useful biomarker for predicting chemoresistance and selecting other or combined intensive chemotherapy regimens in gastric cancer patients.[34] These studies show the diverse roles of miR-1229.

miR-1246 is known as one of the driving miRNAs in several cancer types. Increased expression of miR-1246 in CRC tissues and cell models has been reported by Wang et al.[35] Upregulation of this miRNA has been reported to enhance the proliferation, colonization, invasion, angiogenesis, and migration of CRC cells and also inhibit their apoptosis.[30],[35] Furthermore, serum miR-1246 expression has been demonstrated as a novel diagnostic and prognostic biomarker for oesophageal squamous cell carcinoma.[36] In this study, we showed that overexpression of miR-1246 was associated with TNM stage III and also LNM.

Wang et al. showed that miR-1246 negatively regulated the expression of cyclin G2, promoting growth and metastasis of CRC cells.[35] The levels of serum miR-1246 can predict LNM in cervical squamous cell carcinoma.[37] In this cancer, miR-1246 induces cell proliferation, invasion and migration by targeting THBS2.[38] miR-1246 is also important in differentiation, invasion, and metastasis of esophageal squamous cell carcinoma and hepatocellular carcinoma.[39],[40],[41] miR-1246 is considered to be responsible for proangiogenic function by activating Smad1/5/8 signaling in CRC.[30] However, other biofunctions remain to be elucidated.

Using DIANA-miRPath v3. 0 and also miRTargetLink, we proposed TAOK1 as a potential target for miR-1229 and miR-1246. This gene plays an undeniable role in apoptosis.[42] TAOK1 also plays important roles in negatively regulating inteleukins (e.g., inteleukin-17) and inflammatory responses.[43] Moreover this protein can induce cell death, and inhibit cell growth.[44] All in all, we conjecture that miR-1229 and miR-1246 can exert their functions in CRC through modulating TAOK1; however, it needs further investigation to be confirmed.

Importantly, the calculated AUC by the ROC curve analysis showed that circulating miR-1229 and miR-1246 are suitable to be considered as possible diagnostic biomarkers which can discriminate CRC patients from healthy subjects. Besides, data from the Kaplan–Meier plotter online database showed that high expressions of miR-1229 and miR-1246 are associated with poor survival, suggesting their prognostic values in patients with CRC.

In sum, the up-regulation of circulating serum miR-1229 and miR-1246 in CRC patients is proposed as a potential biomarker for the CRC diagnosis. Moreover, the correlation between the elevated levels of these miRNAs with some clinicopathological features of CRC may offer the opportunity to apply them as promising biomarkers in CRC identification and also surveying their prognosis.

Acknowledgment

We gratefully thank the participants for their contribution to this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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