Prediction of biomarker miRNAs signature in colorectal cancer metastasis to liver cancer

Introduction: microRNAs (miRNAs) are frequently dysregulated in colorectal cancer (CRC) primary tumors vs. metastasic to the liver. Objective: Our aim was to prediction of biomarker miRNAs signature of CRC Metastasis to liver cancer. Material and Methods: mRNA and miRNA expression profiles of CRC primary tumors to metastases formed in the liver were downloaded from NCBI Gene Expression Omnibus (GEO) database. miRNAs targets were predicted using Targetscan algorithm. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analysis were performed using DAVID online tool, since mRNA expression profile of liver and colon cancer primary tumors vs. metastasis to the liver were used in background of these analysis. Results: 43 and 58 downand up-regulated miRNAs were obtained from GSE98406 (p-value < 0.05). mRNA expression profile include of 1,048 mRNAs differentially expressed in metastatic and non-metastatic CRC to liver from GSE40367 (p-value < 0.05). The some of the down-regulated miRNAs were significantly enriched in migration signaling and cancer stem cell signaling. Moreover, the some of the up-regulated miRNAs were significantly enriched in negative regulation of cell migration. Conclusions: Some of the miRNAs are many number of target genes that some of these are oncogenes and tumor suppressor genes. It is concluded that differentially expressed miRNAs in metastatic vs. non-metastatic CRC to the liver take part in cell migration and cancer stem cell signaling pathways.


INTRODUCTION
MicroRNAs (miRNA) are short, 18-25 nucleotide-long, noncoding RNA molecules that regulate gene expression by suppressing mRNA translation whose dysregulation has been implicated in most of the cancers (1).miRNAs bind to the 3' noncoding region of the target mRNA and inhibit the expression of multiple target genes (2).Generally, oncogenic miRNAs (oncomiRs) are overexpressed while tumor-suppressive miRNAs are underexpressed in cancers (3).When these oncomiRs or tumor-suppressor miRNAs are stimulated or inhibited, respectively, cancer cell proliferation, metastasis, and/or survival may be significantly induced (3).
The incidence of colorectal cancer (CRC) is one of the highest rates of morbidity and mortality worldwide (4).Many studies are actively pursuing molecular biological analyses of the mechanisms involved in progression of colon cancer (4).
Biomarkers, such as coding and non-coding RNA are more remarkable and play significant roles in many biological processes (5).Many additional researches demonstrated that miRNAs are one of the most important types of cancer biomarkers and are therapeutic targets for CRC (5).Dysregulation of miRNAs were identified in CRC liver metastasis compared to primary CRC (6).
The biggest problem of cancer treatment is the spread of malignant cells from a primary tumor to distant sites or metastasis that is the most common cause of cancer-related mortality (7).Metastasis involves several continuous steps through which cancer cells disseminate and spread from a primary tumor to distant sites and forming secondary tumors in other tissues (7).Many of miRNAs play a critical role in pathological conditions, including tumorigenesis and metastasis (8).Specific metastasis-regulating miRNAs, "metastamirs", govern molecular processes and pathways in malignant progression (8).However, research on the functions and mechanisms of microRNAs (miRNAs) in metastasis has only recently begun (8).So far, it has not been investigated CRC liver metastasis is being regulated by which important miRNAs.Therefore, our aim was to predict the miRNAs and molecular mechanisms of CRC liver metastasis.

Data Sources
The mRNA expression profile of GSE40367 ( in metastatic vs. non-metastatic CRC to liver) was obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo), that is based on the GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array.On the other hand, comparing miRNA Signature of CRC primary tumors compare to metastases formed in the liver of GSE98406 was obtained from the GEO database, based on Affymetrix Multispecies miRNA-3 array.Description of microarray datasets was shown in Table 1.The row data were analyzed with GEO2R.

miRNA Target Prediction
Target genes of all miRNAs were predicted by Targetscan database with context score <−0.2 (http://www.targetscan.org).The predicted targets that became in GSE40367 were used for analysis.

Pathway and Functional Enrichment Analyses
Kyoto encyclopedia of genes and genomes (KEGG) pathway ( 9) and gene ontology (GO) (10) functional analyses were performed to identify significantly enriched pathways and the biological processes of miRNAs target genes, respectively, using the online tool of the database for annotation, visualization and integrated discovery (DAVID, http://david.abcc.ncifcrf.gov/)(11).mRNAs list in series GSE40367 (p-value < 0.05) were uploaded into DAVID database as background tab.miRNAs target genes lists were uploaded into DAVID database.The entire human genome and mRNAs list in series GSE40367 were selected for the background control and we filtered out the specific gene expressed in metastatic vs. non-metastatic CRC to liver of series GSE40367 (p-value < 0.05).

Differentially Expressed mRNAs and miRNAs in Metastatic vs. Non-metastatic CRC to Liver
In this study we collected the miRNA expression profile (Accession: GSE98406) and mRNA expression profiling (Accession: GSE40367) in GEO database.miRNA expression profile include of 43 and 58 down-and up-regulated miRNAs, respectively.mRNA expression profile include of 1,048 mRNAs differentially expressed in metastatic and non-metastatic CRC to liver (p-value < 0.05).The microarray datasets were descripted in Table 1 and miRNAs list and log FC for each miRNAs were shown in Table 2.

Identification of miRNAs Target Genes
Target genes of miRNAs were identified by Targetscan database (with total context score <−0.2).The number of miRNAs target genes were 30,630 and 24,435 for up-and down-regulated miRNAs in metastatic vs. non-metastatic CRC to liver, respectively.

Biological Process Analysis
In order to investigate the roles of miRNAs target genes, gene ontology (GO ) biological processes (BP) module of the DAVID database were used.The GO BP results indicate that the some of the target genes of some down-regulated miRNAs are enriched to leukocyte migration, fibroblast migration, neuron migration, germ cell migration and regulation of endothelial cell migration.For example, ATP1B3, SLC16A1, KITLG and CD44, the target genes of mir-603, mir-4727-5p, mir-3201 and mir-204-3p, respectively, take part in leukocyte migration.The some of the results were shown in Figure 1.
Many of target genes were targeted by both up-and down-regulated miRNAs and involved in both negative and positive cell migration.The complete GO BP results, miRNAs and their target genes were shown in Table 3.

Signaling Pathways Analysis
We performed the KEGG pathways enrichment analysis for miRNAs target genes that up-or down-expressed in metastatic vs. non-metastatic CRC to liver.KEGG pathway indicated that some of the up-regulated miRNAs such as mir-182-3p, mir-660-3p, mir, mir-4711-3p and mir-25 take part in tight junction, gap junction and adherens junction.Some of the up-regulated miRNAs such as mir-1291 and mir 1244 involved in cancer stem cells signaling as MAPK signaling pathway and PI3K-Akt signaling pathway.
But some of the down-regulated miRNAs in metastatic vs. non-metastatic CRC to liver such as mir-4742-3p, mir-887-5p, mir-4468, mir-933, mir-1911 and mir-4668 involved in MAPK, Notch, Wnt and PI3K-Akt signaling pathway.Moreover, some of the down-regulated miRNAs act in cell junctions.The complete results of KEGG pathway for each up-and downregulated miRNAs target genes were shown in Table 4.

DISCUSSION
According to the gene expression profile analysis between metastatic vs. non-metastatic CRC to liver groups, 58 upregulated and 55 down-regulated miRNAs in metastatic vs. non-metastatic CRC to liver were obtained (P.Value < 0.05).On the other hand, 1,048 mRNAs differentially expressed in metastatic vs. non-metastatic CRC to liver were selected (P.Value < 0.05).After target prediction by targetscan algorithm, the miRNAs target genes that become in series GSE40367 were selected and GO BO and signaling pathways were analyzed by DAVID database.Since multiple miRNAs target the same gene, that determines the expression of miRNA target genes, one of the miRNAs targeted many numbers of mRNAs.Recent study indicated that hsa-mir-21 is an oncomir in which in turn regulates the development of CRC (12).These results indicated that after up-regulation of miR-21 expression, the levels of AKT and PI3K protein expression significantly increased (12).Our results were shown that has-mir-21 upregulated in CRC and take part in cell migration.Evidences showed that has-mir-17 contributed to the proliferation and invasion of colorectal cancer and played a critical role in the proliferation and invasion of colorectal cancer (13).We showed that has-mir-17 were up-regulated in CRC and involved in cell migration (14).Moreover, levels of miR-21, and miR-155 increased significantly in human colon tumor samples, compared with normal tissues that have been associated with tumor growth (14) .In these studies, results indicated that has-mir-155 was up-regulated in CRC and take part in PI3K-Akt signaling pathway and cell migration.Jun Qin et al. suggested that has-mir-221 promotes CRC cell invasion and metastasis (15).In our study, has-mir-221 as an up-regulated miRNAs was enriched in some biological processes in cell migration.Our results were shown that some of the miRNAs are many number of target genes that some of these are oncogenes and some are tumor suppressor genes.Also, our results indicated that differentially expressed miRNAs in metastatic vs. nonmetastatic CRC to the liver take part in cell migration and cancer stem cell signaling pathways.The target genes of some of the miRNAs promote negative regulation of cell migration.The results of bioinformatics analysis indicated that many of up-and down-regulated miRNAs involve in cancer stem cell processing in metastatic vs. non-metastatic CRC to liver that were shown in Table 3 and 4.
In conclusion, our study showed that differentially expressed miRNAs in metastatic vs. non-metastatic CRC to the liver take part in cell migration and cancer stem cell signaling pathways.

Figure 1 :
Figure 1: Overall survival time by stage

Table 2 :
ID, names and fold change of up-and down-regulated miRNAs of metastatic vs. non-metastatic CRC to liver

Table 3 :
MiRNAs, their target genes and GO BP results of up-and down-regulated miRNAs of metastatic vs. non-metastatic CRC to liver

Table 3 (
continued): MiRNAs, their target genes and GO BP results of up-and down-regulated miRNAs of metastatic vs. non-metastatic CRC to liver

Table 3 (continued):
MiRNAs, their target genes and GO BP results of up-and down-regulated miRNAs of metastatic vs. non-metastatic CRC to liver

Table 4 :
MiRNAs, their target genes and KEGG pathway results of up-and down-regulated miRNAs of metastatic vs. nonmetastatic CRC to liver

Table 4 (
continued): MiRNAs, their target genes and KEGG pathway results of up-and down-regulated miRNAs of metastatic vs. non-metastatic CRC to liver

Table 4 (
continued): MiRNAs, their target genes and KEGG pathway results of up-and down-regulated miRNAs of metastatic vs. non-metastatic CRC to liver

Table 4 (
continued): MiRNAs, their target genes and KEGG pathway results of up-and down-regulated miRNAs of metastatic vs. non-metastatic CRC to liver