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BACKGROUND: Altered expression of certain microRNAs is known to occur during colorectal carcinogenesis, and they have been demonstrated to result in the acquisition of more aggressive phenotypes. However, little is known about the genome-wide alterations in microRNAs during the neoplastic progression of colorectal cancers.METHODS: Using an ABI microRNA array platform, we simultaneously evaluated the expression of 668 microRNAs in fresh frozen primary colonic adenocarcinomas across the spectrum of AJCC stages (I-IV). Prediction analysis for microarrays (PAM) was used to compare microRNA expression across AJCC stages and build a microRNA stage classifier. Cross-validation was used to evaluate performance of prediction error rate. False discovery rate (FDR) was controlled at 1%. Biological functions of selected microRNAs of interest were evaluated by overexpressing transfections and subsequent in vitro invasion assays. RESULTS: Primary fresh frozen tissues from 65 patients (40 male and 25 female) with a mean age 65 +/- 13 years and with AJCC Stage I (n=7), Stage II (n=22), Stage III (n=18) and Stage IV (n=18) colon cancers, underwent RNA extraction and microRNA array analysis. We identified a seven-microRNA expression signature to differentiate early cancers (Stage I) from those associated with metastatic disease (Stage IV) (Table). We then demonstrated that this signature was able to allow discrimination between Stage II and III primary colon cancers (Table). Interestingly 6 of the 7 differentially expressed microRNAs were downregulated with tumor progression, suggesting tumor suppressive functions. Moreover, all 7 microRNAs were noted to be complementary strand microRNAs (designated by *) which, until only recently, were thought to be non-functional byproducts of microRNA metabolism. In order to evaluate the biologic function of the downregulated microRNAs of our signature, we separately transfected HCT-116, a highly invasive colon cancer cell line, with corresponding precursor microRNAs and confirmed overexpression by quantitative RT-PCR. We demonstrated that overexpressing transfections of 3 of the microRNAs (miR200c*, miR143*, and miR424*) significantly abrogated invasive potential in Matrigel assays. CONCLUSION: With broad high-throughput evaluation of microRNA expression across the spectrum of colon cancer stages, we have identified a seven-microRNA signature that is associated with more aggressive disease. In addition, forced overexpression of 3 of the microRNAs resulted in an attenuation of in vitro invasion, suggesting direct tumor suppressive function and further supporting the biologic importance of complementary strand microRNAs.
Table

		AJCC Stage IV vs. I	AJCC Stage IV vs. I	AJCC Stage IV vs. I	AJCC Stage III vs. II	AJCC Stage III vs. II	AJCC Stage III vs. II	Correlation with Stage Progression (I to IV)	Correlation with Stage Progression (I to IV)
	Regulation	Fold Change	p-value^	ROC	Fold Change	p-value^	ROC	r	p-value^^
7-microRNA signature		0.05	0.0002	0.98	0.31	0.0081	0.80	-0.63	<0.0001
miR-200c*	Down	0.07	0.0004	0.99	0.77	0.5396	0.53	-0.38	0.0016
miR-15b*	Down	0.02	0.0002	0.96	0.12	0.0021	0.75	-0.59	<0.0001
miR-424*	Down	0.08	0.0036	0.91	0.45	0.0445	0.67	-0.53	<0.0001
miR-143*	Down	0.05	0.0220	0.84	0.33	0.1155	0.63	-0.48	0.0001
miR-135a*	Up	11.05	0.0026	0.94	2.06	0.0902	0.66	0.49	<0.0001
miR-378*	Down	0.10	<0.0001	0.98	0.16	0.0001	0.84	-0.60	<0.0001
miR-106b*	Down	0.05	<0.0001	0.96	0.11	0.0007	0.80	-0.55	<0.0001

^ two-sample test
^^ Pearson correlation test
r=correlation coefficient
ROC= Receiver operator characteristic curve