Our understanding of the molecular mechanisms by which miRNAs modulate cellular signaling pathways will remain incomplete until the full set of miRNA targets is identified and validated. Strategies based on detecting mRNA expression changes, such as microarray analysis, have been used to identify miRNA targets. Because a miRNA may regulate the expression of its targets only through translational inhibition, such mRNA approaches may be inherently limited. Here, we demonstrate the utility of quantitative proteomic approaches to discover targets of miRNAs in an unbiased fashion. Our mRNA based gene expression array data clearly show that miR-143 regulates many of its targets without affecting mRNA abundance (). However, Baek et al observed that mRNA destabilization played a major role in the repression of miRNA targets.10
The reason for the discrepancy may be that different miRNAs in different cells have different mechanisms to regulate their targets.
In total, we identified 93 potential targets of miR-143, which showed ≥ 2-fold downregulation by miR-143 mimic. For example, MAPKAP kinase 2 (MAPKAPK2
), also called MK2, is a serine/threonine kinase. It is a major substrate of p38 MAPK and has been shown to play a pivotal role in inflammatory processes.32
MK2 knockout mice were shown to develop significantly fewer skin tumors compared with wild-type mice.33
Overexpression of miR-143 resulted in 25-fold decreased expression of MK2, which indicates that miR-143 may inhibit tumor growth through inhibiting MK2 expression. MutS homolog 6 (MSH6
) protein is similar to MutS protein in E. coli, which helps in the recognition of mismatched nucleotides in DNA mismatch repair.34
Mutations in the MSH6
gene have been identified in individuals with hereditary nonpolyposis colon cancer (HNPCC) and endometrial cancer.35
protein showed > 4-fold downregulation by miR-143, which suggests that miR-143 might modulate mismatch repair. Tripartite motif-containing 23 (TRIM23
) is a member of the tripartite motif (TRIM) family. TRIM23 is also a member of the ADP ribosylation factor family, which plays a role in the formation of intracellular transport vesicles. Recently, TRIM23
was identified as a cofactor involved in the regulation of NF-kappaB by human cytomegalovirus.36
protein was observed to be downregulated >3-fold by miR-143. Our results also uncover an unexpected link between miR-143 and miRNA transport. Exportin 5 (XPO5
) mediates the export of pre-miRNA from the nucleus to cytoplasm, an essential step in miRNA biogenesis. XPO5
protein not only acts as an export factor but also protects pre-miRNAs from digestion by nucleases.37,38
protein showed almost 3-fold downregulation upon miR-143 overexpression. This result raises the intriguing possibility that miR-143 may regulate miRNA biogenesis in some settings.
Although miRNAs are largely known to downregulate gene expression, a few studies have recently demonstrated that miRNAs can also upregulate their targets under certain circumstances.39–42
For example, Vasudevan et al have shown that miRNA let-7 and synthetic miRNA miRcxcr4 could increase translation of their targets upon cell cycle arrest.42
In another study, Tsai at al found that miR-346 activated activity of receptor-interacting protein 14 by increasing its protein expression.39
In our study, we observed 28 proteins to be upregulated > 1.5-fold and one protein to be elevated > 2-fold by miR-143 - these upregulated molecules could also represent potential targets of miR-143. It is also possible that the upregulation of those molecules may due to indirect effects of miR-143.
Surprisingly, there are two genes showing a change in the opposite direction in luciferase activity compared with protein levels. Both regulator of chromosome condensation 2 (RCC2) and microtubule-associated protein, RP/EB family, member 1 (MAPRE1) are downregulated by miR-143 at protein level, However, both genes showed upregulation in luciferase activity when co-transfected with miR-143 mimic (). This could perhaps be due to some secondary effect of miR-143.
In conclusion, we have identified 93 genes as candidate targets of miR-143 using a global quantitative proteomic approach. Ten of 34 tested target genes are likely to be direct targets of miR-143 as shown by luciferase assays - 8 of these contain perfect 6-mer miR-143 seed matches. These data coupled with results from gene expression arrays, clearly show that miR-143 regulates many of its targets at the translational level without affecting mRNA abundance.