MicroRNA biomarkers predicting risk, initiation and progression of colorectal cancer

Table 1 Summary of experiments relevant to microRNA detection of colorectal cancer

miRNA	Sample size		Findings	Specimen type	Ref.
	Cases (n)	Controls (n)
miR-21 miR-106a miR-17 miR-143 miR-622 miR-654-3p	9 non-advanced adenomas and AA 10 CRC	10 controls (normal colonoscopy)	miR-21, miR-106a: Colorectal neoplasia (adenoma, CRC) patients had higher stool expression of these two miRNA compared to normal colonoscopy subjects (P < 0.05). Adenoma patients had higher stool miR-21 and miR-106a expression compared to CRC patients miR-17, miR-143, miR-622, miR-654-3p: No differences between groups	Stool	Link et al[23]
miR-21 miR-92a miR-31 miR-18a miR-106a	50 AA 200 CRC	80 controls (do not have a current or previous malignancy or inflammatory condition)	miR-21, miR-92a: miR-21 and miR-92a levels in CRC patients and AA patients were significantly higher compared to controls (all P < 0.05). miR-21 yielded an AUC of 0.709 in differentiating AA from controls. miR-92a yielded an AUC of 0.701 in differentiating AA from controls. Both miRNA together yielded an AUC of 0.722 in differentiating AA from controls miR-18a, miR-31, and miR-106a: No significant differences between groups	Serum	Liu et al[24]
miR-21 miR-31	43 AA 60 postoperative patients 186 CRC	53 controls (negative colonoscopic examination, no prior diagnosis of any other malignancy)	miR-21: Serum levels were increased in adenomatous polyp patients compared with controls (P < 0.001). Serum miR-21 levels yielded an AUC of 0.803 (95%CI: 0.669-0.869) in differentiating AA from controls. The sensitivity, specificity, positive predictive value and negative predictive values were 76.8 % and 81.1%, 76.7%, and 81.1%, respectively, at a cut-off value of 0.0013	Serum	Toiyama et al[25]
miR-92a miR-21	44 patients with minor polyp (defined as hyperplastic polyp or adenoma less than 1 cm in diameter) 13 AA 88 CRC	101 controls (asymptomatic individuals)	miR-92a: Stool miR-92a was significantly increased in polyp patients compared with controls (P < 0.0001). Sensitivity of 56.1% for polyp, specificity of 73.3%. Higher sensitivity for AA than minor polyps (P < 0.05). The removal of AA led to a decrease in stool miR-92a level (P < 0.05). miR-21: No difference between polyps and controls	Stool	Wu et al[26]
miR-29a, miR -106b, miR -133a, miR -342-3p, miR -532-3p miR-18a, miR -20a, miR -21, miR -92a, miR -143, miR -145, miR -181b	Marker validation phase 50 AA	Marker validation phase 50 controls (free of colorectal neoplasms)	No statistically significant differences between AA patients and controls for any of the investigated miRNA	Plasma	Luo et al[27]
miR-10a, miR-29a, miR-31, miR-92a, miR-100, miR-125b, miR-184, miR-187, miR-196a, miR-200b, miR-203, miR-17-3p	73 non-advanced adenoma 43 AA 8 CRC	48 controls (polyp-free)	No statistically significant associations with non-advanced adenoma or AA for any of the investigated miRNA	Plasma	Adams et al[28]
miR-34a miR-150 miR-923	Discovery set 8 polyp 16 adenoma 8 CRC (stage I/II) 8 CRC (stage III/IV)	Discovery set 8 controls	miR-34a: Validation cohort: Significantly higher in adenoma group compared to controls (FC 2.09, P = 0.028). Significantly higher in adenoma group compared to the polyp group (FC 2.71, P = 0.002). miR-923: Validation cohort: No significantly different levels	Plasma	Aherne et al[29]
	Validation set 20 polyp 20 adenoma 23 CRC (stage I/II) 14 CRC (stage III/IV)	Validation set 20 controls
miR-18a miR-15b miR-19a miR-19b miR-29a miR-335	Set 1 20 AA 21 CRC Set 2 40 AA 42 CRC	Set 1 20 controls Set 2 53 controls	miR-18a: Set 1 and Set 2: Significantly overexpressed in AA patients compared to controls in both sets. Set 1: Good discriminative capacity in AA patients (AUROC, 0.84; 95%CI: 0.72-0.96; sensitivity [S], 80%; specificity [Sp], 80%). Set 2: Lower discriminative capacity in AA patients (AUROC, 0.64; 95%CI: 0.52- 0.75; S, 72%; Sp, 57%)	Plasma	Giráldez et al[30]
miR-29a, miR-92a,	Large-scale validation 37 AA 100 CRC	Large-scale validation 59 controls (negative results of health examination including blood test, chest X-ray, abdominal ultrasound examination, fecal occult-blood testing, rectal touch, CT scan and colonoscopy. None of these controls had previously been diagnosed with any types of malignancy previously)	miR-29a and miR-92a: Significantly higher in AA compared to controls (P < 0.0001 for miR-29a, P < 0.0001 for miR-92a). Both miRNAs together yielded an AUC of 0.773 (95%CI: 0.669-0.877), sensitivity 73.0% and specificity 79.7%, in discriminating AA. miR-29a: Yielded an AUC of 0.769 (95%CI: 0.669-0.869) for differentiating AA from controls. The sensitivity was 62.2% and specificity 84.7%, at a cut-off value of 1.210 for miR-29a. The odds ratio for cases with miR-29a > 1.210 being associated with AA was 12.20 (95%CI: 4.350-34.237). miR-92a: Yielded an AUC of 0.749 (95%CI: 0.642-0.856) for differentiating AA from controls. Sensitivity 64.9% and specificity 81.4%, at a cut-off value of 1.682 for miR-92a. The odds ratio for cases with miR-92a > 1.682 being associated with AA was 4.56 (95%CI: 1.893-10.988)	Plasma	Huang et al[31]
A panel of 8 miRNAs miR-532-3p + miR-331 + miR-195 + miR-17 + miR-142-3p + miR-15b + miR-532 + miR-652	Initial Screening 9 adenoma 20 CRC (stage III/IV) Validation 16 adenoma 15 CRC (stage I/II) 15 CRC (stage III) 15 CRC (stage IV)	Initial Screening 12 controls (without CR neoplasia) Validation 26 controls (without CR neoplasia)	Initial Screening 15 out of 380 screened miRNAs most dys-regulated in plasma of adenoma patients compared to controls (P < 0.05, FDR: 5%). Validation A panel of 8 plasma miRNAs yielded an AUC of 0.868 (95%CI: 0.76-0.98), sensitivity 88% and specificity 64% in differentiating adenoma from controls	Plasma	Kanaan et al[32]
miR-601 miR-760	Large scale validation 43 AA 90 CRC	Large scale validation 58 controls	miR-601: AUC of 0.638, sensitivity of 72.1% and specificity of 51.7% in differentiating AA from controls miR-760: AUC of 0.682, sensitivity of 69.8% and specificity of 62.1% in differentiating AA from controls miR-601 + miR-760: Significantly decreased in colorectal neoplasia (AA and CRC) compared to controls. Both miRNAs together yielded AUC of 0.683, sensitivity 72.1% and specificity 62.1% in differentiating AA from controls	Plasma	Wang et al[33]
miR-135b miR-31	110 adenomas < 1 cm in size 59 AA 42 IBD 104 CRC	109 controls (normal colonoscopy)	miR-135b: Significantly increased in adenoma subjects (median, 28.4; IQR, 0.2-79.7; P < 0.0001) compared to controls (median, 0; IQR, 0-30.8). No significant difference in IBD subjects compared to controls. AUC of 0.71 for detection of adenoma. Sensitivity of 73% for AA, 61% for adenoma < 1 cm in diameter, 65% for any adenoma and specificity of 68%, at a cut-off of 14 copies/ng of stool RNA. Sensitivity of 44% for adenoma < 1 cm, 46% for AA, and specificity of 80%, at a cut-off of 38 copies/ng of stool RNA. Removal of AA or CRC resulted in a significant reduction of stool miR-135b. miR-31: No significant differences between groups	Stool	Wu et al[34]
miR-18a miR-221	151 adenoma 48 AA 198 CRC	198 controls (normal colonoscopy)	miR-18a, miR-221: No significant up-regulation in adenoma or AA	Stool	Yau et al[35]
A panel of 4 miRNAs miR-19a-3p + miR-223-3p + miR-92a-3p + miR-422a	Validation of the diagnostic performance of the miRNA panel: 73 adenoma 117 CRC	Validation of the diagnostic performance of the miRNA panel: 102 controls (healthy individuals seeking a routine health check- up)	Validation of the miRNA panel The miRNA panel yielded an AUC of 0.765 (95%CI: 0.669-0.845) in differentiating adenoma from controls	Serum	Zheng et al[36]

CRC: Colorectal cancer; AA: Advanced adenomas; IBD: Inflammatory bowel disease.

Table 2 Summary of dietary regulation of microRNAs, potentially relevant to colorectal cancer

MicroRNA	Study population	Diet or nutrient	Analysis method	Findings	Specimen type	Ref.
miR-16 miR-21 miR-34a miR-92a miR-106a miR-146 miR-222	Italian-based 8 vegans 8 vegetarians 8 omnivores	Meat, processed meat, fish, cheese	Food frequency and lifestyle questionnaire	miR-92a was significantly decreased by meat and dairy products, and associated with low body mass index. Weaker associations found between miR-21 levels and vegetable intake	Plasma and stool	Tarallo et al[38]
Let-7d miR-15b miR-107 miR-191 miR-324-5	Sprague-Dawley rats, treated with saline or the carcinogen, azoxymethane	Corn oil vs fish oil in the diet	Effects of diets on the expression of 368 miRNAs in the colonic mucosa	The five identified miRNAs were the most strongly affected by diet X carcinogen actions. The fish fed animals showed the smallest number of differentially expressed miRNAs - interpreted as due to a reduction in inflammation	Colonic mucosa	Davidson et al[39]
miR-1903 miR-467c miR-368 miR-927c	Female athymic nude mice, injected with HT-29 colon cancer cells	Corn oil vs ground walnuts in the diet	Effects of the diets on the expression of four microRNAs in the colon tumours	The first three of these microRNAs were down-regulated and the latter up-regulated in expression. These data were related to significant increases in α-linolenic, eicosapentaenoic, docosahexaenoic and total omega-3 acids, and a decrease in arachidonic acid in the walnut fed mice	Colorectal tumour tissue	Tsoukas et al[40]
miR-155	Young subjects (22 + 2 yr), smokers and non-smokers	High dose vitamin C daily for 8 wk	Expression level of miR-155 in HDL3	miR-155 reduced in HDL fraction by 49% in non-smokers and 75% in smokers after 8 wk supplementation. This effect was related to a reduction in reactive oxygen species	Serum lipoprotein levels	Kim et al[43]
miR-98 miR-92a miR-30e miR-140-5p miR-138	7 different prostate cell models including malignant and non-malignant	30 min treatment with 1a,25(OH)2D3	MiRNA microarray analyses	111 miRNAs showed changed expression levels, but only 5 were seen affected in more than one cell line and only 3 were changed in the same direction	Total mRNA and miRNA from each cell line.	Singh et al[44]
miR-22 miR-29ab miR-134 miR-1207-5p miR-371-5p miR-17 miR-20a	LNCaP human prostate cancer cells	48 h treatment with 100 nmol/L 1,25(OH)2D3 compared with non-treated control, cells	Agilent human microRNA v3 microarrays to measure microRNA expression	Four hundred and twenty genes were up-regulated and 413 genes down-regulated in the 1,25(OH)2D3-treated cells. The most strongly affected are those identified in column 1 (the last two of these miRNAs is downregulated)	Integrative network-based analysis using a publicly available data set	Kutmon et al[45]
miR-155	RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS)	24 h in the presence of EtOH or 20 nmol/L 1,25(OH)2D3	miRNA profiling by microarrays	Several miRNAs were induced by LPS and suppressed by 1,25(OH)2D3, of which miR-155 was on the top of the list, suppressing about 50% of the LPS induction	Total mRNA and miRNA from each cell line	Li et al[46]
miR-22	SW480-ADH and HCT116 colon cancer cells	10-7 mol/L 1,25(OH)2D3 for 24, 48 or 96 h	miRNA profiling by microarrays	Although there were 12 microRNAs that showed differential expression with and without vitamin D, miR-22 showed the most consistent differences	Total miRNA from each cell line	Alvarez-Díaz et al[48]
Let-7f Let-7a miR-151-5p miR-22 miR-221 miR-28-5p miR-552-3p miR-766 miR-99b	Males, generally in good health, with no diabetes or other concomitant diseases	High dose vitamin D3 (20000-40000 IU per week)	Quantitative real-time PCR	In 10 pilot subjects, 136 miRNAs were changed in expression in one or more plasma samples drawn at baseline and after 12 mo of vitamin D supplementation. The twelve miRNAs that showed the greatest change in expression in the pilots were further measured in RNA from baseline and 12 mo plasma samples in 40 subjects given vitamin D and 37 subjects given placebo	Plasma	Jorde et al[47]
miR-122a miR-125b	Fischer 344 rats	0, 12 or 24 mg/kg	Quantitative real-time PCR	Vitamin E sufficiency resulted in increased concentrations of miRNA-122a and miRNA-125b	Liver tissue	Gaedicke et al[49]
miR-625 miR-492 miR-373 miR-22, miR-532-5p miR-106b miR-30b miR-185 miR-203 miR1308 miR-28-5p miR-10b	CaCO2 human colon cancer cells	Selenium-deficient or sufficient medium	Microarray validated with quantitative real-time PCR	Selenium deficiency resulted in altered expression of 12 genes	Total miRNA from combined cells of each treatment	Maciel-Dominguez et al[50]
miR-21	U251 human glioblastoma cells	10, 50 or 100 umol/L Resveratrol for 72 h	Quantitative real-time PCR	Resveratrol inhibited miR-21 expression which in turn suppressed NF-κB activity. However, over-expression of miR-21 could reverse the effect of resveratrol on NF-κB activity and apoptosis	Cell extracts	Li et al[54]
miR-21	Estrogen-dependent MCF-7 and estrogen receptor-negative p53 mutant MDA-MB-468 human breast cancer cells	0, 30 or 60 umol/L	Quantitative real-time PCR	Cells were studied either in tissue culture or as a xenograft in BALB/C female athymic mice miR-21 was up-regulated in DIM-treated MCF-7 cells, but not in the ER negative, p53 mutant MDA-MB-468 cells	Cell extracts	Jin[55]
miR-30b miR-1224-3p miR-197 miR-523-3	HepG2 human hepatocarcinoma cells	3-3’-Diindoyl-methane for 24-96 h 50 mg/L of epigallocatechin gallate (EGCG), 100 mg/L of grape seed extract (GSPE) or 100 mg/L of cocoa proanthocyanidin extract (CPE)	Microarray analysis validated by quantitative real-time PCR	MiR-30b was downregulated by all three treatments, while treatment with GSPE or CPE upregulated miR-1224-3p, miR-197 and miR-532-3p	Cell extracts	Arola-Arnal et al[57]
miR-210 (plus 13 other miRNAs upregulated and 7 down-regulated)	Tobacco carcinogen-induced lung cancer in A/J mice	Purified mouse chow containing 0.4% EGCG	Microarray analysis validated by quantitative real-time PCR	MiR-210 had been previously found upregulated by EGCG in in vitro experiments, but this ranked behind the 13 most strongly upregulated miRNAs (miR-2137, miR-449a, miR-144, miR-486, miR-3107, miR-193, miR-5130, miR-2861, miR-511-3p, miR-763, miR-3473, miR-211, miR-210) or seven most down regulated in this in vivo study	Tumour tissue, all tumours from a single mouse combined to a single sample	Zhou et al[58]