Introduction

Colorectal cancer (CRC) is the fourth most common cancer type in men and the third most common cancer type in women worldwide [1]. Accounting for approximately one million new types of cancers and one-half million deaths, CRC represents 10 % of cancer deaths [2]. Increasing over the last 10 years, colorectal cancer has the highest rate in Australia, New Zealand, Europe, and North America, and has the lowest rate in Asia and Africa [3]. Its rate has also increased in the Middle East and Iran in recent years [4]. East Azerbaijan, also, has a high incidence of cancer in gastrointestinal tract [5]. Therefore, knowing more about the biology and nature of colorectal cancer, it is possible to design effective prognostic, diagnostic, and treatment plans to help reduce the rate of this disease [6].

MicroRNAs are a class of small (18–24) non-coding RNAs that regulate gene expression at post translation levels [7, 8]. This molecular RNAs regulate many biological processes [9].miRNAs expression profiling is now popularized as they have an important role in gene expression networks and are now proved to be biomarkers for disease [10]. Recent studies have shown misregulation of miRNAs in human tumors indicating a role for these molecules in cancer pathogenesis, including cancer onset, progression, and metastasis [11]. They are found to be a new class of cell cycle regulators acting as biomarkers in cancer. Nevertheless, the discovery of microRNAs is a noteworthy progress in medical fields [12]. Recently, their expression has been shown to be specific to tissue and cancer types where they act as tumor suppressors or oncogenes [13]. Clearly, identifying cancer-related genes and understanding their contribution to tumorigenesis are critical steps in controlling cancer [14]. In this regard, given that microRNAs are aberrantly expressed in colorectal cancer (colorectal cancer is a disease in huge need of a biomarker), they are good candidates for extensive investigation [15].

Hsa-miR-1287 is located on chromosome 10q24.2. Research has shown that miR-1287 was downregulated in larynx carcinoma [16]. In another study, it was shown that expression of miR-1287 was upregulated in GC cell line [17]. However, as far as the authors of the present research work are concerned, the expression pattern, clinical relationship, and role of miR-1287 in CRC still remain unknown. Therefore, the present study attempted to investigate the expression levels of miR-1287 in CRC tissues.

Materials and Methods

A total of 40 CRC samples and normal adjacent tissues were collected from 40 patients diagnosed with CRC (i.e., 18 males and 22 females) following colonoscopy and sigmoidoscopy at Imam Reza Hospital (Tabriz, Iran), the first affiliated hospital of Tabriz University of Medical Sciences. All study participants were born in East Azerbaijan, Iran, and none of them had taken chemotherapy or radiotherapy and adjuvant therapy. The study was accepted by the Research Ethics Committee of Imam Reza Hospital in conformity with institutional protocol, and informed consents were obtained from all patients. All samples were collected from November, 2014 to June 2015.

It is noteworthy that the non-tumor counterparts were obtained from a section of the resected specimen at the farthest distance from tumor (>2 cm from tumor). Resected specimens were routinely processed for histopathological assessment.

Sample Preparation and RNA Isolation

Phenol-based RNA extraction was applied using TRIzol reagent (Invitrogen Carlsbad, CA) for all fresh tissue samples, according to the manufacturer’s instructions. To degrade any DNA contamination in extracted RNAs, the researchers performed a 10-μl DNase I treatment reaction by kit (bought from Fermentas, Canada).

Reverse Transcription and Quantitative Real-Time PCR

Reverse transcription was carried out on 120 ng of total RNA in a final volume of 10 μl reaction system. The 10 μl RT reaction mixture was incubated at 37 °C for 60 min, at 85 °C for 5 s, and then held at 4 °C using PrimeScript (R) miRNA cDNA Synthesis Kit (bought from ParsGenom, Iran), according to the manufacturers’ instructions. Then, 90 μl of the RNase-free water was added to dilute the RT product. Real-time PCR was performed using SYBR® Green (ParsGenom, Iran). MiR-1287 and 5 s rRNA (as a control RNA) primers were also purchased from ParsGenom. All PCR reactions, including non-template controls, were run in triplicate using Rotor-Gene Q—QIAGEN Real-time PCR Detection System. Finally, the raw data were analyzed by REST2009 Software. All samples were processed in triplicate. The threshold cycle (CT) was defined as the cycle number at which the fluorescence passed the fixed threshold. A control without a template was included in each experiment. The final products of real-time PCR were confirmed by polyacrylamide gel electrophoresis (PAGE).

Statistical Analysis

The relative expression analysis of miR-1287 was performed by a randomization test using Relative Expression Software Tool (REST) 2009 (http://gene-quantification.com/rest-2009.html). 2-ΔΔCt method was employed to analyze the expression levels of miR-1287 in CRC tissues relative to their matched non-tumor counterparts. The threshold cycle (Ct) of fluorescence for each sample was determined. ΔCt indicated the difference in expression levels with the Ct value between miR-1287 and 5 s rRNA (ΔCt = Ct miR-1287 − Ct 5 s). It, also, confirmed the difference in the ΔCt value between cancer tissues and the matched control (ΔΔCt = ΔCt cancer − ΔCt control). The 2-ΔΔCt value (fold value) was also calculated. It was found that when the fold value was >1, there was a high expression of miR-1287 in the cancer tissues compared to their non-tumorous counterparts. The fold change more than one in expression was defined as increased expression. It should be noted that, in the present study, the expression of miR-1287 was assessed regarding its associations with clinicopathological characteristics using ANOVA test. All the analyses were performed using SPSS 18.0 software (Chicago, IL, USA). All cited P values were two-sided and P values <0.05 were judged as statistically significant. Receiver operating characteristic (ROC) curve was also constructed to evaluate the specificity and sensitivity of predicting CRCs and normal tissues by miR-1287 expression levels. The sensitivity/specificity at various cutoff values was calculated using SigmaPlot 12.5, and P values less than 0.05 were considered to indicate a statistically significant difference.

Fig. 1
figure 1

Differential expression of miR-1287. miR-1287 expression in tumor samples showed a significant increase (3.1 times) compared to normal samples with confidence interval of 95 % (CI = 95 %), P value = 0.03, and P < 0.05

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The Expression Levels of miR-1287 in CRC and Adjacent Noncancerous Tissue (Fig. 1)

To further compare the overall level of miR-1287 expression in colorectal cancer tissues linked to normal tissues, the researchers analyzed the Ct values of all the samples by REST 2009 software. The results of the randomization test showed that mir-1287 expression in tumor samples was 3.1 times more than normal tissues; however, it does not mean that this microRNA can be considered as a tumor marker. With respect to the confidence interval of 95 % (CI = 95 %), P value = 0.03, and P < 0.05, it was revealed that miR-1287 was significantly upregulated in colorectal cancer tissues compared to normal tissues.

The Expression of miR-1287 and Capability of miR-1287 to Function as CRC Tumor Marker (Fig. 2)

As stated before, ROC curves indicated ROC area of 34 %. The expression of miR-1287 with a value of 0.34, P value = 1.98, and P > 0.05 revealed that this microRNA has a low sensitivity and specificity to be regarded as a tumor marker (Fig. 2).

Fig. 2
figure 2

Receiver operating characteristic (ROC) for biomarker in detection of CRCs. The ROC curve was automatically generated from 40 points of cutoff values set by the software SigmaPlot 12.5. The area under the ROC curve (AROC) is 0.034 out of 1, P value = 1.98, and P > 0.05

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The Relationship Between the Expression of miR-1287 and Clinicopathological Features

The results of assessing the relationship between miR-1287 expression and clinicopathological features are shown in Table 1. As shown in Table 1, no significant relationship was observed between the miR-1287 expression and gender (P = 0.241), age (P = 0.699), and histologic grades (P = 0.761).

Table 1 The relationship between miR-1287 expression levels in cancer tissue samples obtained from patients with CRC and clinicopathological characteristics
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Discussion

MicroRNAs have a clear role in the initiation and progression of CRC [18]. Recent studies have shown that the expression of miRNAs is important in colorectal and other types of cancer [19]. Given the importance of the studied issue and considering the fact that CRC rate is increasing in the world, it seems critical to search for appropriate diagnostic tools for the prevention and treatment of this type of cancer in its early stages.

The tumor-specific hypermethylation-mediated silencing of miR-1287 is a relatively frequent molecular event in cervical and breast cancer [20, 21]. Research has also shown that miR-1287 expression increased in most cases of follicular lymphoma [21]. It has been found that miR-1287 was systematically downregulated in tumor tissues and tumor serum samples from patients with breast cancer, compared to adjacent normal tissues [22]. It was reported in another study that the expression of miR-1287 was downregulated in familial ovarian cancer [23]. Because of high sensitivity and specificity of mir-1287 in differentiating early laryngeal carcinoma from normal samples, this classifier was proposed to act as a potential biomarker for the early diagnosis of larynx cancer [16]. Moreover, miR-1287 was reported to be upregulated in the PTX-resistant GC cell line compared to the PTX-sensitive GC cell line [17]. However, its thorough functional and regulating role in all human cancers has not yet been clearly explained.

Conclusion

As far as the authors are concerned, this study is the first report on the expression pattern of miR-1287 in CRC tissues. Present results indicated that miR-1287 was significantly upregulated in CRC samples compared to their normal counterparts. It was revealed that this miRNA was upregulated in CRC samples 3.1 times more than normal samples; however, due to low sensitivity and specificity, it cannot be regarded as a marker. Further investigations are required to clarify its potential clinical diagnostic and therapeutic roles. Moreover, to find the association between these microRNAs and clinicopathological features in detail, other studies should be conducted with numerous sample size. The researchers believe that it is just the beginning of a long and still unknown way of highly promising investigation of mir-1287.