Abstract

Objective

Though the fecal occult blood test is used for colorectal cancer screening worldwide, it does not have a particularly high sensitivity for detecting colorectal cancer. Here we investigated the applicability of the fecal microRNA test to fecal samples that had been used for a previous fecal occult blood test and stored under various conditions.

Methods

Five colorectal cancer patients and five healthy volunteers were enrolled. Fecal samples were stored for 0–5 days at 4°C, room temperature or 37°C. Total RNA was extracted from the fecal occult blood test residuum and microRNA expression was analyzed by real-time reverse transcription polymerase chain reaction.

Results

There were no remarkable differences either in colorectal cancer patients or in controls with regard to the concentration of RNA extracted from the fecal occult blood test residuum in any of the storage groups compared with the samples prepared on day 0 (Group 0). Ribosomal RNA stored at room temperature or 37°C degraded rapidly. In contrast, the ribosomal RNA stored at 4°C remained intact for at least 5 days. The microRNAs in samples stored at 4°C and room temperature were conserved; however, the microRNAs stored at 37°C were significantly degraded compared with Group 0 (P < 0.05). In the residuum stored at 4°C up to 5 days, the relative quantification of miR-106a normalized with miR-24 in colorectal cancer patients was significantly higher than those in healthy volunteers (P < 0.05). In contrast, the quantification of normalized miR-106a was remarkably low in samples stored at room temperature and 37°C.

Conclusions

Fecal microRNA of sufficient quality for reverse transcription polymerase chain reaction analysis was extracted from the fecal occult blood test residuum stored at 4°C for up to 5 days.

INTRODUCTION

Colorectal cancer (CRC) is the third leading cause of cancer-related mortality worldwide (1). To reduce the mortality rate of CRC, the fecal occult blood test (FOBT) has been widely used as a screening test for CRC (2–4). However, large-scale studies have shown that the sensitivity of FOBT is not very high in all subjects using total colonoscopy as a reference standard (5–8). Therefore, several methods for the early detection of CRC have been developed.

MicroRNAs (miRNAs), which are small (18–25 nucleotides in size) non-coding RNA molecules, are known to regulate the activity of specific mRNAs and play a major role in cancer progression. The function of miRNAs is to downregulate the expression of multiple target genes by degrading their mRNAs or blocking their translation into protein through RNA interference (9,10). Several recent studies have reported that fecal miRNA is relatively stable (11), and several fecal miRNAs, such as miRNA-21 (miR-21) (12,13), miR-92a (13), miR-106a (12) and miR-144* (14), are expressed more highly in CRC patients than in healthy volunteers. In addition, we have developed a fecal miRNA test using colonocyte RNA (15).

Hemoglobin in fecal samples stored at 4°C remained stable (16,17); however, hemoglobin degraded rapidly in feces stored at room temperature (RT) or 37°C (18). Thus, the FOBT may be influenced by the storage temperature (19). In contrast, fecal miRNA stored at RT was stable for up to 72 h (13,14) and may be useful for CRC screening. When FOBT is positive, we expect that an auxiliary test using same stool samples can improve the accuracy. Thus, we planned this pilot study.

In the present study, we investigated whether miRNA could be extracted from residual FOBT samples and assessed the suitability of the FOBT samples stored under several conditions for the fecal miRNA test.

METHODS

Study Participants

In the present study, we enrolled five male patients with histologically confirmed CRC, and five healthy volunteers between May 2011 and December 2011 (Table 1). CRC patients with a positive result of FOBT were selected. All CRC patients underwent surgical resection of their primary cancer at the National Cancer Center Hospital East, Kashiwa, Japan. The median age of the patients was 52 years (range, 39–68 years). The primary tumors were located in the sigmoid colon in two patients and the rectum in three patients. The median diameter of the primary tumors was 35 mm (range, 18–45 mm). The clinical stage of the patients according to Dukes' classification was stage A in two patients, stage B in two patients and stage C in one patient. The healthy volunteers consisted of two men and three women without any symptoms or evidence of abnormalities such as adenoma or carcinoma (including hyperplastic polyp), as determined by screening colonoscopy performed at the Research Center for Cancer Prevention and Screening, National Cancer Center, Tokyo. The median age of the volunteers was 57 years (range, 41–73 years). All the patients and all the volunteers were provided with detailed information about this study and provided written consent for participating in the study, which was approved by the institutional review board of the National Cancer Center, Japan.

Table 1.

Characteristics of patients and healthy volunteers

Characteristics Patients (n = 5) Healthy volunteers (n = 5) P value 
Age, years 
 Median 54 57 0.17 
 Range 39–68 41–73 
Sex, no. (%) 
 Male/female 5/0 2/3 0.038 
Tumor location, no. (%) 
 Sigmoid colon 2 (40)   
 Rectum 3 (60)   
Tumor size, mm 
 Median 23   
 Range 18–45   
Histology, no. (%) 
 W/D 3 (60)   
 M/D 2 (40)   
Tumor depth, no. (%) 
 T1 3 (60)   
 T2 1 (20)   
 T3 1 (20)   
 T4   
Dukes' stage, no. (%) 
 A 2 (40)   
 B 2 (40)   
 C 1 (20)   
 D   
FOBT, no. (%) 
 + 5 (100) 0.0016 
 − 5 (100) 
RNA (μg) 
 Median 32.2 24.1 0.089 
 Range 17.7–65.9 16.3–74.4 
Characteristics Patients (n = 5) Healthy volunteers (n = 5) P value 
Age, years 
 Median 54 57 0.17 
 Range 39–68 41–73 
Sex, no. (%) 
 Male/female 5/0 2/3 0.038 
Tumor location, no. (%) 
 Sigmoid colon 2 (40)   
 Rectum 3 (60)   
Tumor size, mm 
 Median 23   
 Range 18–45   
Histology, no. (%) 
 W/D 3 (60)   
 M/D 2 (40)   
Tumor depth, no. (%) 
 T1 3 (60)   
 T2 1 (20)   
 T3 1 (20)   
 T4   
Dukes' stage, no. (%) 
 A 2 (40)   
 B 2 (40)   
 C 1 (20)   
 D   
FOBT, no. (%) 
 + 5 (100) 0.0016 
 − 5 (100) 
RNA (μg) 
 Median 32.2 24.1 0.089 
 Range 17.7–65.9 16.3–74.4 

W/D, well-differentiated adenocarcinoma; M/D, moderately differentiated adenocarcinoma; RNA, RNA extracted from the fecal occult blood test (FOBT) residuum. The differences were analyzed by the Mann–Whitney U test or the χ2 test. P < 0.05 denotes a statistically significant difference.

Fecal Samples and Storage Conditions

Naturally evacuated fecal samples were obtained from the CRC patients prior to surgical resection. Fecal samples were also obtained from the healthy volunteers ∼2 weeks after they underwent a total colonoscopy. All the patients and volunteers were instructed to evacuate the feces into a disposable 5 × 10 cm polystyrene tray (AS ONE, Osaka, Japan). All fecal samples were immediately cooled at 4°C and delivered to our laboratory.

The fecal samples were transferred into the fecal sampling container provided with the FOBT kit, OC-Hemocatch (Eiken Chemical, Tokyo, Japan). The fecal samples were collected from several sites of feces, because the feces were heterogeneous. The fecal samples were divided into 16 groups, one being prepared on the day (day 0) of arrival at the laboratory and the others being stored for 1, 2, 3, 4 or 5 days at 4°C, RT or 37°C. Thus, the storage conditions for the fecal fragments were as follows: Group 0 (processed on the day of arrival); Group 1A (4°C, 1 day); Group 1B (RT, 1 day); Group 1C (37°C, 1 day); Group 2A (4°C, 2 days); Group 2B (RT, 2 days); Group 2C (37°C, 2 days); Group 3A (4°C, 3 days); Group 3B (RT, 3 days); Group 3C (37°C, 3 days); Group 4A (4°C, 4 days); Group 4B (RT, 4 days); Group 4C (37°C, 4 days); Group 5A (4°C, 5 days); Group 1B (RT, 5 days) and Group 5C (37°C, 5 days) (Table 2).

Table 2.

Storage conditions of fecal samples

Storage durations after evacuation (days)
 
Temperature (°C) 
Group 0 1A 2A 3A 4A 5A 
1B 2B 3B 4B 5B RT 
1C 2C 3C 4C 5C 37 
Storage durations after evacuation (days)
 
Temperature (°C) 
Group 0 1A 2A 3A 4A 5A 
1B 2B 3B 4B 5B RT 
1C 2C 3C 4C 5C 37 

RT, room temperature.

Fecal Occult Blood Test

Initially, an immunochemical FOBT was performed using OC-Hemocatch in accordance with the manufacturer's instructions. Briefly, 100 μl samples of the dissolved feces were incubated with the FOBT kit reagents at RT for 5 min. After incubation, a blue line appeared in the control window if the FOBT was performed correctly. The samples in which a blue line appeared clearly in the test window were designated as positive, and the samples in which a line did not appear in the test window were designated as negative. We defined the FOBT scores as positive = 1 and negative = 0. The cut-off value for the concentration of hemoglobin in the FOBT kit was 50 ng/ml.

Total RNA Extraction from the Residuum of FOBT

The solution remaining after FOBT analysis was collected in a fresh 2 ml tube and centrifuged at 700 g for 5 min at 4°C, following which the fecal pellet was collected by removal of the supernatant. Total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Briefly, each fecal pellet was resuspended in 1 ml of QIAzol (Qiagen). Thereafter, 200 μl of chloroform was added, the mixture was vortexed vigorously for 15 s and incubated for 3 min at RT and then centrifuged at 13 000 g for 15 min at 4°C. The upper aqueous phase was transferred to a fresh 1.5 ml tube, and 1.5 volumes of 100% ethanol were added. The solution was mixed thoroughly by pipetting and transferred to an miRNeasy Mini column. After washing with buffer RWT and buffer RPE, total RNA was extracted in 100 μl of RNase-free water. The miRNA was stored at −80°C until use.

The concentration of RNA was measured with a NanoDrop (Thermo Scientific, Wilmington, DE, USA) and the quality of RNA was measured with a 2100 Bioanalyzer (Aligent Technologies, Santa Clara, CA, USA).

cDNA Synthesis and Real-time PCR

cDNA was synthesized using a High-Capacity TaqMan MicroRNA RT Kit (Applied Biosystems, Foster, CA, USA) in accordance with the manufacturer's instructions. The reaction mixture consisted of 5 ng of total RNA, 0.5 μl of 10× RT buffer, 1 μl of 5× specific primer, 0.05 μl of 25× dNTPs (100 mM), 0.06 μl of RNase Inhibitor (20 U/μl) and 0.33 μl of MultiScribe Reverse Transcriptase (50 U/μl) in a final reaction volume of 5 μl. The thermal cycling conditions were as follows: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, followed by incubation at 4°C.

We analyzed the expression of three miRNAs: miR-24, miR-92a and miR-106a. For all of these miRNAs, we used the commercially available TaqMan MicroRNA Assay (Applied Biosystems). The reaction mixture for real-time PCR consisted of 4 μl of template cDNA, 10 μl of TaqMan Fast Universal PCR Master Mix (Applied Biosystems) and 1 μl of 20× primers and probe mixture in a total reaction volume of 20 μl. Real-time PCR was performed with precycling heat activation at 95°C for 20 s, followed by 40 cycles of denaturation at 95°C for 3 s and annealing/extension at 60°C for 30 s, using a 7500 Fast Real-time PCR System (Applied Biosystems).

Statistical Analysis

The miRNA expression data were analyzed using the comparative Ct (threshold cycle) method. In this analysis, the formula for the relative quantification of each gene was as follows: (dCt of each miRNA) = (Ct of each miRNA) − (Ct of each miRNA in Group 0), and (relative quantification of each miRNA) = 2−(dCt of each miRNA). Differences between the two groups were analyzed using the Mann–Whitney U- test or χ2 test, whereas differences among more than two groups were analyzed using Dunnett's test. Statistical analyses were performed using JMP 10 (SAS Institute, Inc., Cary, NC, USA). A P value of <0.05 was considered statistically significant.

RESULTS

Determination of FOBT Score in Samples Stored Under Various Conditions

Initially, FOBT was performed with fecal samples stored under various conditions. The FOBT scores for samples in Group 0 from CRC patients and healthy volunteers were 0.73 ± 0.46 (mean ± SD) and 0, respectively (Table 1). In CRC patients, the FOBT scores for fecal samples stored at 37°C for 1, 4 and 5 days were significantly lower than the score for fecal samples prepared on the day of the arrival (day 0) (P < 0.05), while no significant differences were noted in FOBT scores for samples stored at 4°C and RT compared with Group 0 samples (Fig. 1). Thus, FOBT was stable in the samples stored at 4°C and RT at least for 5 days.

Figure 1.

Fecal occult blood test (FOBT) scores determined using samples stored under various conditions from colorectal cancer (CRC) patients. The FOBT positive and negative scores were 1 and 0, respectively. The differences were analyzed by Dunnett's test compared with Group 0. P < 0.05 denotes a statistically significant difference. *P < 0.05.

Figure 1.

Fecal occult blood test (FOBT) scores determined using samples stored under various conditions from colorectal cancer (CRC) patients. The FOBT positive and negative scores were 1 and 0, respectively. The differences were analyzed by Dunnett's test compared with Group 0. P < 0.05 denotes a statistically significant difference. *P < 0.05.

Total RNA Extracted from FOBT Residuum

Next, the concentration and quality of RNA extracted from the FOBT residuum were analyzed. The median amounts of total RNA extracted from the FOBT residuum of Group 0 samples for CRC patients and healthy volunteers were 32.2 μg (range, 17.7–65.9 μg) and 24.1 μg (range, 6.0–74.4 μg), respectively (Table 1). In CRC patients, no significant differences in RNA concentration were noted in all groups compared with Group 0 samples (Fig. 2A). Moreover, no significant differences in RNA concentration were observed in healthy volunteers in all groups compared with Group 0 (Fig. 2A). Small RNAs, including miRNAs, and degraded RNA and ribosomal RNA (rRNA) from enterobacteria including 16S and 23S rRNA, were detected in RNA extracted from fecal samples for both CRC patients and healthy volunteers (Fig. 2B). RNA stored at 4°C for 5 days was stable compared with that of Group 0 samples. In contrast, 16S and 23S rRNA were not detected in RNA stored at RT or 37°C (Fig. 2B). These results indicated that fecal RNA stored at 4°C was stable up to 5 days after evacuation and that fecal RNA stored at RT and 37°C degraded rapidly.

Figure 2.

RNA concentration and miRNA stability in samples stored under various conditions. (A) Relative RNA concentration compared with Group 0. There were no differences between any of the groups. The differences were analyzed by Dunnett's test compared with Group 0. (B) quality of fecal RNA determined using the Bioanalyzer. Three peaks representing small RNAs, 16s rRNA and 23s rRNA are observed. rRNAs were degraded rapidly when stored at RT and 37°C. (C) miRNA stability compared with Group 0 in CRC patients. miRNA stored at 37°C was significantly more degraded compared with Group 0. The differences were analyzed by Dunnett's test compared with Group 0. P < 0.05 denotes a statistically significant difference. (D) miRNA stability compared with Group 0 in healthy volunteers. miRNA stored at 37°C was significantly more degraded compared with Group 0. The differences were analyzed by Dunnett's test compared with Group 0. P < 0.05 denotes a statistically significant difference.

Figure 2.

RNA concentration and miRNA stability in samples stored under various conditions. (A) Relative RNA concentration compared with Group 0. There were no differences between any of the groups. The differences were analyzed by Dunnett's test compared with Group 0. (B) quality of fecal RNA determined using the Bioanalyzer. Three peaks representing small RNAs, 16s rRNA and 23s rRNA are observed. rRNAs were degraded rapidly when stored at RT and 37°C. (C) miRNA stability compared with Group 0 in CRC patients. miRNA stored at 37°C was significantly more degraded compared with Group 0. The differences were analyzed by Dunnett's test compared with Group 0. P < 0.05 denotes a statistically significant difference. (D) miRNA stability compared with Group 0 in healthy volunteers. miRNA stored at 37°C was significantly more degraded compared with Group 0. The differences were analyzed by Dunnett's test compared with Group 0. P < 0.05 denotes a statistically significant difference.

Stability of miRNA in Samples Stored under Various Conditions

As discussed above, rRNA stored at RT or 37°C was degraded. We further analyzed the stability of miRNA by real-time PCR. All miRNAs in samples stored at 4°C and RT were conserved compared with those in Group 0 samples up to 5 days. However, miRNAs of CRC patients stored at 37°C were significantly degraded in Group 1C, 3C and 5C compared with those in Group 0 samples (Fig. 2C), and those of healthy volunteers were significantly degraded in Group 3C and 5C compared with those in Group 0 samples (Fig. 2D).

Relative Quantification of miRNA by Normalization to miR-24

When gene expression is analyzed by real-time PCR, the target genes are normalized with an internal control gene such as GAPDH or β-2-microglobulin (20). Similarly, miRNA should be normalized with an internal control when performing miRNA expression analysis. However, a universal miRNA suitable for use as an internal control has not yet been determined. In the present study, miR-24 was used as an internal control according to our previous study (15). The relative quantification of miR-92a in fecal RNA extracted from CRC patients was not significant compared with that from healthy volunteers at any storage conditions (P = 0.13) (Table 3 and Fig. 3A). On the other hand, the relative quantification of miR-106a in CRC patients was significantly higher than that in healthy volunteers at 4°C up to 5 days (P < 0.05). The difference at RT and 37°C was not significant between healthy volunteers and CRC patients (Table 3 and Fig. 3B).

Table 3.

Relative quantification of microRNA (miRNA) normalized with miR-24 in Group 0

 CRC patients (mean ± SD) Healthy volunteers (mean ± SD) P value 
miR-92a 0.92 ± 0.48 0.65 ± 0.23 0.13 
miR-106a 0.59 ± 0.18 0.33 ± 0.09 0.0003 
 CRC patients (mean ± SD) Healthy volunteers (mean ± SD) P value 
miR-92a 0.92 ± 0.48 0.65 ± 0.23 0.13 
miR-106a 0.59 ± 0.18 0.33 ± 0.09 0.0003 

CRC, colorectal cancer. The differences were analyzed by the Mann–Whitney U test or the χ2 test. P < 0.05 denotes a statistically significant difference.

Figure 3.

Relative quantification of each miRNA. (A) Relative quantification of miR-92a normalized with miR-24. The relative quantification of miR-92a in CRC patients was no significant difference compared with the relative quantification in healthy volunteers. (B) Relative quantification of miR-106a normalized with miR-24. The relative quantification of miR-106a in CRC patients was significantly higher than the relative quantification in healthy volunteers. The differences were analyzed by the Mann–Whitney U test. P < 0.05 denotes a statistically significant difference. Closed circle and red line, CRC patients; closed rhombus and blue line, healthy volunteers. *P < 0.05.

Figure 3.

Relative quantification of each miRNA. (A) Relative quantification of miR-92a normalized with miR-24. The relative quantification of miR-92a in CRC patients was no significant difference compared with the relative quantification in healthy volunteers. (B) Relative quantification of miR-106a normalized with miR-24. The relative quantification of miR-106a in CRC patients was significantly higher than the relative quantification in healthy volunteers. The differences were analyzed by the Mann–Whitney U test. P < 0.05 denotes a statistically significant difference. Closed circle and red line, CRC patients; closed rhombus and blue line, healthy volunteers. *P < 0.05.

DISCUSSION

The use of fecal DNA testing to improve upon the unsatisfactory sensitivity of FOBT for CRC screening has been investigated since the 1990s (21,22). A large-scale study using this methodology was reported in 2004 (5), yielding results from which the American Cancer Society subsequently updated 2008 guidelines to include the fecal DNA test as a recommended screening test for CRC (23). To date, the fecal DNA test is the only commercially available method of fecal analysis based on molecular biology techniques (24). Regarding the fecal RNA test, fecal COX2 analysis for detecting CRC was reported for the first time in 2004 (25). Recently, various studies in which CRC was detected using fecal miRNA have been reported (12–14,26,27). In Japan, the immunochemical FOBT has been used for CRC screening and initially appeared to have higher sensitivity and specificity than Guaiac FOBT; however, the sensitivity was reported to be 26–69% (3,6,7,28–30). Thus, to improve the sensitivity of FOBT, we evaluated the suitability of fecal miRNA extracted from the FOBT residuum in this study. In the previous study, the detections of fecal miRNA expression were performed from stool samples or colonocytes (12–15,20,27,31). In the present study, we examined the miRNA expression in total RNA extracted from residual FOBT samples.

The immunochemical FOBT has a high sensitivity for the detection of human hemoglobin in feces. However, the detection rate varies according to storage conditions; for example, rates are lower when samples are stored at 37°C than at 4°C or RT. Long RNAs, such as 16s and 23s rRNAs, degraded rapidly at RT and 37°C. In contrast, fecal miRNA was stable at 4°C and RT. Fecal miRNA degraded at 37°C; however, PCR amplification remained successful under these conditions. Indeed, it has been previously reported that miRNA extracted from formalin-fixed paraffin-embedded tissue stored for over 20 years can still be analyzed (32). The relative quantification of miRNAs normalized with an internal control in fecal samples stored at 4°C for up to 5 days was similar to those in Group 0. In contrast, in samples stored at RT and 37°C, the relative quantification of miRNA was significantly reduced significantly. Thus, fecal miRNA extracted from FOBT residuum should be stored under appropriate conditions.

To improve upon the unsatisfactory sensitivity of the FOBT, several tests, including fecal transferrin analysis, have been combined with it. In the present study, the fecal miR-106a normalized with miR-24 in CRC patients was higher than that in healthy volunteers (Fig. 3). Thus, the fecal miRNA test using total RNA extracted from FOBT residuum may potentially assist in improving the sensitivity of CRC screening.

To our knowledge, this is the first study indicating that it is possible to extract miRNA from residual samples after FOBT. Under sub-optimal storage conditions, RNA degraded rapidly; but, we demonstrated that the fecal miRNA test using relative quantification was able to be performed efficiently. However, the present data are preliminary because only five CRC patients were analyzed. Therefore, further evaluation on a large scale must be needed.

Funding

This work was supported by a Grant-in-Aid for the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO) of Japan (Y.K.); Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y.K.); the Innovation Promotion Program from the New Energy and Industrial Technology Development Organization of Japan (Y.M.); the Japan Society for the Promotion of Science (JSPS) through the ‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program)’ initiated by the Council for Science and Technology Policy (CSTP) (Y.M.) and National Cancer Center Research and Development Fund (Y.M.).

Conflict of interest statement

None declared.

Acknowledgements

We thank Ms Noriko F. Abe and Ms Masae Ohmaru for their technical assistance and Ms Kaoru Shiina for secretarial assistance.

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